A comparison of two laboratory methods of maintaining field-collected Lymnaea tomentosa for the production of Fasciola hepatica metacercariae

Four replicates of 50 field-collected Lymnaea tomentosa were maintained in two laboratory culture systems and their efficiency and practicability for the production of Fasciola hepatica metacercariae were assessed. There was no significant difference in the total number of metacercariae produced (56 552 from 64 snails in the closed container system and 54 073 from 82 snails in the shallow aquarium system).There was no significant difference between culture systems as assessed by snail survival, the number of infected snails or the number of infected snails harvested for the recovery of metacercariae. Within both systems there was a significant effect of snail size on the number of metacercariae produced. Optimum survival and production of metacercariae was achieved in young adult snails of 5–8 mm shell length at the time of infection.The collection of L. tomentosa from the field habitat and their maintenance in either culture system was shown to be a practical alternative to the breeding of laboratory adapted strains of L. tomentosa for the regular production of large numbers of F. hepatica metacercariae.     

Determining the Prevalence and Seasonality of Fasciola hepatica in Pasture-based Dairy herds in Ireland using a Bulk Tank Milk ELISA

Background

Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected.

Results

A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %–75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving.

Conclusion

This study demonstrates that F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.     

The influence of watering practices on the transmission of Fasciola among sheep in the Ethiopian highlands

As many ewes and lambs watered at concrete ponds in well-drained sites acquired fluke infection as those watered at natural ponds in areas suitable for the snail intermediate hosts of Fasciola hepatica. Fasciola transmission, as evidenced by the increasing number of seropositive hosts in an enzyme-linked immunosorbent assay (ELISA), occurred during the heavy rainy period of July–September and declined with the onset of the dry season, being succeeded by the emergence of faecal positive hosts. This indicates the superiority of ELISA over coprological techniques in the early diagnosis and seroepidemiology of this parasitic infection. Host productivity, as reflected in weight gains and haematological parameters, was not significantly affected by the fluke infections.     

Natural trematode infestation in feral Nebrodi Black pigs: Pathological investigations

Few studies describe the parasites of pigs bred in the wild state, although pigs are a known reservoir of trematode infestation. This article reports the results of a retrospective study carried out from January 2003 to June 2007 on 3021 Nebrodi Black male and female pigs, regularly slaughtered, aged between 8 months and 4 years. Fasciola hepatica and Dicrocoelium dendriticum flukes were detected in 143 (4.37%) of 3021 livers. The predominant histological features were multifocal to diffuse chronic hepatitis, with fibrosis and severely thickened walls of the bile ducts and chronic parietal, sometimes nodular inflammation. F. hepatica infestation was frequently associated with marked hyperplasia and hypertrophy of the submucosal glands. The study results confirm the important role swine play in the transmission of trematode infestations, indicate the prevalence of these parasites in the Nebrodi Park area, and draw attention to the need for a prophylaxis plan to prevent the spread of infestation to ruminants and humans living in the area.     

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11 wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4–7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3–6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33–66 adults), while it was for F. gigantica infection (burden of 17–69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.     

Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10 mg/kg live weight and ketoconazole at a dosage of 10 mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ + inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.     

Macrophages, myofibroblasts and mast cells in a rat liver infected with Capillaria hepatica

We trapped a rat (Rattus norvegicus) infected with Capillaria hepatica. At necropsy, grossly yellowish-white nodules (2-3 mm in diameter) were noted to be scattered on the liver''s surface. Microscopically, granulomatous and fibrotic nodules that contained the eggs and/or adult worms of Capillaria hepatica were detected in the liver. Septal fibrosis was diffusely formed throughout the liver. There were a number of ED1-positive macrophages located in the sinusoids of the pseudolobules. On the double staining, myofibroblasts and mast cells were generally observed within the fibrous septa with the mast cells in close proximity to the myofibroblasts. We suggest that the interactions between macrophages, myofibroblasts and mast cells play a role in the septal fibrosis observed in rats infected by Capillaria hepatica.     

Fascioliasis in New Zealand: A review

Intermediate hosts of Fasciola hepatica in New Zealand are the native species Lymnaea tomentosa, and introduced species, L. columella and L. truncatula. The latter is of little significance in New Zealand. Fascioliasis has become more widespread since 1950 following colonisation of much of the North Island by L. columella. L. tomentosa can produce eggs at 5°C and L. columella at 2°C. L. columella produces about the three times as many eggs as L. tomentosa and the eggs of the former develop more rapidly and over a wider range of temperatures. Both species inhabit ponds and marshes, although L. columella is better suited to pond habitats. Neither species will survive in marsh habitats that dry out in summer. Both species tolerate wide ranges of Ca and Mg concentrations and pH. Marsh habitats of the two species are indistinguishable though there is evidence of subtle differences in habitat preferences. Snail populations are minimal in winter and fluctuate irregularly throughout the rest of the year. In general, L. tomentosa populations contain proportionally more mature snails than those of L. columella. The epidemiology of fascioliasis in New Zealand is discussed. The absence of outbreaks of acute disease, the multiplicity of snail hosts and variations in climate pose problems for investigators. Overwintering infections of snails have been observed in L. tomentosa, but not in L. columella. With the latter host, transmission of Fasciola occurred over 6–7 months from mid-summer to mid-winter.The rate of infection of tracer sheep over 5 years varied but was not related to temperature or increased summer rainfall. Infection rates tended to increase when summers were dry and animals forced to graze the snails habitats. Rates in infection were not related to snail densities but to the numbers of mature snails on habitats. The infection rate of L. columella was extremely low ($\text{1}\text{790}$ snails examined). Sheep appear to be more important than cattle in maintaining Fasciolainfections from year to year. The control of fascioliasis is discussed briefly. It is argued that prevention of infection is preferable to removing infections already acquired, and low this might be achieved by a strategic anthelmintic treatment when L. columella is involved is described. Further studies on the prevalence and epidemiology of fascioliasis in New Zealand are required.     

Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.     

Fasciola hepatica: Attempts to immunise rats using fluke eggs and in vitro culture products

Rats were sensitised on days 0 and 12 by subcutaneous injection of Fasciola hepatica ova and/or the excretion/ secretion products obtained from in vitro maintained adult flukes and then challenged orally with 30 F. hepatica metacercariae on day 14. Worm burdens were determined 3 and 8 weeks after infection. No resistance to challenge infection was detected, but those fluke recovered from rats sensitised with excretion/secretion products were smaller than those from control groups.     

The role of the gut in acquired resistance to Fasciola hepatica in the rat

The role of the gut in acquired resistance to Fasciola hepatica in the rat was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal challenge infection of male outbred Wistar rats previously infected by the oral or intraperitoneal routes.Previous infection given by either route generated signigicant protection against both oral and intraperitoneal challenge. The rats were resistant to Fasciola challenge in the presence of the primary infection or after its removal by anthelmintic treatment.It was concluded that passage of juvenile flukes through the gut was not essential for either the acquisition or the expression of acquired resistance to F. hepatica in the rat.     

Gastrointestinal parasites in greater rheas (Rhea americana) and lesser rheas (Rhea pennata) from Argentina

Few data exist on the parasites of ratites, especially from regions within their natural range. It is only recently that extensive studies on the parasites of ostriches (Struthio camelus) have been published, mainly from European countries where commercial farming has expanded. Two species of ratites are native in South America: the lesser rhea also known as Darwin's rhea (Rhea pennata) and the greater rhea (Rhea americana). Both species are considered near threatened by the IUCN and are included in the CITES’ Appendices I and II, respectively. Parasitological studies have conservation implications, as they allow us to assess the risk of transmission of pathogens from farmed ratites to wild populations. In this study 92 faecal samples from greater rheas and 55 faecal samples from lesser rheas from different localities in Argentine were analyzed to determine their gastrointestinal parasites. In greater rheas the protozoa (Balantidium coli-like and Entamoeba spp.) and helminths (Fasciola hepatica and Deletrocephalus spp.). The protozoa had not previously been cited as parasites of greater rheas in South America. Cysts and/or trophozoites of B. coli-like were found in 16.3% of the samples, while the prevalence of the remaining parasites was below 10%. Lesser rheas harbored the protozoa B. coli-like, Entamoeba spp. and Chilomastix spp. as well as F. hepatica and nematode eggs and larvae. B. coli-like cysts were found in 20.0% of the samples, while the prevalence of the other parasites remained below 5%. Some of them had not been cited as infecting lesser rheas yet.     

The potential impact of exotic Lymnaea spp. on fascioliasis in Australasia

The presence of the following exotic Lymnaea spp. has been recently confirmed in Australia: L. columella (North and South America, South Africa, New Zealand,) L. viridis (China, Japan, Philippines, New Guinea, Pacific Area), L. auricularia rubiginosa (Indochina, Malaysia, Indonesia) and L. peregra (Europe).In the present studies the relative susceptibility of the exotic snails noted above to Fasciola hepatica was investigated. Two of the species, L. columella and L. viridis, were found to be highly susceptible to the Australian strain of F. hepatica and a substantial number of viable metacercariae were produced. However a strong disparity in the host— parasite relationship was evident when the parthenogenetic development of the fluke in the two exotic snails was compared with that in the native L. tomentosa. It was concluded that L. tomentosa is a more suitable intermediate host for F. hepatica than any of the introduced exotic species, but it has to be considered that the host—parasite relationships between new combinations of Fasciola and Lymnaea spp. may improve by adaptation. In view of the results of the present studies the potential danger of fascioliasis spreading to areas currently not endemic in Australasia through introduced exotic snails is discussed with particular reference to Queensland, Western Australia and the Northern Territory.There is strong circumstantial evidence that the exotic snails were introduced with aquatic plants through the relatively free overseas and Australian trade. Similarly, through the introduction of infected snails, dangerous parasites such as F. gigantica of cattle and sheep or Schistosoma ssp. which cause serious diseases of man and animals could enter the subtropical or tropical regions of Australia. Appropriate State and Federal quarantine regulations are essential to prevent further introduction and spread of the snails, and monitoring of the distribution of established exotic snails in Australasia must be continuous.     

The effect of age of host in resistance expressed at the gut level in rats infected with Fasciola hepatica

The development of oral and intraperitoneal infections with Fasciola hepatica in young and old rats showed that the gut was involved in the expression of age resistance. The role of the gut was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal infection of 2-, 6-, 15-, and 35-week-old male Wistar rats and 6- and 15-week-old male DA rats with encysted metacarcariae of F. hepatica.Rats of both strains behaved similarly in their response to F. hepatica infection. For both routes of infection the number of flukes recovered decreased as host age increased. In 22- and 6-week-old rats equal numbers of flukes were recovered at 4 and 10 weeks after oral or intraperitoneal infection. In 15-week-old rats, fluke burdens 4 weeks after infection were significantly greater following intraperitoneal infection than oral infection. A significant loss of flukes from the intraperitoneal infection of 15-week-old Wistar rats occured between 4 and 10 weeks after infection. In 35-week-old Wistar rats there was no significant effect for route or age of infection.As intraperitoneal infection (to by-pass rejection at the gut level) only partially eliminates the age response, additional age related mechanism(s), able to reject flukes at some time after they have entered the peritoneal cavity, must be operative in the peritoneal cavity and/or the liver.     

Wild fallow deer (Dama dama) as definitive hosts of Fasciola hepatica (liver fluke) in alpine New South Wales

To determine the extent to which wild deer are contributing in the transmission of Fasciola hepatica (liver fluke) livers from deer shot by hunters, farmers undertaking population control on their farms and vertebrate pest controllers were collected and frozen. The livers were later thawed, sliced and examined for the presence of adult flukes or evidence of past infection. Livers from 19 deer were examined (18 fallow [Dama dama] and one sambar [Rusa unicolor]). Seventeen of the fallow deer were animals collected on farms near Jindabyne, New South Wales. The remaining fallow deer was collected in the Australian Capital Territory and one sambar deer was collected in north-eastern Victoria. Nine of the 17 deer (53%) from the Jindabyne area were either infected with Fasciola hepatica (liver fluke) or had thickened bile ducts indicating past infection. Infection levels in the infected animals varied widely from 3 liver fluke to over 50 per liver. No sign of infection was present in the deer from the Australian Capital Territory or Victoria. Fallow deer are wide-spread in the Jindabyne area and their population is increasing. It is likely their contribution to the maintenance and distribution of F. hepatica to livestock in the Jindabyne area, and in other livestock rearing areas of south-eastern Australia, is important and increasing.     

Detection of Fasciola hepatica antigen in cattle faeces by a monoclonal antibody-based sandwich immunoassay

A monoclonal antibody-based sandwich immunoassay (mAb sandwich ELISA) was developed for the detection of Fasciola hepatica antigen in the faeces of cattle. The assay was applied to samples from 100 cattle infected with F hepatica, 56 animals with parasitologically proven infections of other parasites and 100 uninfected animals. F hepatica antigen was detected in all the faecal samples from animals with fasciolosis, but none of the samples from the uninfected animals or from those with other parasitic infections had significant levels of F hepatica antigens. The results indicate that the mAb sandwich ELISA is a rapid, simple and useful method for the diagnosis of active F hepatica infection in cattle.     

The prevalence and economic significance of Fasciola gigantica and Stilesia hepatica in slaughtered animals in the semi-arid coastal Kenya

Fasciola gigantica and Stilesia hepatica constrain ruminant productivity. The prevalence and economic losses caused by F. gigantica and S. hepatica in the ruminant production systems of Taveta division of Kenya were estimated in a retrospective appraisal of the slaughter records on the total number of animals slaughtered and livers condemned over the period 1989 to 2004. Only liver condemnations based on infestation by F. gigantica and S. hepatica were considered for purposes of this study. Liver condemnation rates differed significantly between bovines, caprines and ovines (p≤ 0.05) for F. gigantica (26%, 6.6% and 5.2%, respectively) and for S. hepatica (0.4%, 22% and 28%, respectively). The total loss through condemnation of both F. gigantica and S. hepatica infested livers was 4 408 272 KES (Kenyan shillings) (US$ 72 272). The proportion of loss in bovines, caprines and ovines, was 76%, 17% and 7%, respectively. Fasciolosis contributed 3 505 410 KES (79.5%) and S. hepatica infestation 903 210 KES (20.5%) to the total losses due to liver condemnations. The authors feel strongly that there is a need for more work on the two parasites using live animals to determine the prevalence in animals of various ages, species and breeds and the economics of disease control at farm level.     

A preliminary study to understand the effect of Fasciola hepatica tegument on naïve macrophages and humoral responses in an ovine model

Fasciola hepatica, the liver fluke, is a highly evolved endo-parasite that uses various mechanisms to evade the host immune system. The immunosuppressive capabilities of the parasite's excretory/secretory products have been well demonstrated by previous independent studies. However, the role of the parasite's tegument in the immune responses remains to be investigated. In this study, the effect of the tegument and other fractions of adult F. hepatica (excretory/secretory, liver fluke homogenate and liver fluke homogenate without tegument) in the activation of naïve macrophages in vitro was investigated using an ovine model. In addition, an immunoproteomic approach was used to investigate the characteristics of humoral antibody responses developed in sheep against the tegument fraction. The results indicated significantly increased arginase expression in macrophages incubated with the tegument and excretory/secretory fractions. Two dimensional gel electrophoresis of the tegument demonstrated approximately 100 protein spots, with only four of these spots were highly reactive with the positive serum as determined by 2-DE immunoblotting. These results give a preliminary indication that the liver fluke tegument may play role in avoiding hosts’ protective immune responses against itself.     

Fasciola hepatica in wild boar (Sus scrofa) from Italy

Fasciola hepatica is a trematode infecting ruminants worldwide, occasionally reported in a wide range of animal species, including humans. According to the WHO, fasciolosis is recognized as a re-emerging neglected tropical disease, responsible for endemic and epidemic outbreaks in humans. Although the main hosts of the parasite are represented by cattle, sheep and goats, wildlife may be involved in its circulation. Here we firstly report F. hepatica in a wild boar from Italy (southern area) and characterize it both morphologically and molecularly. The nad1 gene analysis of specimens analyzed, revealed a high genetic similarity with those of humans from Iran and Peru, as well as a close phylogenetic relationship to those in ruminants from Brazil, Ecuador and Egypt. Considering the increase in the wild boar populations in urban and peri-urban areas, a potential role of this ungulate in the circulation of this zoonotic trematode is suggested.