Early onset of changes to the reproductive system of Fasciola hepatica following in vivo treatment with triclabendazole

Lambs infected with the Cullompton triclabendazole (TCBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3h, 24h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis' gland, vitellaria, ovary and testis. At 3h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24h pt. At this time period, the cells of the Mehlis' gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24h, demonstrating that this process is affected very rapidly pt.     

Impact of compound alpha treatment in vivo on egg production by the liver fluke, Fasciola hepatica

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48 h and 72 h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis' gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs. Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48 h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72 h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48 h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72 h pt. In the Mehlis' gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48 h to 72 h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action.     

Time-dependent changes to the tegumental system and gastrodermis of adult Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host

Eight indoor-reared cross-bred sheep with no pre-exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. At 12 weeks post-infection, sheep were dosed with 10mg/kg triclabendazole. Two sheep per time period were euthanized at 48 h, 72 h and 96 h post-treatment (pt). Two control sheep were euthanized alongside the 96 h triclabendazole-treated sheep. Flukes were recovered from each of the sheeps liver and, if present, from the gall bladder and they were processed for transmission electron microscopy (TEM). Disruption to the ultrastructure of the tegument became increasingly severe over time pt. Flukes recovered at 48 h pt showed widespread blebbing of the apical plasma membrane and swelling of the mucopolysaccharide masses surrounding the basal infolds. There was evidence of reduced secretory activity in the tegumental cells and spacing between the cells. Sloughing of the tegumental syncytium was observed at 72 h pt. The subtegumental musculature, parenchyma and tegumental cells were severely disrupted. At 96 h pt, all of the flukes were totally devoid of tegument. Disruption to the subtegumental tissue and somatic musculature was severe, and was so extreme in some specimens that the tegumental cells were barely discernible. Disruption to the gastrodermis was also progressive, though not as severe as disruption to the tegument. There was a general decline of secretory activity with time pt. Autophagic activity was apparent from 48 h pt and became more widespread with increasing time, culminating in breakdown of the gastrodermal cell cytoplasm. The mitochondria were swollen and electron-lucent and the cisternae of the granular endoplasmic reticulum were dilated and fragmented from 72 h pt.     

Tegumental surface changes in juvenile Fasciola hepatica in response to treatment in vivo with triclabendazole

Eight indoor-reared crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection with 10 mg/kg triclabendazole. Two treated sheep were euthanized at 48 h, 72 h and 96 h post-treatment with triclabendazole. Two control sheep were euthanized alongside the 48 h triclabendazole-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for scanning electron microscopy (SEM). Flukes were still active 48 h post-treatment and displayed limited morphological disruption. There was some blebbing and sloughing of the tegument around the oral sucker. In several of the specimens, an extra layer had been deposited on the fluke surface, giving it a flattened appearance. At 72 h post-treatment, only one fluke remained alive and the disruption varied in degree. In the majority of flukes, there was severe swelling of the tegument, accompanied by isolated areas of flattening along the lateral margins of the flukes and in the tail region. Limited areas of sloughing occurred in the tail region. In more seriously affected specimens, the syncytium had been stripped away to reveal the basal lamina and some deeper lesions were also observed. By 96 h post-treatment, all the flukes were dead and were grossly disrupted. They were totally devoid of tegument and deep lesions exposed the internal tissues of the fluke.     

Enhancement of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica by co-treatment with ketoconazole

An in vivo study in the laboratory rat model was carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from combination treatment of triclabendazole (TCBZ) and the metabolic inhibitor, ketoconazole (KTZ). Rats were infected with the TCBZ-resistant Oberon isolate of F. hepatica and divided into 3 groups at 12 weeks post-infection. The first group was dosed orally with TCBZ at a dosage of 10mg/kg and KTZ at a dosage of 10mg/kg. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.). A second group of rats was treated with TCBZ alone (10mg/kg) and sacrificed at 96 h p.t. The third group acted as untreated controls. Surface changes were monitored by scanning electron microscopy (SEM). In flukes from the TCBZ+KTZ-treated group, the results showed a progressive and time-dependent increase in the level of disruption to the tegumental syncytium. Swelling, furrowing, blebbing and sloughing of the syncytium increased with time p.t. Another feature seen was a thick layer of tegumental shedding in some fluke samples at different times p.t. By comparison, flukes treated with TCBZ alone remained unaffected. The results demonstrated that the Oberon isolate is only sensitive to drug action in the presence of ketoconazole, indicating that combining triclabendazole with a metabolic inhibitor could be used to preserve the effectiveness of the drug against TCBZ-resistant populations of F. hepatica.     

Comparison of two assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to triclabendazole in Fasciola hepatica in sheep

A sheep trial was performed to evaluate two diagnostic assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance of Fasciola hepatica to triclabendazole (TCBZ). The FECRT defines successful TCBZ treatment as a 95% or greater reduction in fluke faecal egg counts (FECs) at 14 days post-treatment (dpt). The CRT defines effective TCBZ treatment as faeces negative for Fasciola coproantigens at 14dpt, as measured by the commercial BIO K201 coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium). Forty-nine indoor-reared sheep were split into four trial groups and each sheep was infected with 200 metacercariae of 1 of 4 F. hepatica isolates, previously described as susceptible (Cullompton and Fairhurst) and resistant (Leon and Oberon) to TCBZ action, respectively. TCBZ treatment was administered at 12 weeks post-infection (wpi) to one sub-group in each infected sheep group, and these sheep were culled at 4 weeks post-treatment (wpt). Untreated sheep sub-groups, were culled at a parallel time-point, that is, at 16wpi. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period, sub-sampled and examined by a standardised egg sedimentation protocol and by the BIO K201 ELISA. Results supported the use of both the FECRT and the CRT for the diagnosis of resistance of F. hepatica to TCBZ. In addition, the study confirmed the TCBZ susceptibility of the Cullompton and Fairhurst F. hepatica isolates and the TCBZ resistance of the Oberon F. hepatica isolate. However, the Leon F. hepatica isolate was found to be susceptible, rather than resistant, to TCBZ action.     

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes.     

Reproductive disruption in Fasciola hepatica associated with incomplete efficacy of a new experimental formulation of triclabendazole

The objective of the present study was to analyse the reproductive viability (using histopathologic studies) of Fasciola hepatica from cattle artificially infected and treated subcutaneously with a new experimental formulation of triclabendazole (8mg/kg b.w.). The results of the efficacy controlled test, which only takes into account the presence of live adult flukes, indicated that, whilst in the control group (n=7) 533 live specimens were recovered, in the test groups (doses of 8 and 12mg/kg b.w.) only 195 and 47 adults were recovered, respectively. These numbers indicate efficacies of 69% and 95.6%, respectively. It was observed in that dose of 8mg/kg b.w. some specimens remained viable, but they were infertile, which severely compromises the biological cycle of the trematode. In the testis tubules of flukes treated with the low dose of TCBZ (8mg/kg), very few cells were present and the vitelline follicles were markedly reduced in size and each follicle contained very few cells. This would have direct implications for the pathogenesis of the parasitosis since the remaining parasites would produce little clinical-productive manifestations, would stimulate the immune response and would find it difficult to establish future re-infestations/re-infections. Consequently, these observations will also prompt a review of certain methodological and interpretative aspects related to efficacy tests, where the only discriminative factor is the reduction of the adult parasite load. On one hand, histopathological studies could be complementary to the efficacy controlled test for TCBZ or other BZD formulations.     

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.     

Development of an egg hatch assay for the diagnosis of triclabendazole resistance in Fasciola hepatica: proof of concept

The aim of this study was to develop an Egg Hatch Assay (EHA) test for the detection of triclabendazole (TCBZ) resistance in Fasciola hepatica. A number of fluke isolates were used, of differing sensitivity to TCBZ. Eggs were exposed to solutions of triclabendazole sulphoxide (TCBZ.SO) for 14 days, then triggered to hatch. Egg development was divided into 6 distinct and easily identifiable stages: dead, empty, unembryonated, cell division, eye spot and hatched. The number of eggs reaching those stages was recorded. Initially, the discriminating dose (1% hatch) was determined for the Cullompton isolate, used as TCBZ-susceptible (TCBZ-S) standard. Once this concentration had been resolved, the response of different isolates to this concentration was examined. The hatch rate of the Fairhurst isolate was not significantly different from that of the Cullompton isolate, confirming its TCBZ-S status. The Patagonia isolate has not been exposed to TCBZ in the field and should be TCBZ-S: the results of the EHA supported this. The egg hatch response of the Oberon and Dutch isolates differed significantly from that of the Cullompton isolate; the former isolates are regarded as TCBZ-resistant (TCBZ-R) and the results confirmed this. Another isolate, the Leon isolate, was originally described as being TCBZ-R, but has since been shown to be TCBZ-S. There was no difference in its response to TCBZ.SO in the EHA from the Cullompton (and Fairhurst and Patagonia) isolate(s), further indicating its TCBZ-S status. The impact of TCBZ.SO treatment on the component stages of egg development was determined and revealed differences between the isolates. In conclusion, the results of the study have shown that it is possible to discriminate between TCBZ-S and TCBZ-R isolates of F. hepatica on the basis of the response of their eggs to an EHA and the test could be used to evaluate the TCBZ sensitivity of unknown field isolates.     

Inhibition of cytochrome P450 activity enhances the systemic availability of triclabendazole metabolites in sheep

Understanding the disposition kinetics and the pattern of metabolism is critical to optimise the flukicidal activity of triclabendazole (TCBZ) in ruminants. TCBZ is metabolised by both flavin-monooxygenase (FMO) and cytochrome P450 (P450) in the liver. Interference with these metabolic pathways may be useful to increase the systemic availabilities of TCBZ metabolites, which may improve the efficacy against Fasciola hepatica . The plasma disposition of TCBZ metabolites was evaluated following TCBZ co-administration with FMO [methimazole (MTZ)] and P450 [piperonyl butoxyde (PB) and ketoconazole (KTZ)] inhibitors in sheep. Twenty (20) healthy Corriedale x Merino weaned female lambs were randomly allocated into four experimental groups. Animals of each group were treated as follow: Group A, TCBZ alone (5 mg/kg, IV route); Group B, TCBZ (5 mg/kg, IV) + MTZ (3 mg/kg, IV); Group C, TCBZ (5 mg/kg, IV) + PB (30 mg/kg, IV) and Group D, TCBZ (5 mg/kg, IV) + KTZ (10 mg/kg, orally). Blood samples were taken over 240 h post-treatment and analysed by HPLC. TCBZ sulphoxide and sulphone were the main metabolites recovered in plasma. MTZ did not affect TCBZ disposition kinetics. TCBZ sulphoxide Cmax values were significantly increased ( P  < 0.05) after the TCBZ + PB (62%) and TCBZ + KTZ (37%) treatments compared to those measured in the TCBZ alone treatment. TCBZ sulphoxide plasma AUCs were higher ( P  < 0.05) in the presence of both PB (99%) and KTZ (41%). Inhibition of TCBZ P450-mediated oxidation in the liver accounted for the increased systemic availability of its active metabolite TCBZ sulphoxide. This work contributes to the search of different strategies to improve the use of this flukicidal drug in ruminants.     

Combined use of ivermectin and triclabendazole in sheep: in vitro and in vivo characterisation of their pharmacological interaction

This study evaluated the pharmacokinetic properties of ivermectin (IVM) and triclabendazole (TCBZ) given either separately or co-administered to sheep. Corriedale sheep received IVM alone, TCBZ alone or a combination of IVM and TCBZ intravenously. Ivermectin elimination was delayed and its plasma availability was 3-fold higher when co-administered with TCBZ. Similarly, plasma concentrations of TCBZ and its metabolites were influenced by the co-administration of IVM. Higher peak plasma concentrations of TCBZ metabolites were detected after the co-administration of TCBZ and IVM compared to those obtained following TCBZ treatment in isolation. Complementary in vitro assays were carried out to assess the influence of TCBZ on the P-glycoprotein-mediated intestinal transport of IVM, using the everted gut sac technique. Enhanced accumulation of IVM in the intestinal wall occurred after co-incubation with TCBZ.     

Composite faecal egg count reduction test to detect resistance to triclabendazole in Fasciola hepatica

A faecal egg count reduction test, using composite samples, was developed in order to assess the efficacy of the flukicide, triclabendazole (TCBZ) on commercial sheep farms in England and Wales. First, a comparison between individual counts and composite counts was conducted using sheep on two farms with different levels of infection. Faecal samples were collected from 50 sheep on each farm at the time of TCBZ treatment and 21 days later. The results showed that a composite fluke egg count (CFEC) was as sensitive as using individual samples, and the test was subsequently validated on an additional 18 sheep farms. The pre- and post-treatment CFECs using five composite samples were subjected to bootstrap analysis. The variance in fluke egg counts of composite samples was high, but analyses indicated that 20 individual samples analysed as two composites was as sensitive as using five composites. The two-composite assay was evaluated on another five farms. On seven out of 25 farms sampled, egg counts either did not decrease significantly or increased following treatment, suggesting TCBZ resistance on these farms. This assay represents a practical field test that can be used in the first instance to evaluate the efficacy of TCBZ on sheep farms where resistance is suspected.     

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.     

The effect of feed quality on the kinetic disposition of orally administered triclabendazole in sheep

The effect of two qualities of feed on the kinetic disposition of triclabendazole (TCBZ) metabolites was investigated in sheep (n = 4) following oral administration of TCBZ at 10 mg/kg body weight. The same sheep were given sequentially two qualitatively different diets: a low-quality (LQ) diet based on wheat straw ad libitum, and a high-quality (HQ) diet based on barley+alfalfa. The triclabendazole sulphoxide (TCBZSO) and triclabendazole sulphone (TCBZSO₂) concentrations were determined in blood samples taken serially from the jugular vein between 5 min and 9 days after TCBZ administration. The parent drug TCBZ was not detected in any of the samples. The quality of feed affected the kinetics of both TCBZ metabolites. The rate of appearance (T_lag and T_max) in the jugular blood was slower and the formed amount (AUC) of TCBZSO was slightly higher when the sheep were on the LQ diet (T_lag = 7.74 h; T_max = 27.91 h; AUC = 1042 g.h/ml) than when they were offered the HQ diet (T_lag = 1.90 h; T_max = 16.01 h; AUC = 832.4 g.h/ml). The MRT of TCBZSO was about 40% longer with the LQ diet than with the HQ diet. Similarly, the rate of appearance of TCBZSO₂ in plasma of sheep was slower when they were on the LQ diet than when they were on the HQ diet, suggesting an impairment of the hepatic enzymatic activity involved in the oxidation of TCBZSO to TCBZSO₂.     

Monoclonal antibody sandwich immunoassay detection of coproantigen to evaluate the efficacy of treatment in natural ovine fasciolosis

The monoclonal antibody-based sandwich immunoassay (mAb Sandwich ELISA) was used to evaluate the effectiveness of triclabendazole treatment by the detection of coproantigens in Fasciola hepatica naturally infected sheep. Twelve sheep (2 to 5 years of age) were separated into two groups. The first group (three sheep) remained untreated; the other group (nine sheep) was treated with a single dose of 5% triclabendazole at 10 mg kg-1 of body weight. All but one of the treated group had negative optical density values (OD492) after two weeks of treatment, while seven sheep intermittently shed eggs during the course of the study. In all but one of the treated sheep, no F. hepatica infection; the one positive ELISA in 5th week after treatment according to the mAb Sandwich, had one fluke in the liver. The results of the parasitological examinations, as well as OD492 values obtained by the mAb Sandwich ELISA for the detection of coproantigens are described. The findings at necropsy, of the treated group in comparison to the untreated group are shown. The mAb Sandwich ELISA could be a useful and accurate method with which to monitor the efficacy of flukicides in F. hepatica natural infections.     

An experimental study on triclabendazole resistance of Fasciola hepatica in sheep

The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, gamma-GT, ELISA measuring antibodies against recombinant cathepsin-L(1) and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted.The GLDH, gamma-GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands.     

Experiments on anthelmintic control of Fasciola hepatica in Brazil.

Two separate field trials involving naturally infected cattle were carried out on two farms known to have a history of Fasciola hepatica infection. On the first farm, 15 animals per group were allocated as follows: G1, triclabendazole (TCBZ) four times a year; G2, TCBZ twice a year (May and September); G3, untreated control. All groups grazed together and after 3.5 years the animals were slaughtered and their livers examined by federal meat inspectors who condemned 100% of livers in G3 and 8.3% in G2 owing to the presence of lesions of fasciolosis. In G1 no livers were condemned. Significant differences in weight gains were not detected and fluke counts remained at low levels in the treated groups. Also, in the control group, egg counts started to decrease when animals were 2 years old. On the second farm, groups of 20 animals were treated as follows: G1, TCBZ three times a year (May, September and December); G2, TCBZ twice a year (May and September); G3, nitroxynil twice a year (May and September); G4, rafoxanide twice a year (May and September); G5, untreated controls. All animals were weighed and faecal samples examined at approximately 28-day intervals. During the period of the study, larger weight gains were detected in the TCBZ treated groups than in the others. TCBZ treatment kept F. hepatica egg counts at a lower level for longer periods than the other drugs and significant differences in weight gains were only obtained between the group receiving TCBZ three times a year and the control group.     

The kinetics of triclabendazole disposition in sheep

To investigate whether the disposition of triclabendazole (TCBZ) and its metabolites in blood or bile influenced its flukicidal potency, TCBZ was administered intraruminally at 10 mg kg-1 to sheep surgically fitted with a permanent re-entrant bile duct cannula. The profiles of TCBZ metabolites in peripheral plasma and bile were determined using high performance liquid chromatography. In plasma, only TCBZ sulphoxide (TCBZ-SO) and TCBZ sulphone were present and reached their maximum concentrations (greater than 13 micrograms ml-1) at 18 and 36 h, respectively, after administration. TCBZ metabolites were specifically bound to plasma albumin, which is believed to exert a major influence on the duration of plasma TCBZ metabolite concentrations and consequent exposure of liver fluke. In bile, the major TCBZ metabolites were hydroxylated in the 4' position and secreted predominantly as sulphate esters with lesser proportions as glucuronide conjugates. The major biliary metabolite was conjugated hydroxy TCBZ-SO which reached a maximum concentration in excess of 40 micrograms ml-1 and contributed almost half the total conjugated metabolites. The major free biliary metabolite was TCBZ-SO. Of the administered TCBZ dose, 9.7% was secreted as free metabolites in bile whereas 35.8% was secreted as conjugated metabolites. Approximately 6.5% of the dose was excreted in urine.     

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 (3n=30), whereas the Sligo and wild-type flukes are diploid (2n=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.