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Fasciolosis caused by Fasciola hepatica severely affects the efficiency of livestock production systems worldwide. In addition to the economic impact inflicted on livestock farmers, fasciolosis is an emergent zoonosis. This review emphasizes different aspects of the disease in South America. Available data on epidemiology in bovines and ovines in different countries, as well as a growing body of information on other domestic and wildlife definitive hosts, are summarized. The issue of drug resistance that compromises the long-term sustainability of current pharmacological strategies is examined from a regional perspective. Finally, efforts to develop a single-antigen recombinant vaccine in ruminants are reviewed, focusing on the cases of leucine aminopeptidase or thioredoxin glutathione reductase.

An in vivo study in the laboratory rat model has been carried out to monitor changes to the spermatogenic cells in the testis tubules of adult Fasciola hepatica following treatment with the artemisinins, artemether and artesunate. Rats infected with the triclabendazole (TCBZ)-resistant Sligo isolate were dosed orally with artemether at a concentration of 200 mg/kg and flukes recovered at 24, 48 and 72 h post treatment (pt). Rats infected with the TCBZ-resistant Oberon isolate were dosed orally with artesunate at a concentration of 200 mg/kg and flukes recovered 24, 48, 72 and 96 h pt. The flukes were processed for histological and transmission electron microscope (TEM) examination. Changes to the spermatogenic cells were evident at 24 h pt with artemether. The spermatogonial and spermatocyte cells contained abnormal mitochondria, there were fewer spermatids and spermatozoa in the tubules than normal, and a number of cells showed signs of apoptosis. There was a further decline in cell numbers at 48 h pt and the organization of the spermatocyte and spermatid rosettes was atypical. Sperm formation had become abnormal and those spermatozoa present possessed only a single axoneme. By 72 h pt, the testis tubules were vacuolated and filled with abnormal cells and cell debris. Only spermatogonial cells could be identified and there was widespread evidence of apoptosis in the cells. Distinct cellular changes following artesunate treatment did not become apparent until 48 h pt. The changes seen were similar to those described for artemether, but were generally less severe at matching time-periods. The fine structural changes occurring in the spermatogenic cells were compared to those observed in other cell types and fluke tissues and the overall information was collated to identify the cellular targets for artemisinin action and to establish the time-line for drug action.

Experimental infections of three Egyptian Pseudosuccinea columella populations with sympatric miracidia of Fasciola sp., coming from cattle- or sheep-collected eggs, were carried out to determine the capacity of this lymnaeid to support larval development of the parasite. Using microsatellite markers, the isolates of Egyptian miracidia were identified as Fasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia of F. hepatica were significantly higher than values of 30.4 to 42.2% in snails with bi-miracidial infections. The number of metacercariae ranged from 243 to 472 per cercarial-shedding snail and was independent of snail origin, parasite origin and miracidial dose used for infection. If P. columella was subjected to two successive bi-miracidial infections with F. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported for Radix natalensis infected with the same parasite and the same protocol. Successful experimental infection of P. columella with F. hepatica suggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.

Experimental infections of Galba sp. (origin, Colombia) with allopatric isolates of Fasciola hepatica from France or Fascioloides magna from the Czech Republic were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of either parasite. In snails exposed to F. hepatica, 7 of 400 snails harboured several rediae and only two snails contained a small number of free cercariae on day 50 post-exposure. In contrast, the intensity of F. magna infection in Galba sp. progressively increased from the F1 to F5 generations. Spontaneous cercarial shedding of F. magna occurred in 7 of 100 Galba sp. belonging to the F5 generation and the number of shed cercariae did not differ significantly from that noted in control Galba truncatula of French origin. Galba sp. from Colombia can be added to the list of potential intermediate hosts for F. magna.

Experimental infections of Lymnaea glabra (two populations) with Fasciola hepatica were carried out during seven successive snail generations, to determine if prevalence and intensity of snail infection increased over time through descendants of snails already infected with F. hepatica. Controls were descendants coming from uninfected parents and infected according to the same protocol. No larval forms were found in the bodies of control snails coming from uninfected parents. In contrast, prevalence and intensity of F. hepatica infection in snails originating from infected parents progressively increased from the F2 or F3 to the F6 generation of L. glabra. In another experiment carried out with the F7 generations of L. glabra and a single generation of Galba truncatula (as controls), the prevalence of F. hepatica infection and the total number of cercariae were lower in L. glabra (without significant differences between both populations). If the number of cercariae shed by infected snails was compared to overall cercarial production noted in snails containing cercariae but dying without emission, the percentage was greater in G. truncatula (69% instead of 52-54% in L. glabra). Even if most characteristics of F. hepatica infection were lower in L. glabra, prevalence and intensity of parasite infection increased with snail generation when tested snails came from infected parents. This mode of snail infection with F. hepatica suggests an explanation for cases of fasciolosis occurring in cattle-breeding farms where paramphistomosis is lacking and G. truncatula is absent.

The motility parameters of Fasciola hepatica miracidia were assessed at different temperatures and times post-hatching using computer-assisted sperm analysis. Eggs were incubated at 22 °C or 25 °C for 14 days. Five motion parameters were evaluated at different incubation temperatures up to 10 h post-hatching. No differences were observed in the percentage that hatched after incubation at the two different temperatures. However, the straight-line velocity of miracidia following incubation at 22 °C was significantly different from that observed at 25 °C (P< 0.01). All miracidium motion parameters at different post-hatching temperatures showed an overall decrease at the end of the experiment. Those miracidia hatching from eggs incubated at 25 °C had a higher velocity of 1673.3 μm/s compared with 1553.3 μm/s at 22 °C. Velocity parameters increased as the post-hatching temperature increased from 22 °C to 37 °C.

In Cuba, only two lymnaeid snails, Galba cubensis and Pseudosuccinea columella, with different ecology and distribution patterns, are intermediate hosts for Fasciola hepatica. The compatibility of these two species as hosts was analysed through their rates of infection, the production of rediae and survivorship when exposed to F. hepatica miracidia. Ten populations of G. cubensis, eight of P. columella collected from various habitats and six isolates of F. hepatica sampled in slaughterhouses from different localities were tested. Our results clearly demonstrate that G. cubensis is a more compatible host for F. hepatica in Cuba when compared with P. columella. However, the role that P. columella may have in fascioliasis transmission under certain conditions should not be disregarded. Variation in infectivity among isolates of F. hepatica were also observed and may explain why some regions in Cuba are more commonly subjected to fascioliasis outbreaks.

The main goal of the current work was to develop and validate an in vitro fluke egg hatch test, as a method for the detection of albendazole (ABZ) resistance in the liver fluke, Fasciola hepatica. Fluke eggs (200/ml, n= 5) from six different isolates were used in the current experimental work. They were obtained from different geographical locations and named Cullompton (UK), CEDIVE (Chascomus, Argentina), INTA-Bariloche (Bariloche, Argentina), Rubino (Uruguay), Cajamarca (Perú) and Río Chico (Catamarca, Argentina). The fluke eggs were incubated (25 °C) for a 12-h period in the presence of either ABZ or its sulphoxide metabolite (ABZ.SO) (5, 0.5 or 0.05 nmol/ml). Untreated eggs were incubated as a control. Incubated eggs (with or without drug present) were kept in darkness at 25 °C for 15 days. Afterwards, the trematode eggs were exposed to daylight over a 2-h period. Hatched and unhatched eggs were evaluated using an optical microscope, and the ovicidal activity was assessed for each fluke isolate. A very low ovicidal activity ( ≤ 13.4%) was observed in the ABZ-resistant CEDIVE isolate for both ABZ and ABZ.SO. Conversely, in the INTA-Bariloche and Río Chico isolates, which are suspected to be susceptible to ABZ, ovicidal activities ≥ 70.3% were observed after incubation with ABZ at the lowest concentration tested (0.05 nmol/ml). This finding correlates with that previously described for the ABZ-susceptible Cullompton. Finally, the Cajamarca and Rubino isolates behaved as ABZ resistant, since no ovicidal activity was observed after eggs were incubated with ABZ at 0.5 nmol/ml. Considering the specific results obtained for each isolate under assessment, the egg hatch test described here may be a suitable method for detection of ABZ resistance in F. hepatica.

The development of Fasciola hepatica from two species of definitive hosts, i.e. cattle (Bos taurus) and a marmoset (Callithrix penicillata) in the snail Lymnaea columella was determined based on the production of rediae and cercariae and snail survival rate. More rediae and cercariae at 60-74 days post-infection were produced by snails infected by cattle-derived miracidia (cattle group) than by those infected by marmoset-derived miracidia (marmoset group). Among the L. columella parasitized by the marmoset group, the survival rate and the percentage of positive snails were higher than among those parasitized by the cattle group. Eggs of F. hepatica released in cattle faeces were significantly bigger than those released in marmoset faeces. Miracidia originating from parasites that completed their development in cattle were more efficient in infecting the intermediate host. These results suggest that vertebrate-host origin influences the eggs produced by the parasite and the infection rates in the snail host L. columella.

Experimental infections of Lymnaea columella with Fasciola hepatica were carried out to determine the influence of shell size on the infection rate and on the outcome of rediae and cercariae. Snails were divided into seven groups according to shell size: 2-4 mm, 5-6 mm, 7-8 mm, 9-10 mm, 11-12 mm, 13-14 mm and 15 mm or more. One hundred snails in each group were infected by using four miracidia for each snail. Snails with larger shell size showed a lower infection rate, the groups presenting the highest (79%) and lowest (2%) proportions of positives being those of 5-6 mm and 15 mm or more, respectively. Cercariae were present in 21% of them at 31 days post-infection, and cercarial shedding was observed 61 days post-infection. It was concluded that there is a non-linear negative association between shell size and infection rate.

Erythrograms determined from whole blood analyses and serum analyses for aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) activities, and iron concentration, were used in infected and uninfected cattle to determine the type of anaemia and degree of hepatic damage caused by Fasciola hepatica. Blood samples from 86 infected and 30 uninfected cattle were taken at slaughter. Haematological analyses revealed decreased levels of packed cell volume (PCV), haemoglobin concentration, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in infected compared with uninfected cattle (P < 0.05). A decrease in the concentration of serum iron was also observed in infected cattle compared with uninfected cattle (P < 0.05). Significant increases in AST, GGT and ALP activities were observed in cattle infected with F. hepatica when compared with uninfected cattle (P < 0.05). It was concluded that the anaemia observed in cattle infected with F. hepatica is a normocytic, hypochromic anaemia and the most important aetiology of the anaemia is the chronic blood loss due to the blood-sucking activity of the adult flukes and leakage of blood from the bile duct to the intestine, which results in iron deficiency. The increased activities of serum enzymes indicated chronic hepatic and bile duct injuries associated with chronic infection with F. hepatica.

Laboratory investigations on three aquatic populations of Galba truncatula, originating from the Peruvian Altiplano and French Massif Central, were carried out during three successive snail generations to determine if these populations might be successfully used for the metacercarial production of Fasciola hepatica under experimental conditions. High numbers of surviving snails at day 30 post-exposure (>70%), high prevalences of F. hepatica infections (>60%), and prolonged productions of cercariae for a mean period of 35 to 47 days were observed in the three populations, whatever the snail generation. In the Peruvian population, metacercariae of F. hepatica significantly decreased in numbers from a mean of 251 in the parent snails to 124 per snail in the F2 generation, whereas no significant variation was observed in the two French populations. As these aquatic snails rarely emerged out of water, the use of these populations for the commercial production of F. hepatica metacercariae was of great interest, because the daily time spent watching the breeding boxes of snails was clearly shorter, thereby reducing the cost of producing metacercariae compared with using amphibious snails reared with romaine lettuce.

Studies on the population dynamics and natural infection of Lymnaea columella by Fasciola hepatica were carried out from September 1999 to December 2000 in a low-lying area near Itajubá in the Brazilian State of Minas Gerais. A total of 626 snails were collected monthly at nine different sites, counted, and dissected to search for larvae of F. hepatica. The highest populations of L. columella were reached in October of 1999 and August of 2000, and the highest natural infection rates of snails by F. hepatica were reached in September 1999 (5.2%) and July 2000 (3.9%). The removal by farmers of aquatic plants from the drainage furrows caused a drastic reduction in this snail population.

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.

Rapid freezing and substitution with fixative prior to scanning electron microscopy was used to demonstrate the pattern of beat and recovery of the cilia of free swimming miracidia of Fasciola hepatica. There were stages of dexioplectic metachronal co-ordination and the power stroke was approximately 15 degrees anticlockwise from the anterior-posterior axis. Around the circumference of the body of the miracidia there were approximately 12 metachronal waves of power and recovery. Free-swimming cercariae were recorded by time-lapse photography and, after conventional fixation, by scanning electron microscopy. Cercarial tail-beats were to the posterior of the body in the lateral plane at a rate of 8 Hz. The tail has paired lateral ridges positioned to act as leading edges. There is an array of 32 sensory papillae on the mid-ventral surface of the tail. The tegument of the most distal part of the tail is described: it is free of sensory endings and the surface shows a spiral pattern.

Fasciola hepatica miracidia were experimentally introduced into five sites colonized by Lymnaea palustris over a period of 4 or 6 years. In the first four ponds, a progressive increase in the prevalence of the spring-generation juvenile snails was observed (from 0.4 to 18.1%), with a corresponding increase in the shell height of infected snails (from 3.6 to 7.8 mm). In the fifth habitat, the pond dried in 1990 causing the prevalence to drop as compared with the initial values and to subsequently increase in 1991-1992.

The anterior end of the miracidium of Fasciola hepatica contains a large flask-shaped apical gland and four unicellular lateral glands, all of which have ducts which pass to the tip of the apical papilla. These glands appear to be involved in penetration of the larva into the snail host. The apical gland secretes as the miracidium proves the epidermis of the host before attachment. It seems likely that its secretion is a chemical which lyses the epidermal cells. The lateral glands are PAS-positive and may contain a neutral mucopolysaccharide. They also secrete as the miracidium probes the snail and a layer of PAS-positive material may be seen at the leading edge of the apical papilla as the larva penetrates into the host. Both the apical gland and the lateral glands may be visible in the sporocyst for several days after penetration.