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.     

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.     

The laboratory maintenance of field-collected Lymnaea tomentosa for the production of Fasciola hepatica metacercariae

A culture method is described for the production of Fasciola hepatica metacercariae in Lymnaea tomentosa collected from the field habitat. Two-litre glass jars, filled with artificial spring (A.S.) water and having a substratum of soil-bentonite (S.B.) agar to provide habitat and food, maintain groups of 40 to 100 snails graded according to size. Small quantities of washed lettuce are fed as a supplement. Compressed air, pre-warmed to 25°C, is piped to each jar to maintain thermoregulation, aeration, filtration and circulation of the water. Infection is achieved by reducing the A.S. water level to 3–4 mm and exposing the snails to miracidia for 6 h. During the aquatic developmental phase of 5–6 weeks, twice-weekly changes of A.S. water and the filter minimise proliferation of protozoans and Chaetogaster spp. Accumulation of mature cercariae is achieved by sustaining the infected snails on moist S.B. agar while humidification within the jar is maintained. Alternatively, groups of five to ten snails may be held in disposable petri dishes lined with S.B. agar. Maturation of cercariae may be delayed by refrigeration at 4–5°C. Upon maximum accumulation, the cercariae are harvested by dissection of the snails and metacercariae are stored in plastic containers at 4°C.     

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.     

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.     

Gallus gallus domesticus: Paratenic host of Angiostrongylus vasorum

Angiostrongylus vasorum, a parasite of the cardiorespiratory system in canids, has a heteroxenous biological cycle in which the intermediate hosts are terrestrial and aquatic mollusks. Generally, canids become infected by ingesting the intermediate host or paratenic hosts, such as amphibians, that contain infective larvae (L3). However, there are no reports of birds as paratenic hosts of A. vasorum. To evaluate the susceptibility and viability of Gallus gallus domesticus as a paratenic host of A. vasorum, 17 Cobb chickens were randomly divided into two groups. The animals in group A were inoculated with third stage larvae of A. vasorum, and those in group B ate snails inoculated with A. vasorum L3. At 30 days post-infection, the chickens were killed, and the muscles and organs were placed in a pepsin–HCl solution (1% HCl (37%), 1% pepsin) for 3 h in an oven at 40 °C to recover the L3. In group A, 1863 L3 were recovered per chicken. In group B, 2585 L3 were recovered. A dog that ingested organs and tissues from a chicken from group A released first-stage larvae of A. vasorum in its feces 51 days after infection; the dynamics of this process were monitored for 107 days, when treatment with 25 mg fenbendazole/kg body weight was performed for 21 days. Chickens nourished with infected snails or with infective L3 may be a source of infection for dogs indicate that G. gallus is a potential paratenic host for this parasite.     

Prevalence of Fasciola in cattle and of its intermediate host Lymnaea snails in central Vietnam

The aims of this study were to investigate the prevalence of natural Fasciola infections in both the definitive hosts (cattle) and the intermediate hosts (Lymnaea snails) in central Vietnam. A total of 1,075 fecal samples, randomly collected from cattle in Binh Dinh, Khanh Hoa, and Phu Yen provinces, were examined for Fasciola eggs by a sedimentation method. The overall prevalence of Fasciola was 45.3?%. A subset of the animals (235) was also screened for antibodies against Fasciola by an enzyme-linked immunosorbent assay. Overall, 46.3?% of these animals were shedding Fasciola eggs while 87.2?% were Fasciola seropositive. A lower prevalence of Fasciola was observed in calves ??2?years of age (37.6?%) compared to that in cattle >2?years of age (53.7?%) (p?<?0.05). The prevalence in the rainy season (50.8?%) was significantly different to that in the dry season (38.1?%) (p?<?0.05). Of the 3.269 Lymnaea viridis and 1.128 Lymnaea swinhoei examined, 31 (0.95?%) and seven (0.62?%), respectively, were found to be infected with Fasciola. This appears to be the first epidemiological survey of the prevalence of Fasciola in cattle and snails in these three provinces in central Vietnam.     

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.     

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.     

Looks can deceive: Molecular identity of an intraerythrocytic apicomplexan parasite in Australian gliders

Two yellow-bellied gliders (Petaurus australis) had an intraerythrocytic parasite closely related to the cyst-forming coccidia (Apicomplexa: Sarcocystidae). The parasitaemia persisted for 3 months or more but was observed to clear within 3 years in captivity. The parasite appears not to significantly debilitate its infected host. Traditionally, using morphological identification, the intraerythrocytic parasite would have been classified within the Hepatozoon species typically found in red blood cells. However, molecular diagnostic techniques targeting the parasite's SSU rDNA and LSU rDNA demonstrated the unusual identity of this blood parasite and disputed its identity as a haemogregarine parasite of the genus Hepatozoon. The sequence was compared with available sequences from diverse mammalian and non-mammalian blood parasites (malaria, piroplasms, hemosporidia and sarcosporidia). The intraerythrocytic blood parasite was found to be most closely related to the cyst-forming coccidia including Besnoitia spp., Cystoisospora spp., Hammondia spp., Hyaloklossia lieberkuehni, Neospora caninum, Sarcocystis spp. and Toxoplasma gondii. The life cycle of this intraerythrocytic parasite remains unknown. The presented DNA identification demonstrates its suitability for an improved identification of blood parasites.     

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.     

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.     

A survey of fascioliasis in beef cattle killed at abattoirs in southern Queensland

The overall prevalence of fascioliasis in beef cattle found in an abattoir survey completed in May 1983 in southern Queensland, was 1.1%. Prevalence was 36% in the Stanthorpe Shire, varied from greater than 1 to 10% in 12 shires and was 1% or less in 24 shires. Fascioliasis was not recorded from the other 46 shires in the area of the survey. Prevalence was less than 1% in female cattle of all ages. In males, prevalence increased with age from 0.6% in 17-month-old cattle to 4.7% in 4-year-old cattle. The differences in sex-specific prevalences may be due to differences in susceptibility to the parasite between sexes, differences in susceptibility to the parasite between sexes, differences in management between sexes, or artefacts associated with sampling for a disease of very low prevalence. The presence of the exotic snail species, Lymnaea columella and L. viridis was confirmed in southern Queensland in 1977. These snails are suitable intermediate hosts for Fasciola hepatica and may widen the endemic area for liver fluke because of their more aquatic character and greater tolerance to high temperatures than L. tomentosa, the traditional snail host in Queensland. We suggest that introduced snails have not yet had a substantial impact on the importance of fascioliasis to the beef industry of southern Queensland. The survey provides baseline data for the future monitoring of this potentially important parasitic disease.     

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.     

A three-year study on seasonal transmission and control of Fasciola hepatica of cattle in Louisiana

Seasonal transmission data on sentinel calves, herd prevalence and snail population dynamics over a 3-year period indicate that major transmission of Fasciola hepatica to cattle occurs between February and July in Louisiana. Soil temperature and water budget were used as indicators of microhabitat suitability for development of snail populations and extra-mammalian stages of F. hepatica and were related to wide annual variation in fascioliasis risk.     

Fasciola hepatica infections in livestock flock, guanacos and coypus in two wildlife reserves in Argentina

Between autumn and spring 2006, a coprological survey was performed in two wildlife reserves located in the north of Argentine Patagonia to determine the prevalence of Fasciola hepatica and the number of parasite eggs per gram (epg) of feces in wild guanacos (Lama guanicoe), coypus (Myocastor coypus), and locally born and raised goats and sheep. Snails of the Family Lymnaeidae were collected in freshwater habitats, identified taxonomically and analyzed parasitologically.Prevalence of patent infection was 100% in sheep (n = 69) and coypus (n = 9), 84% in goats (n = 20) and 0.5% in guanacos (n = 224). No significant differences in epg were found among animals, but the median epg of coypus (160) and sheep (160) was higher than that of goats (80). For guanacos and goats, a negative binomial model estimating the population egg-count frequency could be fitted, while for coypus and sheep parasite egg-count frequencies trended toward a normal distribution, indicative of a more even, and much less aggregated distribution across sampled hosts. All snails (n = 175) were Lymnaea truncatula and none of them was found infected. This is the first report of fascioliasis in free-ranging guanacos in Argentina. Coypu appears to be a major wildlife reservoir of F. hepatica, which was presumably introduced locally by livestock.     

PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails

Fasciolosis, caused by the trematode Fasciola hepatica, is a zoonosis of economic importance in livestock that is emerging as a chronic disease in humans. The intermediate hosts are lymnaeid snails, in which diagnosis of infection is traditionally based on cercarial shedding, tissue sectioning and crushing. We developed a PCR assay for the sensitive and specific detection of F. hepatica in field-collected Lymnaea sp. snails. A primer pair was designed to amplify a 405 bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica. The PCR assay showed a limit of detection of 10 pg of genomic F. hepatica DNA. No cross-reactions were observed with samples from other related trematode species or from the snail hosts Lymnaea columella and Lymnaea viatrix. DNA sequencing of the amplicon showed 100% homology with F. hepatica, and 75-89% homology with other trematodes on regions that did not include the entire set of primers. Two samples from Argentina were analysed. For snails in sample 1 (n = 240), identified as L. columella, the infection rate was 17.5 and 51.3% by direct examination and PCR, respectively. For snails in sample 2 (n = 34), identified as L. viatrix, the infection rate was 2.9 and 61.8% by direct examination and PCR, respectively. Differences in infection rates between these diagnosis methods were significant for both samples. Our PCR technique showed to be effective for detecting specific F. hepatica infections of low intensity in the intermediate host, and hence it could be used to study the epidemiological situation in a given area, as well as to assess host suitability for the parasite.     

Isoenzyme patterns of phosphateses and esterases in Fasciola hepatica and Dicrocoelium dendriticum

A study was made of alkaline and acid phosphatase isoenzymes and non-specific esterases in homogenates of the trematodes Fasciola hepatica and Dicrocoelium dendriticum obtained from infected tissues of Capra hircus and Ovis aries using horizontal polyacrylamide gel electrophoresis.The esterase patterns in F. hepatica and D. dendriticum were different. Homogenate of F. hepaticafrom both hosts gave four enzyme bands. For D. dendriticum, however, while homogenates of parasites from C. hircus also gave four bands, those of O. aries gave only three bands.Acid and alkaline phosphatases in homogenates of both parasites showed three enzyme bands, but there was a host species difference betwen the enzyme patterns of specimens collected from C. hircus versus O. aries.     

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.     

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.