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Pseudosuccinea columella is considered invasive and has become an important intermediate host of both Fasciola species in many regions of the world. This systematic review assessed the geographical distribution of P. columella, and its implications in the transmission of Fasciola hepatica and Fasciola gigantica, globally. A literature search was conducted on Google Scholar, JSTOR and PubMed databases using Boolean operators in combination with predetermined search terms for thematic analysis. Results show that P. columella has been documented in 22 countries from Europe (3), Africa (8), Oceania (2), North America (3) and South America (6). Furthermore, this snail species has shown to adapt to and inhabit a vast array of freshwater bodies including thermal lakes and ditches with acidic soils. Studies showed that P. columella transmits F. hepatica, with natural and experimental infections documented in sub-Saharan Africa, Europe, South America and North America. Experimental infection studies in Cuba showed the presence of P. columella populations resistant to F. hepatica infection. Furthermore, some populations of this invasive snail collected from F. hepatica endemic locations in Brazil, Venezuela, Australia, South Africa, Colombia and Argentina were found without Fasciola infection. As a result, the role played by this snail in the transmission of Fasciola spp. in these endemic areas is still uncertain. Therefore, further studies to detect natural infections are needed in regions/countries where the snail is deemed invasive to better understand the veterinary and public health importance of this snail species in Fasciola-endemic areas and determine the global dispersion of resistant populations of P. columella.

Across Far East Asia, aspermic Fasciola forms are found endemically. They have abnormal spermatogenesis and oogenesis, and it is presumed that their progeny are produced parthenogenetically and clonally. Because of this, they are also termed parthenogenic Fasciola forms. Currently, there is no evidence that they do indeed reproduce parthenogenetically and clonally. In this study, the multilocus genetic type (MLG) in 12 microsatellite markers of adult flukes and their subsequent progeny larvae were analysed using two laboratory aspermic Fasciola triploid strains. The MLGs of adults and their larvae were identical for all markers evaluated, suggesting that these strains reproduce their progeny clonally. The deviation between theoretical and actual frequency within the larvae genotype of the Fh_6 locus resulted in the inability for self-fertilization within individual adult flukes. These findings strongly suggested that aspermic Fasciola forms reproduce their progeny by means of parthenogenesis, possibly gynogenesis.

Across Far East Asia, aspermic Fasciola forms are found endemically. They have abnormal spermatogenesis and oogenesis, and it is presumed that their progeny are produced parthenogenetically and clonally. Because of this, they are also termed parthenogenic Fasciola forms. Currently, there is no evidence that they do indeed reproduce parthenogenetically and clonally. In this study, the multilocus genetic type (MLG) in 12 microsatellite markers of adult flukes and their subsequent progeny larvae were analysed using two laboratory aspermic Fasciola triploid strains. The MLGs of adults and their larvae were identical for all markers evaluated, suggesting that these strains reproduce their progeny clonally. The deviation between theoretical and actual frequency within the larvae genotype of the Fh_6 locus resulted in the inability for self-fertilization within individual adult flukes. These findings strongly suggested that aspermic Fasciola forms reproduce their progeny by means of parthenogenesis, possibly gynogenesis.

Many members of Fasciolidae are common trematodes in cattle, buffaloes, sheep, elephants, pigs, with some capable of infecting humans also. In this study, the complete or near-complete sequences of ribosomal transcription unit (rTU or rDNA), each ofFasciola hepatica(Australia),Fascioloides jacksoni(Sri Lanka),Fasciolopsis buski(Vietnam) and three isolates ofF. gigantica(Vietnam), were obtained and characterized. The full length of rDNA for eachF. hepatica, ‘hybrid’Fasciolasp.,Fas. jacksoniandFa. Buski, was 7657 bp, 7966 bp, 7781 bp and 8361 bp, with the complete intergenic spacer region (IGS) (862 bp, 1170 bp, 987 bp and 561 bp), respectively. The rDNA of two ‘pure’F. giganticaisolates from Vietnam was 6794 bp with unsequenced IGS. For 28S rRNA genes theFasciolaspp. are equal, 1958 bp for 18S, 160 bp for 5.8S, 3863 bp and 454 bp for ITS1 but ITS2 differ by one nucleotide (Thymine) (359 or 360 bp). The ITS1 of thesensu lato Fa. buskihas some distinguishable features, 286 bp for ITS2, 3862 bp for 28S and four repeat units of 356–361 bp each found in ITS1. The 28S rDNA analysis showed the lowest level of divergence (0–0.57%) betweenF. hepaticaandF. giganticaand higher (2.23–2.62%) and highest (6–6.42%) forFas. jacksoniandFasciolopsis, respectively. The tree of 43 strains/species clearly produced a well-supported phylogeny, where 18 fasciolids consistently grouped, forming a discrete Fasciolidae clade, distinct from Philophthalmidae, Echinostomatidae and Echinochasmidae in Echinostomatoidea.Fascioloides jacksoniis outsideFasciolaspp.: basal withFas. magna, as previously demonstrated.

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

An abattoir study was carried out between May and October 2016 to determine the parasitic causes of organ condemnation during meat inspection and to evaluate the attendant financial losses in Fako abattoirs, in the South-West region of Cameroon. Organs (liver, lungs, heart, tongue, kidney, spleen and intestine) were examined at meat inspection following standard procedures and the financial loss was estimated by considering the total weight of condemned organs and the price per kilogram of marketable organs, obtained from the local market. The organs of 1472 cattle were examined, of which 357 (24.38%) were condemned. The organs condemned because of parasitic infestations were the liver (333) and small intestine (24), and the infections were caused by flukes of Fasciola sp. and proglottids of Moniezia sp., respectively. Hydatid cysts and cysticerci were absent. The prevalence of fasciolosis and monieziosis was 22.62% and 1.63%, respectively. Condemnation deprived the region of 665.457 kg of meat, with an associated financial loss of CFA 1,330,902 (USD 2505), during the study period. Parasitic diseases worsen the food insecurity situation as they result in the withdrawal of a considerable amount of meat from the food chain. Fasciolosis, the leading parasitic cause of meat condemnation in Fako, is also zoonotic. It is therefore important that effective control measures are implemented countrywide against this parasitosis.

Trematode infections cause serious economic losses to livestock worldwide. Global production losses due to fasciolosis alone exceed US$3 billion annually. Many trematode infections are also zoonotic and thus a public health concern. The World Health Organization has estimated that about 56 million people worldwide are infected by at least one zoonotic trematode species, and up to 750 million people are at risk of infection. Fasciolosis caused by the flukeFasciola giganticais endemic in Nigeria and is one of the most common causes of liver condemnation in abattoirs. Total cattle losses fromFasciolainfection in Nigeria have been estimated to cost £32.5 million. Other trematode infections of cattle, including paramphistomosis, dicrocoeliasis and schistosomiasis, have all been reported in various parts of Nigeria, with varying prevalence. Most publications on trematode infections are limited to Nigerian local and national journals, with very few international reports. This paper therefore summarized the current data on distribution, control and zoonotic trematode infections in Nigeria and other African countries. We also identified research gaps and made recommendations for future research and areas for funding for policy/planning.

The main intermediate host of Fasciola gigantica in sub-Saharan Africa is Lymnaea (Radix) natalensis. Lymnaea (Pseudosuccinea) columella is capable of transmitting both F. gigantica and F. hepatica and has been reported to be present in South Africa. To date, no natural infection with F. gigantica has been reported despite the wide distribution of the snail. The aim of this study was to confirm whether L. (P.) columella was transmitting F. gigantica and/or F. hepatica in selected locations of KwaZulu-Natal and Eastern Cape provinces of South Africa. Lymnaea (Pseudosuccinea) columella snails were collected from two locations in two provinces of South Africa and screened for cercariae shedding. This was followed by humanely sacrificing the screened snails, and whole tissue of each individual snail was homogenized and amplified using primers designed to amplify the ITS-1 region of Fasciola spp. No cercariae were shed from the screened snails and molecular analysis showed that snails from the two locations were infected with F. gigantica. This study confirms natural infection of L. (P.) columella with F. gigantica in South Africa, where F. gigantica and F. hepatica have already been reported to coexist. Although L. (P.) columella is able to transmit the two species, surprisingly no infection with F. hepatica was detected from the screened snails. The natural intermediate host of F. gigantica in southern Africa, including South Africa, is Lymnaea (Radix) natalensis and comparative studies are needed to determine the competence of the two snail species in the transmission of F. gigantica.

The main intermediate host ofFasciola giganticain sub-Saharan Africa isLymnaea(Radix)natalensis.Lymnaea(Pseudosuccinea)columellais capable of transmitting bothF. giganticaandF. hepaticaand has been reported to be present in South Africa. To date, no natural infection withF. giganticahas been reported despite the wide distribution of the snail. The aim of this study was to confirm whetherL.(P.)columellawas transmittingF. giganticaand/orF. hepaticain selected locations of KwaZulu-Natal and Eastern Cape provinces of South Africa.Lymnaea(Pseudosuccinea)columellasnails were collected from two locations in two provinces of South Africa and screened for cercariae shedding. This was followed by humanely sacrificing the screened snails, and whole tissue of each individual snail was homogenized and amplified using primers designed to amplify the ITS-1 region ofFasciolaspp. No cercariae were shed from the screened snails and molecular analysis showed that snails from the two locations were infected withF. gigantica. This study confirms natural infection ofL.(P.)columellawithF. giganticain South Africa, whereF. giganticaandF. hepaticahave already been reported to coexist. AlthoughL.(P.)columellais able to transmit the two species, surprisingly no infection withF. hepaticawas detected from the screened snails. The natural intermediate host ofF. giganticain southern Africa, including South Africa, isLymnaea(Radix)natalensisand comparative studies are needed to determine the competence of the two snail species in the transmission ofF. gigantica.

Fascioliasis is an important disease affecting livestock, with great costs to producers worldwide. It has also become a serious issue for human populations in some endemic areas as an emerging zoonotic infection. There are two Fasciola species of liver fluke responsible for this disease, which occur worldwide, Fasciola hepatica and Fasciola gigantica. Identifying these two species on the basis of adult or egg morphology requires specialist knowledge due to the similarity of characters, and may misidentify putative intermediate or hybrid forms. In this study we sequenced the internal transcribed spacer 2 (ITS-2) rDNA of liver flukes collected from multiple species of hosts from seven localities in the Punjab and Baluchistan provinces of Pakistan, to determine the distribution of these two species. All 46 flukes processed in this study, collected from seven sites, showed the rDNA ITS-2 genotype corresponding to F. gigantica, contradicting previous reports, based on adult and egg morphology, that both species are present in Pakistan, with F. hepatica being the more common.

Experimental infections of three EgyptianPseudosuccinea columellapopulations with sympatric miracidia ofFasciolasp., 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 asFasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia ofF. hepaticawere 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. IfP. columellawas subjected to two successive bi-miracidial infections withF. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported forRadix natalensisinfected with the same parasite and the same protocol. Successful experimental infection ofP. columellawithF. hepaticasuggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.

Although the efficacy of triclabendazole treatment against Fasciola gigantica has been shown before in cattle and buffaloes, there appears to be no published report on the efficacy of triclabendazole in impala or other antelope species. As part of a health monitoring programme at Mulungushi International Conference Centre, Lusaka, Zambia, a coprological examination was undertaken to investigate the helminthological status of captive impala (Aepyceros melampus). Of 39 fresh coprological samples, 46% contained F. gigantica. The source of infection was identified to be a fountain within the grounds of the conference centre. Lymnaea natalensis, collected from the study site, were induced to shed cercariae, and were thus confirmed as the snail intermediate host. In managing this disease, triclabendazole at 6 mg/kg was administered together with the feed bait. Water from the pond was drained; vegetation within it cleared and the pond allowed to dry for 1 week before water was replaced. Three weeks post-treatment, faecal examination revealed that the Fasciola had been cleared. To the best of our knowledge, this is a first case of Fasciola spp. infection reported in captive wild impala in Zambia and provides evidence that triclabendazole may be delivered to free-ranging antelope using medicated bait.

Gastrointestinal parasites of livestock cause diseases of important socio-economic concern worldwide. The present study investigated the prevalence of gastrointestinal parasites in sheep and goats in lowland and highland regions of Papua New Guinea (PNG). Faecal samples were collected from a total of 165 small ruminants (110 sheep and 55 goats) from February to April 2011. Analysis by a modified McMaster technique revealed that 128 animals (72% of sheep and 89% of goats) were infected with one or more species of gastrointestinal parasites. The gastrointestinal parasites found and their prevalences in sheep (S) and in goats (G) were as follows: strongyle 67.3% (S), 85.5% (G); Eimeria 17.3% (S), 16.4% (G); Strongyloides, 8.2% (S), 23.6% (G); Fasciola, 5.5% (S), 18.2% (G); Trichuris, 1.8% (S), 3.6% (G); and Nematodirus, 1.8% (S), 3.6% (G). Two additional genera were found in goats: Moniezia (9.1%) and Dictocaulus (3.6%). This is the first study to quantitatively examine the prevalence of gastrointestinal parasites in goats in PNG. The high rates of parasitism observed in the present study are likely to be associated with poor farming management practices, including lack of pasture recovery time, lack of parasite control measures and poor-quality feed.

Fasciola giganticafatty acid binding protein (FABP) was evaluated for evoking an immune response in mice, by delivering the gene coding for this protein with mannosylated-polyethylenimine (PEI) to peritoneal cells. Mice were immunized with 50 μg recombinant plasmid DNA (Group I) or DNA-PEI-mannose (a 22 kDa linear cationic polymer with mannose ligand) (Group II) via the intraperitoneal route. Antibody studies showed no significant humoral immune response evoked to this DNA immunization with either PEI-mannose-delivered or naked DNA. However, on protein boosting of these DNA-primed mice there was a significant enhancement of antibody titre. Flow cytometric bead array was used to measure quantitites of interleukin (IL)-2, IL-4, IL-5, interferon-γ (IFN-γ) and tumour necrosis factor (TNF) cytokines. Overexpression of T-helper 1 (Th1) cytokines such as IFN-γ and TNF, with a lower but significant expression of the T-helper 2 (Th2) cytokine IL-5 was detected. Gene delivery using polyethylenimine-mannose ligand showed significant expression of IFN-γ and TNF (P P>0.05) cytokine expression was observed between naked-DNA- and mannosylated PEI-DNA-delivered mice. Naked- or PEI-delivered-DNA immunization produced insignificant levels of IL-2 and IL-4 (P>0.05) cytokines in both groups of mice.

Fasciola giganticafatty acid binding protein (FABP) was evaluated for evoking an effective immune response in mice and rabbits, when delivered as a DNA vaccine in muscle cells. Polyethylenimine (PEI), 25 kDa, branched cationic polymer was used as a delivery vehicle for this DNA in the muscle cells of mice and rabbits. Naked DNA evoked mixed Th1 and Th2 responses in mice. PEI condensed DNA, at amine nitrogen over DNA phosphate (N/P) ratios of 4, 6 and 8 and with various DNA concentrations, failed to evoke a significantly higher antibody response compared to naked DNA in mice. Similarly, the humoral immune response to naked DNA administration in rabbit thigh muscles was poor and no boosting of this antibody response on administration of DNA complexed to PEI was observed. On metacercarial challenge, rabbits failed to show any significant protective immune response in both the naked DNA and PEI–DNA immunized groups. Administration of PEI alone (12.5 μg) in mouse thigh muscles caused significant muscle cytotoxicity but condensation of DNA with PEI had less of a toxic effect on muscle cells, which was inversely related to the N/P ratio. Delivery of plasmid DNA encodingF. giganticaFABP by high molecular weight polyethylenimine (branched, 25 kDa) did not boost the effective immune response in both the animal species, which could either be attributed to cytotoxicity associated with this cationic polymer or muscle cells being unsuitable target cells for PEI condensed DNA delivery.

Wild isolates of the egg-parasitic fungiPaecilomyces lilacinusandVerticillium chlamydosporium, obtained from the organic environment of Durg, Chhattisgarh, India, were subjected to screening forin vitrogrowth using different media types, range of incubation temperature and pH, and their predatory activity to the eggs ofFasciola giganticaandGigantocotyle explanatum. Maximum growth ofP. lilacinuswas obtained in corn-meal agar compared to any other media types. The preferred medium for growth ofV. chlamydosporiumwas corn-meal agar, followed by potato-dextrose agar. After initial growth for 16 h of incubation, no growth was observed in water agar for both the fungi. Six different temperatures – 4°C, 10°C, 18°C, 26°C, 34°C and 40°C – were used to observe growth profiles of the fungi in corn-meal agar medium. While no and very little growth ofP. lilacinusandV. chlamydosporiumwas observed at 4°C and 10°C, respectively, growth profiles of both the fungi were optimal at 26–40°C. A range of pH (pH 4–8) supported growth of bothP. lilacinusandV. chlamydosporium. Full-grown plates of the fungi baited with viable eggs ofF. giganticaandG. explanatumrevealed thatV. chlamydosporiumwas more vigorous in its egg-parasitic ability compared toP. lilacinus. Distortion of the eggs started on day 2–3 of egg baiting in culture plates ofV. chlamydosporium, with complete distortion by day 7. On the contrary,P. lilacinusexhibited very limited egg-parasitic ability and some of the baited eggs even showed development of miracidia.

This study focuses on the development of fibrosis of the liver of cattle with Fasciola hepatica infection, correlating with the intensity of infection. Animals with an established diagnosis of chronic F. hepatica infection were identified in a slaughterhouse in Lima, Perú. The study included 24 fresh cattle livers from infected animals and two uninfected controls. Tissues were stored at 4°C for approximately 8 h after which they were brought to a necropsy room and examined. Between 9 and 12 biopsies were randomly obtained from each liver. Histological staining of formalin-fixed liver sections with haematoxylin and eosin (H & E) and Masson's trichrome were performed. Liver samples were examined using a pathology protocol that included 30 items. Histopathologically, 16 out of 30 liver specimens (67.6%) showed diffuse fibrotic lesions (cirrhosis) with a mean number of Fasciola of 116 ± 30 (range 4–435). Pathological data were matched to number of adult parasites and presence of cirrhosis after being reviewed by two independent pathologists. There was concordance between the two pathologists (K = 0.72). The group with cirrhosis showed an average of 116 ± 30 adult parasites whereas the group not showing cirrhosis contained 56 ± 28 flukes (P = 0.2). To measure how number of flukes and diagnosis of cirrhosis are related we used Kendall's tau-b coefficient; the correlation was +0.296 (P = 0.04). Receiver Operating Characteristic (ROC) curve results showed that the best point was 38 parasite adults, which had 93.8% sensitivity and 75% specificity. We conclude that as the number of F. hepatica adult forms increases, the likelihood of developing liver fibrosis will also increase in cattle.

Vietnam is recognized to be endemic for fasciolosis. However, most of the available publications have not been published in international journals. This review is based on national and international Vietnamese publications and highlights the current status of fasciolosis in Vietnam. It also provides some information available for neighbouring countries. Updated data on responsible species, distribution, transmission and control aspects are summarized. The central region of Vietnam is reported as being highly endemic for fasciolosis, with a high number of human patients (more than 20,000 in 2011). Fasciola gigantica is reported to be the main species in Vietnam. However, hybrids between F. gigantica and F. hepatica were identified. Both humans and animals are infected by the ingestion of raw vegetables and possibly contaminated drinking water. Three lymnaeid snail species (Austropeplea viridis, Radix auricularia and Radix rubiginosa) may act as intermediate hosts of Fasciola spp. However, due to the likely misidentification of snail species and cercariae during the past decade the critical analysis of published data is difficult. A better understanding of transmission aspects of fasciolosis would allow the implementation of preventive measures of this important neglected zoonotic disease.

A serological and coprological survey of fasciolosis was conducted in bovine hosts from the Sargodha district, Pakistan using excretory–secretory (ES) antigens of Fasciola gigantica from cattle and buffaloes. Livers, faecal and blood samples of 146 cattle and 184 buffaloes were collected from slaughterhouses and examined for the presence of any Fasciola in bile ducts and ova in faeces. Serum was separated. ES antigens were prepared by incubating adult Fasciola in phosphate-buffered saline for 6–8 h and then filtering using a 0.22-μm syringe filter. Checkerboard titration was performed and optimum concentrations of antigen and serum were determined. Sero-prevalence was found to be 50.00 and 38.35% in buffalo and cattle, respectively. Using liver examination as the gold standard, enzyme-linked immunosorbent assay (ELISA) sensitivity was found to be 100% in both buffalo and cattle as compared with that of coprological examination in buffalo (61.79%) and cattle (54.54%). This indigenous ELISA was also highly specific, with values of 96.84 and 98.90% in buffalo and cattle, respectively. Positive predictive values were calculated as 96.74 and 98.21% in buffalo and cattle, respectively, while negative predictive values were 100%. For the validation of indigenous ELISA in field surveys, faecal and blood samples were collected from six sub-districts (tehsils) in the district of Sargodha. Sera were screened for the presence of anti-fasciola antibodies using both the indigenous and commercial ELISA kits. While both kits were equally sensitive, the indigenous ELISA was found to be more specific. The highest prevalence of fasciolosis was found in December, as ascertained using both serological and coprological examination. Significant differences were found in prevalences of fasciolosis in different sub-districts and age groups, together with feeding and watering systems.

We determined the prevalence and seasonality of infections by Fasciola of goats and bovine species (cattle and water buffalo) in Hubei and Anhui provinces of China. Faecal samples were collected at 2- to 3-month intervals from 200 goats in Hubei province and from 152 bovine species in Anhui province. All faecal samples were examined for the presence of parasites. We determined the nucleotide sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA (rDNA) of 39 Fasciola worms from Anhui province. The prevalence of Fasciola infection in goats ranged between 3.5 and 37.0%, with mean eggs per gram (EPG) ranging between 29.0 and 166.0. Prevalence and EPG exhibited downward trends over time with significant differences. The prevalence of Fasciola infection in cattle ranged between 13.3 and 46.2% (mean EPG, 36.4–100.0), and that of water buffalo ranged between 10.3 and 35.4% (mean EPG, 25.0–89.6), with a higher prevalence of infection and EPG from June to October compared with December to March. Analysis of ITS-1 and ITS-2 sequences revealed that F. hepatica and F. gigantica were present in all bovine species of Anhui province and that F. gigantica mainly infected water buffalo. This is the first demonstration of Fasciola infection in Hubei province and detection of F. hepatica and F. gigantica in Anhui province. The present study of Hubei province shows that mass treatment of livestock with closantel sodium injections in April and August/September controlled Fasciola infection effectively.

Fascioliasis is an important trematode infection of herbivores worldwide with increasing evidence of prevalence as a disease of humans. Vaccination studies with purified native and recombinantFasciolaantigens suggest that this approach to diminished morbidity and mortality and reduced transmission is a realistic goal. Among the major potential vaccine candidates are fatty acid binding protein (FABP), cysteine (cathepsin) proteases, haemoglobulin, leucine aminopeptidase, and a saposin-like protein. In the case ofFasciola hepaticaFABP, cross-reaction and cross-protection againstSchistosoma mansoniis an important feature. In addition to protective effects with significant worm burden reductions, some vaccine candidates also have anti-fecundity (smaller flukes), anti-pathology (less liver lesions), and anti-embryonation effects. Optimism is tempered by the fact that fascioliasis in humans is an orphan disease and in need of governmental and foundation support.

Considered a secondary zoonotic disease until the mid-1990s, human fascioliasis is at present emerging or re-emerging in many countries, including increases of prevalence and intensity and geographical expansion. Research in recent years has justified the inclusion of fascioliasis in the list of important human parasitic diseases. At present, fascioliasis is a vector-borne disease presenting the widest known latitudinal, longitudinal and altitudinal distribution.Fasciola hepaticahas succeeded in expanding from its European original geographical area to colonize five continents, despite theoretical restrictions related to its biology and in turn dependent upon environmental and human activities. Among the different epidemiological situations, human hypo- to hyperendemic areas, including epidemics, are noteworthy. A global analysis of the distribution of human cases shows that the expected correlation between animal and human fascioliasis only appears at a basic level. Areas presenting very high human prevalences and intensities, especially in children and females, have been recently described. In hypo- to hyperendemic areas of Central and South America, Europe, Africa and Asia, human fascioliasis presents a range of epidemiological characteristics related to a wide diversity of environments. Thus far well-known epidemiological patterns of fascioliasis may not always explain the transmission characteristics in any given area and control measures should consider the results of ecoepidemiological studies undertaken in the zones concerned.

Bimiracidial infections ofLymnaea truncatulawith three isolates ofFasciola gigantica, originating from China, Egypt and Madagascar, were carried out to determine the effect of geographic origin of the parasite on the larval productivity of redial generations. The prevalences of experimental infections in snails exposed to strains from Madagascar, China and Egypt were 20.8%, 60.0% and 80.0%, respectively. At day 49 post-exposure (p.e.), the total number of free rediae in snails infected with the Egyptian isolate was significantly higher than that recorded in the Madagascan group. On the other hand, at day 49 p.e., the majority of cercariae in the Chinese and Egyptian groups were produced by R2a rediae (70.6% and 66.6% of cercariae produced by all live rediae), while, in the Madagascan group, the cercariae were produced mainly by the first redial generation. Snails infected with the Egyptian isolate of miracidia developed more live rediae and, consequently, could produce a higher number of cercariae. As a result,L. truncatulasnails were highly adapted to infections with the Egyptian and Chinese isolates ofF. gigantica.

Experimental infections of Lymnaea truncatula with Fasciola hepatica were performed to study the consequences of the presence of predators (sciomyzid larvae or zonitid snails) on the characteristics of larval F. hepatica development in surviving snails. Controls consisted of infected snails that were not subjected to predators. Compared to controls, the survival rate at day 30 post-exposure, the duration of cercarial shedding, and the number of cercariae shed by surviving snails were significantly lower when predators were present in snail breeding boxes, whatever the type of predator used. In contrast, the prevalences of Fasciola infections in snails, and the length of time between exposure and the onset of cercarial shedding showed no significant variation. The progressive development of a stress reaction in surviving snails against predators during the first 30 days of experimental exposure to F. hepatica would influence snail survival during the cercarial shedding period and, consequently, the number of cercariae shed by the snails.

IgG and IgM antibody responses to fluke cysteine proteinases in Paragonimus ohirai- and Fasciola sp.-infected rats were followed by means of cystatin capture ELISA using fluke excretory-secretory products for 10 weeks after infection. The specific IgG antibodies were detectable at week 2 postinfection in all P. ohirai-infected and some Fasciola-infected rats. Levels of specific IgG antibodies increased rapidly between week 2 and 6, and slightly thereafter, in both infected groups. From week 3, specific IgG antibody levels were higher in Fasciola-infected than P. ohirai-infected rats. Sera from infected rats did not react with heterologous cysteine proteinases throughout the infection periods. In both infected groups, the kinetic patterns of specific IgM antibody responses were similar to those of specific IgG antibody responses although the ELISA levels of the IgM antibody responses were much lower. In abnormal infections with P. ohirai metacercariae x-irradiated at 2 krad, the specific IgG antibodies were detectable at week 2 postinfection with similar ELISA values to normal P. ohirai infection, but thereafter increased little. In infections with P. westermani, for which the rat is not a suitable host, even stunted worms induced a comparable specific IgG antibody response, although the response was lower than in normal infections with P. ohirai. These results indicate that cystatin capture ELISA can distinguish clearly between Paragonimus and Fasciola infections which show immunodiagnostic cross-reactivity and is useful even in the early stages of the infection and in the infection of unsuitable hosts.

Field investigations were carried out over a 4-year period in three farms of the Haute-Vienne department (France) in order to determine the prevalence of Fasciola hepatica infection in the annual generations of Lymnaea truncatula. Infection rates found in March were significantly lower than those recorded in July or in September (0.8% to 2.2% compared to 5.7% to 13.5% and 4.4% to 9.3%, respectively). They were always lower in overwintering snails than in other snail generations (summer generation in 1989,1990 and 1991; spring and summer generations in 1992). Experimental infections of L. truncatula by F. hepatica were performed to evaluate the characteristics of Fasciola infection with reference to snail generation and snail activity. Survival of summer generation snails at day 30 postexposure was significantly lower in snails collected in September than in those collected in May and June. In the winter generation of L. truncatula, snail survival was significantly higher in snails collected from December to March than in those from November. The prevalence of infection was significantly lower in snails collected in September (summer generation) than in those collected from December to March (winter generation). The duration of the prepatent period was significantly delayed in snails collected from January to March (a mean of 54.2 to 58.2 days instead of 43.1 to 49 days in the other groups), whereas that of the patent period was significantly shorter (5 to 9.3 days instead of 18.3 to 40.3 days, respectively). In snails collected from January to March, the number of metacercariae was significantly lower than that found from other snail groups (a mean of 21.5 to 42 metacercariae instead of 72.8 to 151.4 cysts, respectively). Fasciola infections of L. truncatula originating from spring and summer generations were more efficient than those from the winter generation of snails.

Fascioliasis is an important food-borne parasitic disease caused by the two trematode species, Fasciola hepatica and Fasciola gigantica. The phenotypic features of fasciolid adults and eggs infecting buffaloes inhabiting the Central Punjab area, Pakistan, have been studied to characterize fasciolid populations involved. Morphometric analyses were made with a computer image analysis system (CIAS) applied on the basis of standardized measurements. Since it is the first study of this kind undertaken in Pakistan, the results are compared to pure fasciolid populations: (a) F. hepatica from the European Mediterranean area; and (b) F. gigantica from Burkina Faso; i.e. geographical areas where both species do not co-exist. Only parasites obtained from bovines were used. The multivariate analysis showed that the characteristics, including egg morphometrics, of fasciolids from Central Punjab, Pakistan, are between F. hepatica and F. gigantica standard populations. Similarly, the morphometric measurements of fasciolid eggs from Central Punjab are also between F. hepatica and F. gigantica standard populations. These results demonstrate the existence of fasciolid intermediate forms in endemic areas in Pakistan.

The 14.5 kDa fatty acid binding protein (FABP) was isolated from the crude extract of adultFasciola giganticaworms. Polyclonal anti-FABP IgG was generated in rabbits immunized with prepared FABP antigen. Sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect coproantigen in stools and circulatingFasciolaantigen (CA) in sera of 126 water buffaloes by using purified and horseradish peroxidase (HRP)-conjugated anti-FABP IgG. Sandwich ELISA sensitivity was 96.97% and 94.95%; while specificity was 94.12% and 82.35% for coproantigen and CA detection, respectively. However, sensitivity and specificity of the Kato–Katz technique was 73.74% and 100%, respectively. The diagnostic efficacy of sandwich ELISA was 96.55% and 93.1% for coproantigen and CA detection, respectively. In contrast, the diagnostic efficacy of the Kato–Katz technique was 77.59%. In conclusion, these results demonstrate that the purified 14.5 kDa FABP provides a more suitable antigen for immunodiagnosis of early and current bubaline fascioliasis by using sandwich ELISA.

Twelve enzymes (encoded by 14 loci) in liver flukes of Fasciola species originating from Japan (parthenogenetic triploids), Korea (parthenogenetic diploids), the United States of America (USA) and Australia (all sexual diploids) were analysed using starch gel electrophoresis. Variation in electrophoretic patterns between samples was detected at five enzyme loci (Ak, Got, Gpi, 6-Pgd and Pgm-2). Japanese worms (31, of which six were established as uniparental laboratory strains), which reproduce by parthenogenesis, exhibited three different isozyme patterns. This indicates that triploidy has arisen more than once in Japanese flukes. Japanese Fasciola sp. can be separated into three types on morphological grounds. For the six laboratory strains of Japanese worms, the parental morphological type was known. Each of the three isozyme patterns observed was restricted to one morphological type. Most alleles detected in the Japanese triploids were also found in diploid worms from the other countries: the only alleles not represented elsewhere were four at the Got locus and two at the Pgm locus. Flukes from a laboratory strain derived from a single Korean diploid worm resembled the Japanese worms in genotype more closely than did American (seven uniparental laboratory strains) or Australian (30 worms) specimens. Worms from the last two countries were closely related.

This paper provides an estimation of the point prevalence of fascioliasis and its economic impact in terms of increased milk yield after chemotherapy of a bovine population from the district of Toba Tek Singh, Punjab, Pakistan. A total of 2400 cattle and buffaloes were examined quantitatively using the McMaster egg-counting technique. Infected cattle and buffaloes (50 of each) were randomly selected and each divided into two groups of 25 animals. Groups A (buffaloes) and C (cattle) were treated with oxyclozanide (orally, 16.6 mg kg− 1 body weight). Groups B and D served as negative controls for buffaloes and cattle, respectively. Pre- and post-treatment milk yield was recorded to determine if there were any changes in milk yield after treatment. Of 2400 faecal samples analysed, 654 (27.25%) were positive for Fasciola spp. with a mean number of eggs per gram (EPG) of 503.2. The point prevalence and worm burden of fascioliasis was significantly higher (OR = 2.13; P F. gigantica (19.88%; 477/2400) was predominant (OR = 3.12; P F. hepatica (7.38%; 177/2400). An average daily increase of 0.67 and 0.87 litres of milk, with 0.41% and 0.37% more fat per animal, was observed in oxyclozanide-treated buffaloes and cattle, respectively. The economic value of reduced production of infected animals was estimated as US$0.33 and 0.32 per animal per day for cattle and buffaloes, respectively.

The Kafue lechwe antelope (Kobus leche kafuensis), a medium-sized, semi-aquatic antelope, grazes extensively on pastures accessed by livestock in and around Lochinvar and Blue Lagoon national parks in the Kafue wetlands of Zambia. This interaction has a potential for bi-modal transmission of a wide range of parasitic helminths between lechwe and domestic ruminants. A survey was conducted to investigate the status of helminths in the Kafue lechwe during the 2008 (July–December) hunting season, involving 65 animals hunted under special research licences. Worm identification was based on morphological features using standard identification keys. Eleven different types of helminths were identified in the animals studied; namely, Oesophagostomum, Bunostomum, Cooperia, Dictyocaulus, Marshallagia, Stilesia, Setaria, Trichuris, Fasciola, amphistomes and Schistosoma. Amphistomes (100%) and Oesophagostomum (60.9%) were the most common while Fasciola (7.8%) and Stilesia (1.6%) were the least of the identified helminths. There was no evidence that helminths, at intensities observed, adversely affected the health of the lechwe. The degree of worm infection was observed to vary between the two study areas, with Blue Lagoon recording higher infection levels compared to Lochinvar. The host range of many of the helminths found in the Kafue lechwe is broad and could serve as a potentially stable source of infection to domestic animals such as goats and cattle. Therefore, issues concerning livestock management and conservation may arise.

This study investigated interactions amongFasciola gigantica,Schistosomaspp. andAmphistomumspp. concurrent natural infections in Zambian cattle, based on egg and worm counts. In the abattoir 315 cattle were screened for worms ofF. giganticain the liver,Schistosomaspp. in mesenteric veins and/orAmphistomumspp. in the rumen. One hundred and thirty-three (42.2%) of the abattoir-examined cattle harboured one, two or all three trematodes. Of 133 cattle, 50 were randomly selected for worm and egg counts. The mean numbers ( ± SD) ofAmphistomum, SchistosomaandFasciolawere 622.08 ( ± 97.87), 33.68 ( ± 7.44) and 19.46 ( ± 4.58), respectively. A total of 32% harboured all the three trematodes, 66% hadF. giganticaandAmphistomumspp. infections, 52% hadSchistosomaspp. andAmphistomumspp. infections while 32% hadF. giganticaandSchistosomainfections. A positive correlation (P = 0.014) was found betweenF. giganticaandAmphistomumworm burdens. There were no correlations betweenAmphistomumandSchistosomaworm burdens and betweenF. giganticaandSchistosomaworm burdens. It may be concluded that there is no significant cross-protection among these trematodes in cattle in endemic areas.

To estimate prevalence of amphistomiasis and its association with bovine Fasciola gigantica infections in Zambia, 709 faeces of cattle presented for slaughter from three major cattle rearing areas of Central, Southern and Western provinces were analysed. The prevalence rate of amphistomes was 51.6%. Egg counts per gram (EPG) of faeces ranged from 0 to 385 with a mean (± SEM) of 11.96±1.07. The origin of the cattle had a significant influence (PFasciola gigantica infections accounted for 46.7% of the cattle examined. The mean EPG count (± SEM) was 6.3±0.66 with a range of 0 to 223. A total of 34.6% were mixed infections while single Fasciola and amphistome infections represented 12.1% and 17.1%, respectively. Significantly more cattle (63.8%) were infected with either single or both trematode infections (Pr2=0.0428) was present in mixed infections. In order to minimize losses, epidemiology and cross-resistance of amphistomiasis and fascioliasis should be studied in different ecological regions of Zambia to formulate efficient control programmes.

The outer cysts (Oc) ofFasciola giganticametacercariae (Mc) were separated in an aqueous two-phase system composed of polyethylene glycol and dextran in distilled water. A large number of separated Mc without Oc were recovered in the bottom layer rich in dextran in the system. Ten separated Mc in the layer were used to inoculate the mouse with a syringe and juveniles were recovered from the animals on the 14th day. When the number of juveniles recovered was compared with that from the mice infected orally with the same number of Mc, there was a significant difference between them (P

Mitochondrial DNA polymorphism was characterized in a triploid form of Fasciola found in Japan in comparison with F. hepatica, F. gigantica and Korean Fasciola worm. Seventy worms of Fasciola from Japan, three of F. hepatica from Uruguay and Australia, two of F. gigantica from Thailand and one of Fasciola from Korea were used in the study. Mitochondrial DNA polymorphism was detected by restriction fragment length polymorphism (RFLP) using eight restriction enzymes, BamH I, Bgl II, Dra I, EcoR I, EcoR V, Hind III, Mfl I and Sca I. Three different types (types 1, 2 and 3) were detected from 76 Fasciola worms used in the study. Eight of 70 Japanese worms were categorized in type 2 (F. gigantica type), and the remaining 62 were in type 3 (F. hepatica type).

Adult Fasciola gigantica are leaf-shaped with tapered anterior and posterior ends and measure about 35 mm in length and 15 mm in width across the mid section. Under the scanning electron microscope its surface appears rough due to the presence of numerous spines and surface foldings. Both oral and ventral suckers have thick rims covered with transverse folds and appear spineless. On the anterior part of the ventral surface of the body, the spines are small and closely-spaced. Each spine has a serrated edge with 16 to 20 sharp points, and measures about 20 μm in width and 30 μm in height. In the mid-region the spines increase in size (up to 54 μm in width and 58 μm in height) and number, especially towards the lateral aspect of the body. Towards the posterior end the spines progressively decrease in both size and number. The tegumental surface between the spines appears highly corrugated with transverse folds alternating with grooves. At higher magnifications the surface of each fold is further increased with a meshwork of small ridges separated by variable-sized pits or slits. There are three types of sensory papillae on the surface. Types 1 and 2 are bulbous, measuring 4–6 μm in diameter at the base with nipple-like tips, and the type 2 also have short cilia. Type 3 papillae are also bulbous and of similar size but with a smooth surface. These sensory papillae usually occur in clusters, each having between 2 and 15 units depending on the region of the body. Clusters of papillae on the lateral aspect (usually types 1 and 2) and around the suckers (type 3) tend to be more numerous and larger in size. The dorsal side of the body exhibits similar surface features, but the spines and papillae appear less numerous and are smaller. Corrugation and invaginations of the surface are also less extensive than on the ventral side of the body.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.