FASCIOLA-HEPATICA - TEGUMENTAL SURFACE CHANGES IN ADULT AND JUVENILE FLUKES FOLLOWING TREATMENT IN-VITRO WITH THE SULFOXIDE METABOLITE OF TRICLABENDAZOLE (FASINEX)

The effects of the novel benzimidazole, triclabendazole (Fasinex, Ciba-Geigy), in its active sulphoxide metabolite form (TCBZ-SX), on the tegumental surface of Fasciola hepatica has been examined in vitro. The tegument of adult flukes incubated in TCBZ-SX (50 mug/ml) appeared swollen and blebbed after only 6 h. In addition, progressive spine loss at the oral cone was evident following 12 h treatment. After 24 h, the tegumental syncytium and spines had completely sloughed away, leaving an exposed basal lamina and empty spine sockets. Juvenile flukes (3 weeks old) also demonstrated tegumental alterations after treatment with TCBZ-SX (20 mug/ml). The syncytium became extremely roughened and corrugated on both dorsal and ventral surfaces after only 3 h. Following 6- and 9-h incubations, there were many deep furrows, which were especially pronounced on the ventral surface, and by 18 h, the juvenile tegument was severely disrupted, especially on the ventral surface. In all cases, the effects were more marked than in the previous incubation periods. The results confirm the potent activity of triclabendazole against F. hepatica and suggest that the tegument of adult and juvenile flukes may be a target organ for this important fasciolicide.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

Differential expression of liver fluke β-tubulin isotypes at selected life cycle stages

We have shown that Fasciola hepatica expresses at least six ß-tubulins in the adult stage of its life cycle, designated F.hep-ß-tub1-6 (Ryan et al., 2008). Here we show that different complements of tubulin isotypes are expressed in different tissues and at different life cycle stages; this information may inform the search for novel anthelmintics. The predominant (as judged by quantitative PCR) isotype transcribed at the adult stage was F.hep-ß-tub1 and immunolocalisation studies revealed that this isotype occurred mainly in mature spermatozoa and vitelline follicles. Quantitative PCR indicated that changes occurred in the transcription levels of ß-tubulin isotypes at certain life cycle stages and may be of importance in the efficacy of benzimidazole-based anthelmintic drugs, but there were no significant differences between the triclabendazole (TCBZ)-susceptible Leon isolate and the TCBZ-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to TCBZ were compared, only one amino acid change resulting from a homozygous coding sequence difference (Gly269Ser) in isotype 4 was observed. However, this change was not predicted to alter the overall structure of the protein. In conclusion, these findings indicate that there is tissue-specific expression of tubulin isotypes in the liver fluke but the development of resistance to TCBZ is not associated with changes in its presumed target molecule.

Nicotinamide Benzimidazolide Dinucleotides, Non-Cyclisable Analogues of NAD+

Benzimidazole-based nucleotides and dinucleotides have been synthesised to increase the range of chemical tools available to probe the NAD+ biology space. They were examined for their reactivity in alkylation-type reactions, where they yielded unstable alkylated heteoaromatic adducts, both chemically and enzymatically. While unsuited for NAD+ cyclases, these NAD+ analogues could be viable substrates for non-adenine modifying NAD+-dependent enzyme classes.

Fasciola hepatica:the effect of the microtubule inhibitors colchicine and tubulozole-C on the ultrastructure of the adult fluke

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of T1 secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.