Metacercariae Of Fasciola Hepatica Research Articles

Immune response in Santa Inês sheep during Fasciola hepatica infection

In the present study, we describe the proliferative response and the IFN-γ, IL-10, IL-4 cytokine expression profiles in peripheral blood, and IgG production, after the experimental infection of Santa Inês sheep with 250 Fasciola hepatica metacercariae that were evaluated at 60 and 210 days post-infection(dpi). The cytokine expression profile at 60 dpi was characterized by the production of IFN-γ and IL-10, which is indicative of an initial mixed Th1/Th2 response. The modulation of the response occurred at 210 dpi with a predominance of IL-10 and IL-4 over IFN-γ. Mononuclear cells from peripheral blood stimulated with F. hepatica antigen exhibited proliferative capacity at 60 dpi, whereas the response was consistent with modulation from Th1 to Th2 in the chronic phase. The IgG antibody response was more marked at 60 days than at 210 dpi, confirming the mixed-response profile. The late modulation of the T lymphocyte response in association with the predominance of IL-10 and IL-4 at 210 dpi suggests the involvement of these cytokines after the establishment of the parasites in the bile ducts. The transition from a mixed to a regulatory response in the chronic phase was also accompanied by a reduced number of eggs per gram of feces.

Vaccination with cathepsin L mimotopes of Fasciola hepatica in goats reduces worm burden, morphometric measurements, and reproductive structures

Fasciolosis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. We conducted an experimental trial with four groups of five goats each, vaccinated with 1 × 1013 phage particles of cathepsin L1 (CLI: DPWWLKQ), CL1 (SGTFLFS), and CL2 (PPIRNGK) mimotopes combined with Quil A adjuvant. Animals received a booster four weeks later. The control group received phosphate-buffered saline. All animals were infected with 200 Fasciola hepatica metacercariae at week six and euthanised 16 weeks later. The percentage of significant worm reduction in CL1 (DPWWLKQ), CL1 (SGTFLFS), and CL2 (PPIRNGK) compared to the control group were 55.40%, 70.42% (P < 0.05), and 32.39%, respectively. Vaccinated animals showed a significant reduction in faecal egg production and egg viability. A significant reduction in the total biomass of parasites recovered was observed in the CL1 (DPWWLKQ) and CL1 (SGTFLFS) groups. In goats vaccinated with CL2 (PPIRNGK), fluke length and width were smaller than those in the control group. Furthermore, animals receiving CL mimotopes showed a significant reduction in the total area of reproductive structures. Goats immunised with phage-displayed mimotopes produced significantly high titres of specific IgG1 and IgG2 isotypes, indicating a mixed Th1/Th2 response. The liver fluke burdens in goats vaccinated with CL1 (DPWWLKQ) and CL1 (SGTFLFS) were significantly correlated with IgG and IgG1 levels.

A review of our current understanding of parasite survival in silage and stored forages, with a focus on Fasciola hepatica metacercariae.

Fasciola hepatica, the parasitic liver fluke, is a re‐emerging zoonotic infection and an important cause of morbidity and mortality in ruminant livestock worldwide. A significant animal welfare concern, fasciolosis also has a detrimental impact on food security, with the effects of sub‐clinical infection on growth rate and milk yield estimated to cost the UK cattle industry £40.4 million annually. It is understood that up to 50% of infective F. hepatica metacercariae may overwinter on pasture and remain viable to infect grazing livestock the following spring. However, the infection risk posed by feeding grass silage to livestock remains largely unknown as the majority of previous studies are outdated in both experimental design and analysis of ensiled metacercariae viability. Anecdotal reports of fasciolosis outbreaks in housed livestock have reignited interest in F. hepatica metacercariae survival during modern ensiling processes and more importantly if they retain viability within stored forages. Consequently, a comprehensive review of the available literature is required in order to identify knowledge gaps and highlight future research opportunities regarding the role of silage in the transmission of F. hepatica.

Immune response of rats vaccinated orally with various plant-expressed recombinant cysteine proteinase constructs when challenged with Fasciola hepatica metacercariae.

BackgroundCysteine proteinases of Fasciola hepatica are important candidates for vaccine antigens because of their role in fluke biology and host-parasite relationships. In our previous experiments, we found that a recombinant cysteine proteinase cloned from adult F. hepatica (CPFhW) can protect rats against liver fluke infections when it is administered intramuscularly or intranasally in the form of cDNA. We also observed considerable protection upon challenge following mucosal vaccination with inclusion bodies containing recombinant CPFhW produced in Escherichia coli.In this study, we explore oral vaccination, which may be the desired method of delivery and is potentially capable of preventing infections at the site of helminth entry. To provide antigen encapsulation and to protect the vaccine antigen from degradation in the intestinal tract, transgenic plant-based systems are used.MethodologyIn the present study, we aimed to evaluate the protective ability of mucosal vaccinations of 12-week-old rats with CPFhW produced in a transgenic-plant-based system. To avoid inducing tolerance and to maximise the immune response induced by oral immunisation, we used the hepatitis B virus (HBV) core protein (HBcAg) as a carrier. Animals were immunised with two doses of the antigen and challenged with 25 or 30 metacercariae of F. hepatica.ConclusionsWe obtained substantial protection after oral administration of the plant-produced hybrids of CPFhW and HBcAg. The highest level of protection (65.4%) was observed in animals immunised with transgenic plants expressing the mature CPFhW enzyme flanked by Gly-rich linkers and inserted into c/e1 epitope of truncated HBcAg. The immunised rats showed clear IgG1 and IgM responses to CPFhW for 4 consecutive weeks after the challenge.

Development of a versatile in vitro method for understanding the migration of Fasciola hepatica newly excysted juveniles.

Fasciola hepatica is a parasitic trematode that causes serious losses to livestock producers, and also zoonotic disease. The limitations of chemotherapy for the control of fasciolosis have led to significant interest in the development of vaccines to protect cattle and sheep from infection. However, relatively few studies have concentrated on the mechanisms of invasion of the gut by newly excysted juvenile liver flukes (NEJ) and the host response triggered by this event. The aim of this work was to develop an in vitro model to study invasion by NEJ, while also reducing the requirement for challenge infections of experimental animals. Fasciola hepatica metacercariae were excysted in vitro and placed into compartments containing rat distal jejunal sheets. Variations in incubation medium, chamber size and incubation temperature were used to identify optimal conditions for NEJ migration across the gut. Histological examination showed increased migration until 120 min post-incubation. The use of RPMI, without gassing at 39 °C, as the incubation medium was found to be optimal, with 40·5% of NEJ migrating after 150 min. This study describes a readily-reproducible method for studying the migration of F. hepatica NEJ within the definitive host. It will be useful for identifying potential drug and vaccine targets.

Immunoexpression of intermediate filaments and morphological changes in the liver and bile duct of rats infected with Fasciola hepatica

We investigated the immunoexpression of the intermediate filament proteins, cytokeratin and desmin, and the morphological changes in the liver of rats during experimental fasciolosis at 4, 7 and 10 weeks post-infection. Rats were infected with 30 Fasciola hepatica metacercariae. Paraffin sections of the liver were stained using H & E, PAS and azan stains. Immunohistochemical reactions were performed using antibodies against cytokeratin and desmin. The experimental F. hepatica infection led to fibrosis and cirrhosis of the liver, and to inflammation of the common bile ducts. The expression of cytokeratin was increased in the epithelial cells of both the liver bile ductules at 4, 7 and 10 weeks post-infection and in the common bile ducts at 7 and 10 weeks post-infection compared to uninfected rats; expression in the common bile ducts was more intense. The myofibroblasts of the liver and smooth myocytes of the interlobular bile ducts and common bile ducts, showed a slight increase in desmin expression compared to the uninfected rats. The increased expression of cytokeratins in the hyperplastic rat common bile duct epithelium during the biliary phase of fasciolosis at 7 and 10 weeks post-infection may be explained by mechanical irritation by the parasite and an inflammatory reaction in the bile duct epithelium and in periductal fibrous tissue.

A highly water soluble benzimidazole derivative useful for the treatment of fasciolosis

This study describes the synthesis of compound (7), a highly hydrosoluble phosphonooxymethyl prodrug of compound alpha (4). Compound (7) improved the aqueous solubility of its precursor compound (4) by 50,000 times and it is stable at neutral pH. The prodrug showed faciolicidal activity when evaluated in vitro against excysted Fasciola hepatica metacercariae. The in vivo evaluation of (7) was carried out via oral, intramuscular and subcutaneous administration in sheep artificially infected with F. hepatica metacercariae. At an intramuscular dose of 4mg/kg, the activity of (7) was similar to that of compound alpha (4) at an oral dose of 15mg/kg.

The production of mammalian trematode infective stages by the snail Galba truncatula

Several experiments on the breeding of trematode-infected Galba truncatula for obtaining and packaging Fasciola hepatica and Paramphistomum daubneyi metacercariae were carried out to determine the more convenient methods to use for commercial production of these infective stages. Compared to the breeding of infected snails in aquaterraria, the use of 14-cm Petri dishes allowed a greater prevalence of snail infection and a higher number of metacercariae. The production of these larvae was still 2.3-3.4 times greater if infected snails were dissected during the patent period. The aspiration of these metacercariae at the extremity of a Pasteur pipette significantly shortens the time necessary for their transfer from Petri dishes to Eppendorf tubes. Using 14-cm Petri dishes, snail dissection and metacercarial aspiration for their transfer strongly reduce the cost price for metacercarial production of the trematodes Fasciola hepatica and Paramphistomum daubneyi.

Experimental Murine Fascioliasis Derives Early Immune Suppression with Increased Levels of TGF-β and IL-4

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19+ B cells was observed as early as week 1 post-infection while CD4+/CD8+ T cells were down-regulated. Accumulation of Mac1+ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1β expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-β were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-β and IL-4 during the early stages of infection.

Evaluation of the comparative efficacy of a moxidectin plus triclabendazole pour-on solution against adult and immature liver fluke, Fasciola hepatica, in cattle

The objective of this study was to evaluate the efficacy of a pour-on solution containing moxidectin plus triclabendazole (MOX plus TCBZ) against immature and adult stages of the liver fluke in cattle and compare the efficacy with other commercially available preparations. To this end, 104 male Holstein-Friesian calves aged between 3 and 4 months, were randomly allocated to 13 groups of eight animals each, and infected with approximately 500 Fasciola hepatica metacercariae. One group remained untreated, four groups were treated with MOX plus TCBZ at a dose rate of 0.1mL/kg, four other groups were treated with ivermectin (IVM) plus clorsulon injectable at a dose rate of 0.02mL/kg, and the remaining four groups were treated with IVM plus closantel pour-on at a dose rate of 0.1mL/kg. Each treatment was applied to one of the groups at 4 weeks, 6 weeks, 8 weeks and 12 weeks after the experimental infection. At necropsy (99–102 days after infection), all untreated animals were infected with a minimum of 30 flukes. The MOX plus TCBZ treated animals had significantly (P<0.0001) lower fluke counts compared to the untreated control animals at all time points after treatment. Efficacy against 8-week old and adult flukes was >99.5%. For 6-week old immature fluke, the efficacy was 98.0% and for 4-week old immature fluke the efficacy was 90.9%. The IVM plus closantel pour-on treated animals had significantly lower fluke counts compared to the untreated control animals for adult and 8-week old flukes (P<0.0001), and for 6-week old flukes (P=0.002). The efficacy was 26.8%, 68.2%, 90.6% and 99.3% against 4-week, 6-week and 8-week old immature flukes, and adult flukes respectively. The IVM plus clorsulon treated animals had significantly lower fluke counts compared to the untreated control animals for adult (P<0.0001) and 8-week old (P<0.05) flukes. The efficacy was 29.7%, 43.4%, 53.2% and 99.2% against 4-week, 6-week and 8-week old immature flukes, and adult flukes respectively. For treatments at 4, 6 and 8 weeks after infection, the fluke counts were significantly (P<0.0001) lower for the MOX plus TCBZ treatment than for IVM plus closantel or IVM plus clorsulon. The results confirm the high efficacy (>90%) of the MOX plus TCBZ pour-on combination against 4-week old to adult liver fluke in cattle. The IVM plus closantel pour-on combination was effective (>90%) against 8-week old and adult flukes, but had low efficacy against 4- and 6-week old fluke. The IVM plus clorsulon injectable combination was effective (>90%) against adult fluke only.

Protection and antibody isotype responses against Fasciola hepatica with specific antibody to pIII-displayed peptide mimotopes of cathepsin L1 in sheep

Fluke burdens, liver fluke size and biomass, fecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep immunized with peptide mimotopes of cathepsin L1 and challenged with Fasciola hepatica metacercariae. Twenty sheep were randomly allocated to four groups of five animals each; groups 1 and 2 were immunized at weeks 0 and 2 with mimotopes YVYRWVEAECVA and FSPAYLRDAALK, respectively; group 3 was immunized with wild-type M13KE phage and the control group received phosphate-buffered saline (PBS). All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. Groups 1 and 2 showed a reduction in fluke burden of 51.7% and 35.9%, respectively, when compared to the control group, but only the former was significant at the 5% level. Vaccinated animals showed a significant reduction in fluke length and width, wet weights and egg output. A significant diminution in the total biomass of parasites recovered was also observed in group 1. Levels of anti-phage total IgG increased rapidly within 2 weeks of the first immunization and were always significantly higher in groups 1 and 2 than in the infected control group. The fluke burden in group 1 was significantly correlated with IgG1 and total IgG. The vaccinated sheep with phage clones produced significantly high titres of IgG1 and IgG2 antibodies indicating a mixed Th1/Th2 response.

MyD88 is required for the recruitment of eosinophils and neutrophils but dispensable for host protective immune responses during Fasciola hepatica infection

Adaptive immunity detects non-self through the recognition of peptide antigens using receptors expressed on the surface of B and T cells, however that how innate immune system recognizes non-self has been remained unclear. Myeloid differentiation factor 88 (MyD88) is a critical adaptor molecule shared by toll-like receptors (TLRs), and has been found crucial on control of Th1 immunity. However, whether a Th2 response against helminth infection requires a signaling through MyD88 has been uncertain. We hypothesized Th2 immune responses induced by helminth require a MyD88-dependent signaling. To test it, a MyD88 knockout mouse model infected with Fasciola hepatica metacercariae was used and unexpected results were exhibited with dispensable role of MyD88 on host protective immune responses marked as increased IgG1, IgE, IL4, IL5, IL9 and IL13 and decreased IgG2a and IFNγ, which could be induced even with intact MyD88 molecule during F. hepatica infection. No differences were found on IL17 and TGFβ production in both infected WT and MyD88 knockout mice. The mRNA levels of transcript factors for Th1 (T-bet), Th2 (GATA3), Th17 (RORγt) and Tregs (FoxP3) were also measured by real-time PCR, showing consistent trends to the productions of Th1 (IFNγ), Th2 (IL4, IL5 and IL13) and IL9 cytokines, and no differences were found on RORγt and FoxP3 in both infected WT and MyD88 knockout mice. However, the numbers and proportions of eosinophils and neutrophils recruited in peritoneal cavity were significantly decreased in mice defecting MyD88 although no differences were found on macrophages, lymphocytes and CD4 positive cells, indicating signaling through MyD88 might be involved in recruitment of immune effector cells during helminth infection. Additionally, no differences were found on the adult worm burden and liver damage in both infected WT and MyD88 knockout mice. Taking together, our experiments showed MyD88 is required for the recruitment of eosinophils and neutrophils but dispensable for host protective immune responses during F. hepatica infection.

Experimental Infection of Liver Flukes, Fasciola hepatica and Fascioloides magna, in Bison (Bison bison)

This experimental study was conducted to evaluate the susceptibility of American bison (Bison bison) to liver flukes, Fascioloides magna and Fasciola hepatica. Six bison were each experimentally inoculated with 600 metacercariae of Fascioloides magna, and three were later treated with triclabendazole suspension at 40 mg/kg of body weight. Four additional bison were each experimentally inoculated with 600 metacercariae of Fasciola hepatica. Five control bison were placebo controls. Two controls and all inoculated bison were euthanized 10 mo (Fascioloides magna) and 7 mo (Fasciola hepatica) after inoculation. None of the control bison or the bison inoculated with Fascioloides magna had flukes or lesions characteristic of fluke infection at necropsy. All four bison inoculated with Fasciola hepatica had characteristic liver fluke lesions at necropsy, and three of four bison contained four, 103, and 111 adult flukes, respectively. Fluke eggs were detected in feces of all Fasciola hepatica-inoculated bison during the experiment, but not from the Fascioloides magna-infected bison or control bison. Clinical signs of infection were not observed during the experiment, but hemoglobin and packed cell volumes were lower in the Fasciola hepatica bison when compared to controls, and eosinophil levels were increased. Triclabendazole at 40 mg/kg of body weight appeared to be safe in bison because no toxic reactions were observed. Results from this study indicated bison are susceptible to infection with Fasciola hepatica and are efficient definitive hosts. Because no Fascioloides magna were recovered, bison may have a decreased susceptibility or innate resistance to Fascioloides magna infection, which may account for a lack of reported infections in this host.

Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis

Use of the rabbit as disease model has long been hampered by a lack of immunological assays specific to this species. In the present study we developed a SYBR Green-based, real-time RT-PCR protocol to quantitate cytokine mRNA in freshly harvested rabbit peripheral mononuclear cells. The method was validated in the course of a vaccination trial in which animals vaccinated with the recombinant antigen FhSAP2 were challenged with Fasciola hepatica metacercariae. Changes in the levels of rabbit interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNFα), and interferon-gamma (IFNγ) mRNA were determined. Messenger RNA from the universally expressed housekeeping gene GAPDH was used as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. Rabbits vaccinated with FhSAP2 showed an 83.3% reduction in liver fluke burden after challenge infection when compared to non-vaccinated controls. All cytokine mRNAs were found at detectable levels; however, the levels of IFNγ, TNFα, IL-2 and IL-10 were significantly higher in the vaccinated group compared to the non-vaccinated group. These results suggest that protection conferred by FhSAP2 protein could be associated with a mixed Th1/Th2 immune response in which Th1 cytokines are dominant. The real-time RT-PCR method described herein can be a useful tool for monitoring changes in basic immune functions in the rabbit model of fascioliasis and may also aid in studies of human diseases for which the rabbit is an important experimental model.

Fasciola hepatica: Histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10 mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd α), a benzimidazole derivative of TCBZ (15 mg/kg). The lambs were euthanised 48, 72 and 96 h after TCBZ treatment, or 24, 48 and 72 h after Cpd α treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanised 24 h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis’ secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis’ gland, histological evidence indicating failure of ovigenesis was evident from 24 h post-treatment onwards. The development of these lesions may be related to the known anti-tubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd α is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings.

Experimental Infection of Bighorn Sheep with Liver Flukes (Fasciola hepatica)

Nine Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were each inoculated orally with 250 metacercariae of Fasciola hepatica. Blood and fecal samples were collected at the time of inoculation and at 5, 10, 14, and 20 wk after inoculation. Numbers of fluke eggs in feces, hemoglobin, packed cell volume, and eosinophil values were determined. Five of the bighorn sheep were treated with triclabendazole at 40 mg/kg of body weight 14 wk after inoculation. Based on fecal evaluations, all bighorns developed patent infections. Six weeks after treatment, fluke eggs were not detected in feces from the five treated animals but were present in two of four untreated animals. One untreated bighorn sheep was euthanized 20 wk after inoculation, and 57 adult F. hepatica were recovered from the liver. Results from this experiment indicated that bighorn sheep are efficient hosts for F. hepatica. Triclabendazole at 40 mg/kg of body weight was safe and, based on fluke egg recovery in feces, apparently an effective treatment. To my knowledge, this is the first published report of F. hepatica in bighorn sheep.

In Silico Fasciolicide Activity of Three Experimental Compounds in Sheep

The aim of the present study was to evaluate the fasciolicide activity of three experimental drugs, selected by an in silico system called TOMOCOMD-CARDD, in sheep. Drugs were identified by the computer system, and, after statistical selection, 24 Pelibuey sheep were infected on days 0 and 30, each with 200 metacercariae of Fasciola hepatica. When the infection reached 8 and 4 weeks of age, respectively, four groups of six animals each were formed. Group 1 received thiacetazone 150 mg/animal/p.o. Group 2 was treated with 3,5,5, trimethyloxazolidine 2,4-dione at 450 mg/animal/p.o. G3 received guanabenz acetate at a dose of 1.5 mg/animal/p.o. G4 served as an untreated control. Monitoring of the animals was followed by individual coprological examinations and slaughter of the animals 15 days after treatment to collect and count flukes from the liver. Efficacy was measured as the reduction in the percentage of flukes of treated animals relative to untreated controls. Results indicated an efficacy of 80.0, 43.8, and 100% for 8-week-old flukes and 62.1, 57.9, and 100% for 4-week-old flukes in the three experimental groups, respectively. Even though guanabenz acetate showed a high efficacy, it was highly toxic since two animals died approximately 24 h after being treated. We conclude that further investigations should be conducted to perform computer-aided prediction of drugs aimed to detect fasciolicide activity.

In Vitro Fasciolicide Activity of Some Plant Extracts against Newly Excysted Flukes

The aim of the present study was to evaluate, under in vitro conditions, the activity of 20 plant extracts against newly excysted Fasciola hepatica metacercariae. The plants were selected on the basis of anthelmintic activity, traditional use, availability, solubility, and permanence in the liver. For the in vitro evaluation, 24-well Nunc-immunoplates containing RPMI-1640 culture medium bovine sera plus antibiotics were used. Extract concentrations of 500 and 50 mg/mL were placed in triplicate. Approximately 10 newly excysted flukes were placed in each well, and the plates were incubated in a CO(2) incubator at 5% atmosphere for 3 days. In each plate four control wells containing nontreated flukes were included. All procedures were undertaken under an air-flow cabinet. Efficacy was measured on days 1 and 3 by considering the motility of the live or dead trematodes evaluated with the aid of an inverted microscope. Results indicated that, from all extracts evaluated, Tilia mexicana showed an average efficacy for concentrations of 500 and 50 mg/mL, respectively, of 100 and 100%; Eriobotrya japonica, 100 and 95%; Coffea arabica, 100 and 100%; Crataegus mexicana, 95 and 90%; and Ternstroemia pringlei 100 and 100%. From this preliminary evaluation, we conclude that further and more detailed evaluations at lower concentrations should be undertaken to understand the real efficacy of these plant extracts.

Fasciola hepatica: Histology of the testis in egg-producing adults of several laboratory-maintained isolates of flukes grown to maturity in cattle and sheep and in flukes from naturally infected hosts

A total of 8 calves approximately 6 months old and 22 lambs of similar age were infected with metacercariae of Fasciola hepatica of various laboratory-maintained isolates including: Cullompton (sensitive to triclabendazole) and Sligo, Oberon and Leon (reported as resistant to triclabendazole). Ten to 16 weeks after infection, flukes were harvested from these experimental animals and the histology of the testis tissue was examined in a representative sample of flukes from each population. Adult wild-type flukes were also collected from 5 chronically infected cattle and 7 chronically infected sheep identified at post-mortem inspection. The testis tissue of these flukes was compared with that of the various laboratory-maintained isolates. Whilst the testes of the wild-type, Oberon and Leon flukes displayed all the usual cell types associated with spermatogenesis in Fasciola hepatica (spermatogonia, spermatocytes, spermatids and mature sperm), the Cullompton flukes from both cattle and sheep showed arrested spermatogenesis, with no stages later than primary spermatocytes represented in the testis profiles. The presence of numerous eosinophilic apoptotic bodies and nuclear fragments suggested that meiotic division was anomalous and incomplete. In contrast to the wild-type flukes, no mature spermatozoa were present in the testes or amongst the shelled eggs in the uterus. A high proportion of the eggs collected from these flukes hatched to release normal-appearing miracidia after an appropriate incubation period, as indeed was the case with all isolates examined and the wild-type flukes. It is concluded that the eggs of Cullompton flukes are capable of development without fertilization, i.e. are parthenogenetic. The implications of this for rapid evolution of resistant clones following an anthelmintic selection event are discussed. Amongst the Sligo flukes examined, two subtypes were recognised, namely, those flukes with all stages of spermatogenesis and mature spermatozoa present in the testes (type 1), and those flukes with all stages of spermatogenesis up to spermatids present, but no maturing spermatozoa in the testes (type 2). Each sheep infected with the Sligo isolate had both type 1 (approximately 60%) and type 2 (approximately 40%) flukes present in the population. Spermatozoa were found amongst the eggs in the uterus in 64% of flukes and this did not necessarily reflect the occurrence of spermatozoa in the testis profiles of particular flukes, suggesting that cross-fertilization had occurred. The apparent disruption of meiosis in the spermatocytes of the Cullompton flukes is consistent with reports that Cullompton flukes are triploid (3 n = 30), whereas the Sligo and wild-type flukes are diploid (2 n = 20). In the Sligo flukes the populations are apparently genetically heterogenous, with a proportion of the flukes unable to produce fully formed spermatozoa perhaps because of a failure in spermiogenesis involving elongation of the nucleus during morphogenesis.

The effect of a parenteral ivermectin/closantel injection on the growth and reproductive development of early immature Fasciola hepatica in cattle

Sixteen calves approximately 6 months old were each infected with 500 metacercariae of Fasciola hepatica. Thirty-two days later they were weighed and divided into two groups, and on day 35 all calves in one of the groups were injected subcutaneously with an ivermectin/closantel combination. Both groups were sacrificed between days 70 and 72 to enable counting and examination of the flukes recovered from the bile ducts. Eggs released by the flukes were collected for incubation, hatching and estimation of egg viability. Flukes were counted, flat-fixed in 70% ethanol, stained with catechol and carmine and measured. The reproductive organs, namely testis, vitelline glands, ovary and uterus, were examined and scored on a 0–3 scale according to their state of development. This was visually assessed on the basis of size, distribution and staining density of their constituent tissues and the abundance of eggs in the uterus. A representative sample of flukes from each animal was fixed in formalin for histological sectioning to enable more detailed examination of the reproductive structures. Treatment of the immature flukes reduced the population in cattle by 42.6% as compared with the controls and as a result of the stunting effect due to the presence of closantel during early development the size of treated flukes was reduced by 43.9%. A bimodal pattern of size and reproductive score was also observed in flukes from treated cattle, suggesting that the stunting effect on individual flukes differed depending on whether or not they had gained access to the bile ducts or were still migrating in the hepatic parenchymal tissue at the time of drug exposure with the effect being greater once the fluke had gained access to the bile ducts. The mean reproductive score for untreated flukes was 8.76 and for treated flukes 5.64, a 35.6% reduction. This difference was highly significant ( p < 0.001). Egg shedding from treated flukes was significantly less than that from controls ( p < 0.05), but there were no differences in hatchability, suggesting that whilst drug treatment reduced the energy supply available for gametogenesis/oogenesis, it did not induce functional defects in the gonads or accessory reproductive organs. Histological examination confirmed that there was a reduction in development of testes, ovaries and vitellaria in treated flukes, with a consequent reduction in egg production. In the treated flukes, early spermatogonia and oogonia were the predominant cell types in the testes and ovary, whilst undifferentiated stem cells were abundant in the vitelline follicles. In untreated flukes, cells representing more advanced stages in gametogenesis and vitellogenesis predominated in the respective organs. It is likely that this inhibition of gametogenesis and vitellogenesis was caused by the effects of closantel treatment on intermediary metabolism in the flukes. Clearly these effects were evident even at a relatively early stage of fluke growth, and because of the impact on egg output may have epidemiological importance in addition to the reduction in fluke numbers.