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.     

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.     

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.     

Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens

Liver flukes produce cathepsin B and cathepsin L in their excretory–secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin L's are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.     

Fasciola hepatica: A comparative survey of adult fluke resistance to triclabendazole,nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting,coproantigen ELISA testing and fluke histology

In order to investigate the incidence and distribution of adult fluke resistance to the fasciolicide tricalbendazole (TCBZ) amongst populations of Fasciola hepatica in sheep flocks in Northern Ireland (NI), individual rectal faeces samples were collected from 3 groups of 20 sheep, before (pre-dose), and 21 days after (post-dose) treatment of the animals with TCBZ, nitroxynil or closantel, on each of 13 well-managed sheep farms distributed across the province. The efficacy of each flukicide was determined for each farm, using faecal egg count reduction (FECRT) and F. hepatica coproantigen ELISA testing. In certain flocks, 2 sheep with high pre-dose faecal egg counts (FEC) were killed 3 days and 21 days respectively after TCBZ treatment, and the histology of the fluke reproductive organs was compared with that of flukes from untreated sheep, and from sheep treated with nitroxynil or closantel 2 days prior to death, using haematoxylin and eosin (H&E) staining and an in situ hybridisation method (TdT-mediated dUDP nick end labelling [TUNEL]) to demonstrate apoptosis. Results from FECRT revealed that in all flocks with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis, and this finding was generally supported by the results of the coproantigen reduction test (CRT). The histology of reproductive organs of flukes from TCBZ-treated sheep in these flocks was normal, when compared with untreated flukes, and this, together with the FECRT and CRT findings, indicated a likely diagnosis of TCBZ resistance in all the flocks with a high fluke burden. In contrast, nitroxynil and closantel were found to be fully effective against TCBZ-resistant flukes in each of the flocks bearing a high chronic fluke burden. All of the flocks with a high fluke burden and TCBZ resistance were managed on lowland in the South and East of NI. Upland flocks, in the North and West, had low fluke burdens, or were clear of infection; and FECs were too low to allow valid resistance testing. The study highlights the high level of penetration of TCBZ resistance throughout F. hepatica populations in areas of intensively managed sheep production with a high level of fluke challenge. Further, it emphasises the importance of pre-emptive chemotherapeutic action against chronic fasciolosis, using flukicides effective against the egg-producing adult flukes to minimise pasture contamination for the next season's lamb crop. This study also exemplifies the use of several complementary methods (FECRT; CRT; fluke histology; comparative anthelmintic efficacy testing) for confirmation of a diagnosis of fluke drug resistance.     

Fasciola hepatica: Histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates,and in field-derived flukes from triclabendazole-treated hosts,using in situ hybridisation to visualise endonuclease-generated DNA strand breaks

Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1–4 days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the testis of untreated Cullompton (TCBZ-S) and Sligo Type 2 (TCBZ-R) flukes, which exhibit abnormal spermatogenesis and spermiogenesis, respectively. This was attributed to apoptosis and to heterophagy of effete germ line cells by the sustentacular tissue. It is concluded that demonstration of apoptosis by in situ hybridisation using the TUNEL method on sections of 1–4 days in vivo TCBZ-treated F. hepatica can contribute to the diagnosis of TCBZ resistance in field outbreaks of fasciolosis.     

Precipitating antibodies against excretory/secretory antigens of Fasciola hepatica in sheep serum

Gel diffusion techniques were used to study antigen-antibody reactions in precipitates forming around juvenile, semi-mature and adult Fasciola hepatica cultured in serum from infected sheep. The number of reactions was analysed in both primary and challenge infections. The number of antigens shared by the various developmental stages of the fluke was also examined.At least 2 antigens were involved in precipitate formation around juvenile flukes. These antigens were also produced by the later stages of development. Two additional antigens were produced by semi-mature flukes and these, together with two others, were produced by adult flukes.Challenge infections had little effect on the number of antigens or antibodies in the precipitate.     

Early Pathological Changes Associated with Fasciola hepatica Infection in White-tailed Deer

Three white-tailed deer (Odocoileus virginianus) were inoculated with 1000 metacercariae of Fasciola hepatica and examined on days 7, 14, and 28 postinoculation to determine the early response of a resistant host to this infection. It was concluded that only small numbers of the metacercariae penetrated the intestinal wall into the peritoneal cavity. Flukes that migrated to the liver penetrated through Glisson's capsule, primarily on the parietal surface. Marked fibroplasia and cellular infiltration of the capsule were induced and flukes were killed and destroyed in granulomas immediately beneath the capsule. Migration in hepatic parenchyma was minimal and immature flukes or migratory tracks were not found. There were infiltrations of eosinophils and mononuclear cells, bile duct hyperplasia and fibroplasia in portal areas. A few flukes penetrated through the diaphragm within 14 days postinoculation and on day 28 granulomas were observed on the dorsal surface of the lung where F. hepatica had penetrated this organ. The early reaction of Glisson's capsule to F. hepatica infection in white-tailed deer has not been described in cattle, sheep or swine infected with this fluke.     

The response of rabbits to repeated infections withFasciola hepatica

Rabbits developed a partial resistance to infection withFasciola hepatica following two previous infections of four weeks duration, which had been terminated by chemotherapy. Serum glutamic dehydrogenase assays 4–5 weeks post infection correlated negatively with both the number of flukes derived from the challenge infection and with the number of previous infections. Peripheral eosinophil counts did not give similar correlations.     

Maintenance in vitro of adult Fasciola hepatica in a continuous-flow system

A continuous-flow apparatus is described for the maintenance of groups of adult Fasciola hepatica. The ammonia produced by the flukes was used as a measure of their metabolic activity and it was shown that this is best maintained in culture fluids containing a large proportion of bovine serum. More ammonia per fluke was consistently produced when the flow rate of the medium was increased but including small pieces of clotted sheep blood or increasing the number of flukes per culture had no significant effects.     

The activity of triclabendazole against immature and adult Fasciola hepatica infections in sheep

SUMMARY The efficiency of a new benzimidazole anthelmintic, triclabendazole, was tested against cumulative infections with Fasciola hepatica aged 1 to 12 weeks in sheep and compared with that of rafoxanide. At 10 mg/kg, triclabendazole was 99% effective in eliminating both immature and adult flukes. At a lower dose rate of 5 mg/kg, triclabendazole was highly effective against adults and significantly reduced the number of early immature flukes with an 87% overall reduction of fluke burden. Rafoxanide at 7.5 mg/kg showed high efficiency against adult fluke, but its effect on immatures was not significant, and overall efficiency was 64%.     

Failure to induce homologous immunity to Fasciola hepatica in sheep vaccinated with irradiated metacercariae

Vaccination of sheep with either 100 or 1000 γ-irradiated (2.5 krad) metacercariae of Fasciola hepatica, on two occasions six weeks apart, did not generate significant protection against intraruminal challenge with F. hepatica six weeks after the second vaccinating dose as measured by recovery of flukes from liver and bile ducts, twenty weeks after challenge. There was, however, a significant increase in the proportion of flukes retarded in the parenchyma of both vaccinated groups. The percentage of retarded flukes was positively correlated with the degree of liver damage and increased weight of the hepatic lymph nodes. It was not possible to determine if the retarded flukes were derived from the vaccine or challenge infections or both.Challenge infection of both vaccinated and unvaccinated sheep significantly increased the numbers of eosinophils and globule leucocytes in the parenchymal bile duct and the numbers of mast cells and globule leucocytes in the abdominal bile duct. In addition the numbers of eosinophils and globule leucocytes in the parenchymal bile duct were significantly correlated with the percentage of retarded flukes in both vaccinated groups. In the abdominal bile duct, only the numbers of eosinophils in the low level vaccination group were significantly correlated with fluke retardation.Vaccination did not protect against the pathogenic effects of challenge infection as measured by reduced packed cell volumes and weight gain.     

The anthelmintic activity of some benzimidazole derivatives against Fasciola hepatica

Micronised mebendazole has been shown to have anthelmintic activity against Fasciola hepatica in rats and sheep. Activity is higher against non-egg-producing F. hepatica in bile ducts than against the migrating larval stages. Parbendazole, cambendazole and thiabendazole have been shown to be much less active, or devoid of activity, against mature and immature F. hepatica in rats, at elevated dose rates.

Reasons for the differences, in terms of molecular structure, are briefly discussed and the pitfalls of structure/activity analyses are considered.     

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.     

Cellular proliferation of bile ductules and gamma‐glutamyl transpeptidase in livers and sera of young cattle following a single infection with Fasciola hepatica

Summary

During the course of experimental single infection with Fasciola hepatica in calves, the origin of the increased concentration of gamma‐glutamyl transpeptidase (γ‐GT) found to be present in the serum during the biliary stage of the disease, was studied.

The enzyme originates from the liver after flukes have caused intermittent and selective cholestasis in the bile‐duct system. The enzyme which normally is present in the bile ducts can under these circumstances enter the surrounding interstitial tissues through broken‐up junctions of the bile drainage system or be added to these from proliferating bile‐ductule epithelium. The multiplication of bile‐ductule cells can be looked upon as a neogenesis of cholangioles, which becomes essential when newly formed perilobular connective tissues prevent restoration of the anatomical connections of the bile capillaries with the biliary tree. The connective tissue growth is induced by bile fluid. An initially regional perilobular fibrosis is eventually distributed diffusely through the liver by migration of flukes.

The continued and extending fibrosis is due to the constant recurrence of intermittent and selective cholestasis. The transient occlusions of branches of the bile ducts are very likely to be attributable to persistent cholangitis. The growth of connective tissues has a considerable effect on the gain in weight of the liver.     

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.     

Abattoir Condemnation due to Parasitic Infections and its Economic Implications in the Region of Trikala,Greece

The prevalence of parasitic infections responsible for the condemnation of carcasses and viscera during meat inspection, and their economic implication, was estimated in a year long abattoir survey of 10 277 slaughtered farm animals in the region of Trikala, Greece. The organs examined for the presence of parasitic lesions during meat inspection were: liver and lungs of all animals, rumen of cattle, small intestine of lambs and kids, and muscles of cattle and swine. The parasitic lesions observed in the lungs of cattle, sheep and goats were caused only by hydatid cysts. No hydatid cysts were observed in the lungs of swine. The parasitic lesions observed in the liver of cattle, sheep and goats were as a result of hydatid cysts and flukes of Fasciola hepatica and Dicrocoelium dendriticum, while those of swine were due to milk spots only. Moniezia sp. proglottids were found in the small intestine of lambs only. The prevalence of parasites responsible for the condemnation of marketable organs was low (0.26%). Parasites were responsible for 22% of the total of condemned organs, and their annual cost was 99 500 GDR (～€292). The parasites most contributing to marketable organ condemnation were hydatid cysts (26%) and D. dendriticum flukes (26%).     

Effect of inoculum on the size and location of Fasciola hepatica subsequently recovered from the livers of rats

Two outbred strains of Sprague-Dawley rats were given, by intraperitoneal injection, 5, 10, 20, 30 or 50 newly excysted juvenile (NEJ) Fasciola hepatica. Ninety days post-infection all rats were killed and their livers teased apart under 10 X magnification for quantitation of the flukes present. There was no significant difference in worm numbers between the rat strains. However, several significant differences (P less than 0.05) between the various infection groups were observed. As the size of the infective dose was increased from 5 to 50 NEJ, the percentage of the infective dose recovered from the livers of the infected rats decreased from 36.7 to 12.1%. With the 5 and 10 NEJ infective doses, the worms recovered were large mature flukes (1.52 and 1.68 cm, respectively) and were found in the common bile ducts. In the rats receiving infections of 30 and 50 NEJ, the flukes were smaller (0.88 and 0.55 cm, respectively), immature, and were primarily located in the liver parenchyma. These findings are important in light of previous studies on the development of resistance in the rat to challenge infections in which immunity was based on both the size and the location of the flukes recovered. The results from our study indicate that in a primary infection of 20 or greater NEJ, many small immature flukes remain in the liver parenchyma, even after several months. When testing for resistance to F. hepatica in rats, these flukes may erroneously be thought to comprise a portion of the challenge infection.     

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.     

Canine Leishmaniasis: Parasitological Follow-up of the Treated Subject

A cross-sectional study was carried out to determine the prevalence of gastrointestinal (GI) nematodes and flukes (Fasciola and amphistomes) infection in communally grazed traditional cattle, zero-grazed small-scale dairy cattle and intensively grazed large-scale dairy cattle through examination of helminth eggs in faeces. Results indicated that the type of management, especially the grazing habit, has a significant influence on the prevalence and intensity of GI nematodes and flukes. The prevalence of GI nematodes in traditional, large-scale dairy and small-scale dairy cattle was 67%, 44.4% and 37%, respectively, with the highest faecal egg counts in calves. The overall prevalence of Fasciola gigantica in traditional, large-scale dairy and small-scale dairy cattle was 63.8%, 46.2% and 28.4%, respectively. The prevalence of amphistomes was 81.9%, 55.5% and 41.1% in traditional, large-scale dairy and small-scale dairy cattle, respectively. The high prevalence of flukes in the traditional system was attributed to communal grazing and watering management practices. Stomach flukes recovered in examined cattle at the abattoir were Calicophoron microbothrium and Cotylophoron jacksoni. About 42.1% of infected animals had both Fasciola and amphistomes. The prevalence of both GI nematodes and flukes varied greatly among villages and farms. The prevalence of both Fasciola and amphistomes was higher in adults (58.5%, 75.2%) than in yearlings (36.5%, 51.5%) or calves (24.9%, 47.2%). The variation in the prevalence of both GI nematodes and flukes among management and age groups within systems can be used as an entry point towards rational use of anthelmintics for each management system. More studies on seasonal transmission pattern of all these parasites are required in order to design rational, economic and locally sustainable parasite control programmes.