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.     

Disruption of egg formation by Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles, Mehlis' gland and ovary. Over the 4-day post-treatment period, there was a progressive reduction in the number of oogonia and oocytes in the ovary and evidence of apoptosis. Vacuolation and a decrease in the number of Mehlis' gland cells were observed from 48 h pt onwards and disruption of the normal role of the gland in egg formation was evident. The vitelline follicles showed a gradual decrease in size and became vacuolated; the population structure in each follicle changed to be one consisting mainly of mature cells and the production of shell protein material declined. The follicle became disorganised as the cells broke down and released their contents into the lumen of the follicle. While the uterus appeared to contain eggs at 48 h pt in whole-mount specimens, no properly-formed eggs were observed in histological sections. By 96 h pt, the uterus was completely devoid of eggs. Overall, egg production was seen to be severely affected by TCBZ treatment and flukes were incapable of producing normal eggs within 2 days of treatment. The implications of this in terms of the epidemiology of the disease are discussed.     

Surface and internal tegumental changes in juvenile Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha

Eight indoor-reared, crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole-susceptible, Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection using 15mg/kg compound alpha. Two treated sheep per time period were euthanized at 24h, 48h and 72h post-treatment with compound alpha. The two sheep from the control group were euthanized alongside the 24h alpha-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for examination by electron microscopy. The surface morphology of the flukes' tegument was assessed using scanning electron microscopy (SEM). The ultrastructure of the tegumental syncytium and underlying tegumental cells and connections and somatic musculature were investigated using transmission electron microscopy (TEM). Both the SEM and TEM results revealed a level of disruption that increased with time, culminating at 72h with extensive tegumental loss and substantial degeneration of the cell bodies. The effects of compound alpha on the surface morphology were not particularly apparent until 48h post-treatment, when disruption included swelling and blebbing of the tegument. At 72h post-treatment, SEM revealed loss of the entire syncytial layer over large areas of the flukes. In the areas where the syncytium was lost and the basal lamina exposed, lesions of varying sizes had developed, revealing underlying tissues. Though minor forms of disruption to the ultrastructure of the syncytium were observed using TEM 24h post-treatment, it was at 48h post-treatment that substantial stress responses occurred. They included the presence of autophagic vacuoles and 'open' bodies at the apex of the syncytium and swelling of the basal infolds. The mitochondria within the syncytium and tegumental cells became progressively more disrupted over the three time periods and, by 72h post-treatment, they were frequently distorted and swollen in appearance, and contained severely swollen cristae. By 72h, the number of secretory bodies, particularly T1 bodies, had become significantly depleted in their respective cell bodies, cytoplasmic processes and in the tegumental syncytium. Both the circular and longitudinal muscle bundles were severely disrupted 72h post-treatment. They frequently contained a reduced number of muscle fibres and, in more severe instances, there was an absence of fibres altogether.     

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.     

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.     

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.     

An in vitro effect of propolis on adult worms of Fasciola gigantica

The effect of Siwa propolis on adult flukes was evaluated using scanning electron microcopy. It gave an overview of the surface architecture of the tegument of Fasciola gigantica apical cone. The base of the spines appeared to be "flaking off" and showed severe blebbing after 24h incubation with 10 micro/ml propolis. This swelling became so sever and the spines were barely visible, on increasing the concentration to 20 micro/ml. Besides, there were many large blebs on the apical cone, a number of which appeared to have burst, causing lesions and the tegument was marked by a number of pits caused by the loss of spines. With the higher concentration of 30 micro/ml, erosion of the surface had occurred to such extent that no tegument remained, only a mass of fibrous structures. The tegumental changes occurred following incubation in propolis were compared with those observed with triclabendazole (TCBZ) "Fasinex" because of its high efficacy against both mature and immature flukes.     

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.     

应用三氯苯唑控制羊春乏季节肝片吸虫病试验

目的为有效预防和控制定西市羊肝片吸虫的发生。方法选用三氯苯唑利用不同剂量和不同时间进行羊肝片吸虫驱治效果及最佳驱治时间的试验,并以硝氯酚驱虫作为对照。结果选用不同剂量的三氯苯唑对各个阶段羊肝片吸虫驱治率均可达到99.8%-100%;三氯苯唑驱虫组分别在10月、11月、12月对肝片吸虫进行驱治,来年4月份肝片吸虫驱净率分别为10%、60%和100%;硝氯酚驱虫组为30%。结论利用三氯苯唑在每年12月份驱治羊肝片吸虫,可达到春季羊体内无肝片吸虫的最佳效果,能彻底控制和消灭羊肝片吸虫病的发生。     

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.     

Interaction of Fasciola hepatica with albendazole and its metabolites

Adult Fasciola hepatica recovered from sheep 12 and 24 h after a single oral dose of albendazole (20 mg/kg) contained significant amounts of two oxidized metabolites of albendazole (ABZ), a sulphoxide (SX) and a sulphone (SO), but not ABZ. Flukes incubated in vitro with 10 microM SX or SO contained these metabolites at a level two to three times the level observed in flukes recovered from sheep 24 h after a curative oral dose of ABZ. The concentration of ABZ in flukes was 10-fold greater than either SX or SO after a 24 h in vitro incubation in 10 microM of the respective drug. Flukes exposed to ABZ in vitro contained two-fold higher SX levels than SX-treated flukes due to a combination of spontaneous oxidation in media and fluke-mediated oxidation of ABZ. Measurement of end-products of glucose metabolism following 24 h incubation in 10 microM of either ABZ, SX or SO did not show a significant difference between treated and untreated flukes.     

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.     

用免疫荧光技术检测细胞培养物中兔出血症病毒

用本所实验室制备的兔出血症病毒（rabbit hemorrhagic disease virus，RHDV）高免血清．按常规方法提纯出抗体（IgG），用异硫氰酸荧光素（FITC）标记IgG。在细胞培养时，培养瓶中放入玻片，当细胞长成单层时，按常规方法接种病毒液，培养24、48、96、120h时取出玻片，用荧光抗体染色，在荧光显微镜下观察不同代次的细胞毒。经观察：兔肾上皮细胞（RK）毒培养到36～48h、羊睾丸细胞（ST）毒培养到72～96h时可观察到特异性荧光，随着培养时间的延长，荧光亮度增强，胞浆内充满特异性荧光。用肝组织强毒病料触片，呈特异性荧光，对照细胞培养48h无荧光出现。证实了两株细胞培养物中有大量的兔出血症病毒存在，从而成功的分离培养出了兔出血症病毒细胞毒。     

用免疫荧光技术检测细胞培养物中兔出血症病毒

用本实验室制备的兔出血症病毒（rabbit hemorrhagic disease virus,RHDV）高免血清,按常规方法提纯出抗体（IgG）,用异硫氰酸荧光素（FITC）标记IgG。在细胞培养时,培养瓶中放入玻片,当细胞长成单层时,按常规方法接种病毒液,培养24、48、96、120h时取出玻片,用荧光抗体染色,在荧光显微镜下观察不同代次的细胞毒。经观察：兔肾上皮细胞（RK）毒培养到36—48h,羊睾丸细胞（ST）毒培养到72—96h时可观察到特异性荧光,随着培养时间的延长,荧光亮度增强,胞浆内充满特异性荧光。用肝组织强毒病料触片,呈特异性荧光,对照细胞培养48h无荧光出现,证实了两株细胞培养物中有大量的兔出血症病毒存在,从而成功的分离培养出了兔出血症病毒细胞毒。     

Pharmacokinetics and efficacy of triclabendazole in goats with induced fascioliasis

The pharmacokinetics of triclabendazole were evaluated in normal goats and in goats artificially infected with Fasciola hepatica. Triclabendazole and its metabolites were determined using a novel high performance liquid chromatographic method with fluorimetric detection after solid-phase extraction. In normal goats triclabendazole given orally was metabolized rapidly to its sulphoxide and sulphone derivatives. The maximum plasma concentrations for the sulphoxide and sulphone were similar ranging from 9 to 19 micrograms/ml and these were attained at an average 12.8 and 25.6 h, respectively, after administration. Both metabolites were eliminated slowly from plasma with elimination half-lives of 22.4 h for the sulphoxide and 19.4 h for the sulphone. They persisted at measurable concentrations in plasma for up to seven days. In milk, the two metabolites occurred in low concentrations and none of them was detectable (sulphoxide less than 0.04 microgram/ml, sulphone less than 0.02 microgram/ml) after seven days. The pharmacokinetic behaviour of triclabendazole was not altered in animals with fascioliasis. Efficacy of the drug against immature (six-week) F. hepatica was 100%.     

Efficacy of a fenbendazole-triclabendazole combination against Fasciola hepatica and gastrointestinal nematodes in sheep

A fenbendazole and triclabendazole combination was tested against experimentally induced 6-and 14-week-old infections of Fasciola hepatica and naturally acquired infections of gastrointestinal nematodes in sheep. Forty-eight sheep were divided randomly into six treatment groups of eight animals each. At 7.5 and 10.0 mg kg-1 body weight (BW) (3.75 and 5.0 mg kg-1 BW of each drug), the drug combination was 98.3 and 99.7% effective against 6-week-old F. hepatica infections, and 100 and 99.9% effective against 14-week-old infections. At the same drug dosages, the drug combination was effective against naturally acquired adult Ostertagia spp. (97.3-100%) and adult Nematodirus spp. (90.3-99.9%). No adverse effects associated with treatment were noted.     

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.     

The efficacy of formulations of triclabendazole and ivermectin in combination against liver fluke (Fasciola hepatica) and gastro-intestinal nematodes in cattle and sheep and sucking lice species in cattle

OBJECTIVE: To assess the efficacy of two formulations of triclabendazole and ivermectin in combination against liver fluke (Fasciola hepatica), gastro-intestinal nematodes and sucking louse species in cattle and sheep. PROCEDURE: A study of 540 cattle and 428 sheep at 18 sites throughout Victoria and New South Wales was undertaken. At each site, one group of cattle or sheep was treated with a combined formulation (Fasimec Cattle or Fasimec Sheep), another received ivermectin and triclabendazole separately. In trials on lice infestation, an additional group remained untreated. Samples for faecal egg counts were collected on days -7, 0 (treatment day), +7, +14 and +21 after treatment. Lice assessments were carried out on days -7, 0, +7, +14, +28, +42 and +56. RESULTS: Both treatments were highly efficacious (> 98% efficacy) against liver fluke in cattle and sheep, against three sucking lice species of cattle and against gastro-intestinal nematodes in sheep. There was also no significant difference between treatments in efficacy. Against gastro-intestinal nematodes, Fasimec Cattle was significantly (P < 0.01) more effective than the separately applied ivermectin and triclabendazole treatment. Mean efficacy for the Fasimec Cattle and Ivomec/Fasinex 120 groups respectively, was 97.6% and 94.2% on Day +7, 98.9% and 91% on Day +14 and 98.5% and 92.6% on Day +21. CONCLUSION: The efficacy of Fasimec' Cattle and Fasimec Sheep was at least equal to that of currently registered products (with the same active ingredients) used to control these parasites.     

The effect of albendazole and triclabendazole on colchicine binding in the liver fluke Fasciola hepatica

Albendazole (ABZ) and its sulfoxide (SX) and sulfone (SO) metabolites inhibit the binding of 3H-colchicine, a ligand with high affinity for tubulin to homogenate preparations of the liver fluke Fasciola hepatica. The relative potency of these compounds is SX greater than ABZ greater than SO. The benzimidazoles (cambendazole, parbendazole, oxibendazole and mebendazole), when tested at a concentration of 10 microM, also inhibited colchicine binding to fluke homogenates. However, a potent new benzimidazole flukacide, triclabendazole (TCB), was without effect on colchicine binding to F. hepatica homogenates. When intact flukes were exposed in vitro to 10(-5)M SX for as little as 5 min the subsequent binding of 3H-colchicine to fluke homogenates was significantly reduced. However, flukes recovered from sheep either 12 or 24 h after treatment with ABZ did not have a decreased ability to bind colchicine, although the non-specific binding was higher in flukes from treated sheep, suggesting some interaction of drug with tubulin in vivo. ABZ, SX and SO were effective in preventing embryonation of fluke eggs at doses as low as 0.01 microM, but TCB was without effect at concentrations as high as 10 microM. The results suggest that ABZ exerts at least part of its anthelmintic effect by interaction with fluke tubulin.