The egg hatch test: A useful tool for albendazole resistance diagnosis in Fasciola hepatica.

In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the "gold standard" controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 µM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 µM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 µM and those of the in vivo assays.

Control of Nematodirus spp. infection by sheep flock owners in Northern Ireland.

BACKGROUND: To address a lack of information on the control of ovine helminth parasites in Northern Ireland (NI), a number of research projects have been undertaken, dealing with gastrointestinal nematodes, tapeworms and liver fluke. This investigation concerns Nematodirus and concentrates on three aspects of disease: farm management strategies for its control, derived from the results of a Questionnaire; the efficacy of treatment used by farmers, as determined by a coprological survey; and the hatching requirements of Nematodirus eggs, that is, whether prolonged chilling is a pre-requisite for hatching. RESULTS: A Questionnaire was sent to 252 sheep farmers in NI in March 2012 (covering the years 2009-2012) and replies were received from 228 farmers. Under-dosing, inaccurate calibration of equipment and inappropriate product choice were poor practices identified. Following this survey, the efficacy of treatment of Nematodirus spp. in sheep flocks was evaluated in April and May 2012. Sampling kits were sent to 51 flock owners, all of whom returned pre- and post-anthelmintic dosing faecal samples to the laboratory for analysis. At the time of treatment, 41 flocks were positive for Nematodirus (as diagnosed by the presence of eggs). Reduced benzimidazole efficacy was detected in 35.7% of flocks tested (n = 28). Although only involving a small number of flocks, reduced efficacy of levamisole treatment was detected in 50%, of avermectins in 33% and of moxidectin in 75% of flocks tested (n = 2, 6 and 4, respectively). In the egg hatch experiment, carried out under "chilled" and "non-chilled" conditions, 43% of the eggs in the "non-chilled" group were able to hatch, compared to 100% in the "chilled" group. CONCLUSIONS: The identification of inefficient control strategies argues for continued education of stockholders, in order to improve their management programmes. This is particularly important where the practices might impact on the development of anthelmintic resistance, which has been shown to exist on NI farms. The appropriate choice of anthelmintic is a vital part of this plan. The ability of eggs to hatch under non-chilled conditions demonstrates a flexibility in hatching behaviour. This may represent an adaptation to climate change and account for the recent emergence of a second, autumnal peak of infection.

Disruption of egg production by triclabendazole-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh (Mirazid).

An in vitro study has been carried out to monitor changes to the female reproductive system in adult triclabendazole (TCBZ)-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh ("Mirazid"). Flukes were immersed for 6 h and 24 h in myrrh extract at a concentration of 200 µg/ml, then processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis' gland, ovary and vitellaria. Egg production had become abnormal at 6 h post-treatment (pt), with the uterine lumen being filled with free vitelline cells and masses of shell protein material; few eggs were present. At 24 h pt, no eggs were present. Distinct changes to the ovary and Mehlis' gland were only observed after 24 h incubation in Mirazid. The ovary contained numbers of apoptotic oogonia and oocytes. In the Mehlis' gland, the S1 cells were disorganised and the processes from them were vacuolated, although the disruption was not significant. More severe changes were observed in the vitelline cells and follicles. After 6 h incubation in Mirazid, although the gross organisation of the vitelline follicles appeared to be normal, nuclear changes indicative of the early stages of apoptosis were observed in the stem cells and shell protein production by the mature cells had decreased. At 24 h pt, a distinct shift in cell population was evident, with the follicles containing mainly mature cells and spaces were present between the cells. The shell globule clusters in the mature cells were disorganised. In more severely-affected follicles, cells were seen to be breaking down, with karyolytic nuclei and disintegrating cytoplasm. Overall, the results have shown that exposure to Mirazid treatment had a severe impact on egg production by TCBZ-resistant flukes, an effect that was mediated by disruption of the vitelline cells and of the mechanism co-ordinating egg formation in the ootype.

Time-course and accumulation of triclabendazole and its metabolites in bile, liver tissues and flukes collected from treated sheep.

The flukicidal compound triclabendazole (TCBZ) has a complex metabolic pattern that includes the systemic presence of its sulphoxide (TCBZ.SO) and sulphone (TCBZ.SO2) metabolites, usually recovered from the bile of treated animals. The aim of the current work was to evaluate the time-course and pattern of in vivo accumulation of TCBZ/metabolites into adult Fasciola hepatica specimens recovered from infected sheep. Twelve (12) healthy Corriedale sheep were orally infected with one hundred (100) metacercariae of the TCBZ-susceptible Cullomptom isolate of F. hepatica. Sixteen weeks after infection, animals were intraruminally treated with TCBZ (10mg/kg). At 3, 24, 48 and 60h post-treatment (pt), animals were sacrificed (n=3/time period) and samples of blood, bile, liver tissue and adult F. hepatica specimens were collected. The concentrations of TCBZ/metabolites were measured by HPLC. TCBZ.SO and TCBZ.SO2 were the only molecules recovered in the bloodstream, with peak plasma concentrations of 10.8µg/mL (TCBZ.SO) and 12.6µg/mL (TCBZ.SO2). The same metabolites were also the main analytes accumulated within the adult flukes, reaching peak concentrations between 6.35µg/g (TCBZ.SO) and 13.9µg/g (TCBZ.SO2) at 24h pt, which was coincident with the time when the maximum plasma concentration was attained. Low levels of TCBZ parent drug (0.14µg/g at 24h pt) were measured within collected flukes. TCBZ parent drug and its sulpho- and hydroxy-derivatives were recovered in bile collected from treated sheep between 3 and 60h pt. Although relatively high concentrations of hydroxy-TCBZ (ranging from 0.86 to 10.1µg/mL) were measured in bile, this metabolite was not recovered within the flukes at any time pt. Finally, TCBZ parent drug was the main compound accumulated in liver tissue over the 60h pt period. The time-course and drug concentration patterns within the adult liver fluke after TCBZ treatment followed a similar trend to those observed in plasma. Overall, the data reported here confirm that oral ingestion is a main route of drug entry into the trematode in vivo exposed to TCBZ/metabolites. However, the presence of TCBZ within the adult fluke (despite being absent in the systemic circulation) may be related to some degree of trans-tegumental diffusion from bile or by a direct oral ingestion from portal blood.

Differential expression of liver fluke ß-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-susceptible Leon isolate and the triclabendazole-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to triclabendazole 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 triclabendazole is not associated with changes in its presumed target molecule.

Mitochondrial DNA haplotype analysis of liver fluke in bison from Bialowieza Primaeval Forest indicates domestic cattle as the likely source of infection.

We have determined the mitochondrial genotype of liver fluke present in Bison (Bison bonasus) from the herd maintained in the Bialowieza National Park in order to determine the origin of the infection. Our results demonstrated that the infrapopulations present in the bison were genetically diverse and were likely to have been derived from the population present in local cattle. From a consideration of the genetic structure of the liver fluke infrapopulations we conclude that the provision of hay at feeding stations may be implicated in the transmission of this parasite to the bison. This information may be of relevance to the successful management of the herd.

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations.

Control of fasciolosis is threatened by the development of anthelmintic resistance. Enhanced triclabendazole (TCBZ) efflux by ABC transporters such as P-glycoprotein (Pgp) has been implicated in this process. A putative full length cDNA coding for a Pgp expressed in adult Fasciola hepatica has been constructed and used to design a primer set capable of amplifying a region encoding part of the second nucleotide binding domain of Pgp when genomic DNA was used as a template. Application of this primer set to genomic DNA from TCBZ-resistant and -susceptible field populations has shown a significant difference in the alleles present. Analysis of an allele occurring at a three-fold higher frequency in the "resistant" population revealed that it was characterised by a serine to arginine substitution at residue 1144. Homology modelling studies have been used to locate this site in the Pgp structure and hence assess its potential to modify functional activity.

Validity and reliability of GPS for measuring instantaneous velocity during acceleration, deceleration, and constant motion.

In this study, we assessed the validity and reliability of 5 and 10 Hz global positioning systems (GPS) for measuring instantaneous velocity during acceleration, deceleration, and constant velocity while straight-line running. Three participants performed 80 running trials while wearing two GPS units each (5 Hz, V2.0 and 10 Hz, V4.0; MinimaxX, Catapult Innovations, Scoresby, VIC, Australia). The criterion measure used to assess GPS validity was instantaneous velocity recorded using a tripod-mounted laser. Validity was established using the standard error of the estimate (± 90% confidence limits). Reliability was determined using typical error (± 90% confidence limits, expressed as coefficient of variation) and Pearson's correlation. The 10 Hz GPS devices were two to three times more accurate than the 5 Hz devices when compared with a criterion value for instantaneous velocity during tasks completed at a range of velocities (coefficient of variation 3.1-11.3%). Similarly, the 10 Hz GPS units were up to six-fold more reliable for measuring instantaneous velocity than the 5 Hz units (coefficient of variation 1.9-6.0%). Newer GPS may provide an acceptable tool for the measurement of constant velocity, acceleration, and deceleration during straight-line running and have sufficient sensitivity for detecting changes in performance in team sport. However, researchers must account for the inherent match-to-match variation reported when using these devices.

Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy?

The liver fluke remains an economically significant parasite of livestock and is emerging as an important zoonotic infection of humans. The incidence of the disease has increased in the last few years, as a possible consequence of changes to the World's climate. Future predictions suggest that this trend is likely to continue. Allied to the changing pattern of disease, reports of resistance to triclabendazole (TCBZ) have appeared in the literature, although they do not all represent genuine cases of resistance. Nevertheless, any reports of resistance are a concern, because triclabendazole is the only drug that has high activity against the migratory and damaging juvenile stages of infection. How to deal with the twin problems (of increasing incidence and drug resistance) is the overall theme of the session on "Trematodes: Fasciola hepatica epidemiology and control" and of this review to introduce the session. Greater knowledge of fluke epidemiology and population genetics will highlight those regions where surveillance is most required and indicate how quickly resistant populations of fluke may arise. Models of disease risk are becoming increasingly sophisticated and precise, with more refined data analysis programmes and Geographic Information Systems (GIS) data. Recent improvements have been made in our understanding of the action of triclabendazole and the ways in which flukes have become resistant to it. While microtubules are the most likely target for drug action, tubulin mutations do not seem to be involved in the resistance mechanism. Rather, upregulation of drug uptake and metabolism processes appear to be more important and the data relating to them will be discussed. The information may help in the design of new treatment strategies or pinpoint potential molecular markers for monitoring fluke populations. Advances in the identification of novel targets for drugs and vaccines will be made by the various "-omics" technologies that are now being applied to Fasciola. A major area of concern in the current control of fasciolosis is the lack of reliable tests for the diagnosis of drug (TCBZ) resistance. This has led to inaccurate reports of resistance, which is hindering successful disease management, as farmers may be encouraged to switch to less effective drugs. Progress with the development of a number of new diagnostic tests will be reviewed.

Fasciola hepatica: time-dependent disruption of spermatogenesis following in vivo treatment with triclabendazole.

Sheep infected with the Cullompton isolate of Fasciola hepatica were treated with triclabendazole at a concentration of 10 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the liver and, where present, from the gall bladder at 48, 72 and 96 h post-treatment (pt). Gross changes to the spermatogenic cells of the testis were examined by histology and ultrastructural alterations were visualised via transmission electron microscopy. Disruption was progressive in nature, with the testis tubules becoming shrunken, vacuolated and gradually more denuded of cellular content over the 96-h time period. From 48 h pt, the number of primary and secondary spermatogonia decreased and multinucleate spermatogonial cells were frequent. Later, developmental stages were uncommon, giving rise to much empty space within the tubules. By 72 h pt, the tubules contained many apoptotic and degraded cells and had an extremely disorganised appearance. At 96 h pt, the tubules were almost completely empty, with the exception of the remains of degraded spermatogenic cells. These results indicate that triclabendazole severely disrupts spermatogenesis in the liver fluke from 48 h pt in vivo.

Comparative proteomic analysis of triclabendazole response in the liver fluke Fasciola hepatica.

Control of Fasciola hepatica infections of livestock in the absence of vaccines depends largely on the chemical triclabendazole (TCBZ) because it is effective against immature and adult parasites. Overdependence on a single drug and improper application is considered a significant factor in increasing global reports of fluke resistant to TCBZ. The mode(s) of action and biological target(s) of TCBZ are not confirmed, delaying detection and the monitoring of early TCBZ resistance. In this study, to further understand liver fluke response to TCBZ, the soluble proteomes of TCBZ-resistant and TCBZ-susceptible isolates of F. hepatica were compared with and without in vitro exposure to the metabolically active form of the parent drug triclabendazole sulphoxide (TCBZ-SO), via two-dimensional gel electrophoresis (2-DE). Gel image analysis revealed proteins displaying altered synthesis patterns and responses both between isolates and under TCBZ-SO exposure. These proteins were identified by mass spectrometry supported by a F. hepatica expressed sequence tag (EST) data set. The TCBZ responding proteins were grouped into three categories; structural proteins, energy metabolism proteins, and "stress" response proteins. This single proteomic investigation supported the reductionist experiments from many laboratories that collectively suggest TCBZ has a range of effects on liver fluke metabolism. Proteomics highlighted differences in the innate proteome profile of different fluke isolates that may influence future therapy and diagnostics design. Two of the TCBZ responding proteins, a glutathione transferase and a fatty acid binding protein, were cloned, produced as recombinants, and both found to bind TCBZ-SO at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance.

Liver fluke ß-tubulin isotype 2 binds albendazole and is thus a probable target of this drug.

Albendazole is a benzimidazole drug which can be used to treat liver fluke (Fasciola hepatica) infections. Its mode of action is believed to be the inhibition of microtubule formation through binding to ß-tubulin. However, F. hepatica expresses at least six different isotypes of ß-tubulin, and this has confused, rather than clarified, understanding of the molecular mechanisms of benzimidazole drugs in this organism. Recombinant F. hepatica ß-tubulin proteins were expressed in, and purified from, Escherichia coli. These proteins were then used in pull-down assays in which albendazole was covalently linked to Sepharose. ß-Tubulin isotype 2 was pulled down in this assay, and this interaction could be reduced by adding competing albendazole. Molecular modelling of ß-tubulin isotypes suggests that changes in the side change conformations of residue 200 in the putative albendazole binding site may be important in determining whether, or not, a particular isotype will bind to the drug. These results, together with previous work demonstrating that albendazole causes disruption of microtubules in the liver fluke, strongly suggest that ß-tubulin isotype 2 is one of the targets of this drug.

Enhancement of the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica using the metabolic inhibitor ketoconazole.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered by using the metabolic inhibitor, ketoconazole (KTZ) to inhibit the cytochrome P450 (CYP 450) system within Fasciola hepatica. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 enzyme system was inhibited by a 2 h pre-incubation in KTZ (40 microM). Flukes were then incubated for a further 22 h in NCTC medium containing either KTZ; KTZ + nicotinamide adenine dinucleotide phosphate (NADPH; 1 nM); KTZ + NADPH + TCBZ (15 microg/ml); or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO;15 microg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible isolate than the TCBZ-resistant isolate. However, co-incubation with KTZ and TCBZ/TCBZ.SO led to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own, with greater swelling and blebbing of the tegument and even the loss of the apical plasma membrane in places. With the Cullompton isolate, there was limited potentiation of drug action in combination with KTZ, and only with TCBZ.SO. The results support the concept of altered drug metabolism within TCBZ-resistant isolates and indicate that this process may play a role in the development of drug resistance.

Binding of serum albumin to the anthelmintic drugs albendazole, triclabendazole and their sulphoxides.

The binding of drugs to plasma proteins--especially serum albumin--is an important factor in controlling the availability and distribution of these drugs. In this study we have investigated the binding of two drugs commonly used to treat liver fluke infections, albendazole (ABZ) and triclabendazole (TCBZ), and their sulphoxide metabolites to bovine serum albumin (BSA). Both ABZ and TCBZ caused shifts in the mobility of BSA in native gel electrophoresis. No such changes were observed with the sulphoxides under identical conditions. The drugs, and their sulphoxides, caused quenching of the intrinsic tryptophan fluorescence of BSA, indicating association between the drugs and this protein. Quantification of this quenching suggested a 5-10-fold reduction in affinity of the sulphoxides compared to the parent compounds. These results are discussed in respect to previous work on the pharmacodynamics of these fasciolicides and will inform the design of novel anthelmintics.

Potentiation of triclabendazole sulphoxide-induced tegumental disruption by methimazole in a triclabendazole-resistant isolate of Fasciola hepatica.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 microM), then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); MTZ+NADPH+TCBZ (15 microg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 microg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than triclabedazole-resistant isolate. However, co-incubation with MTZ+TCBZ, but more particularly MTZ+TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own, with severe swelling of the basal infolds and mucopolysaccharide masses in the syncytium, accompanied by a reduction in numbers of secretory bodies. The synthesis and production of secretory bodies in the tegumental cells was severely affected as well. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes, and this process may play a role in the development of drug resistance.

Unchanged triclabendazole kinetics after co-administration with ivermectin and methimazole: failure of its therapeutic activity against triclabendazole-resistant liver flukes.

BACKGROUND: The reduced drug accumulation based on enhanced drug efflux and metabolic capacity, identified in triclabendazole (TCBZ)-resistant Fasciola hepatica may contribute to the development of resistance to TCBZ. The aim of this work was to evaluate the pharmacokinetics and clinical efficacy of TCBZ administered alone or co-administered with ivermectin (IVM, efflux modulator) and methimazole (MTZ, metabolic inhibitor) in TCBZ-resistant F. hepatica-parasitized sheep. Sheep infected with TCBZ-resistant F. hepatica (Sligo isolate) were divided into three groups (n = 4): untreated control, TCBZ-treated (i.r. at 10 mg/kg) and TCBZ+IVM+MTZ treated sheep (10 i.r., 0.2 s.c. and 1.5 i.m. mg/kg, respectively). Plasma samples were collected and analysed by HPLC. In the clinical efficacy study, the animals were sacrificed at 15 days post-treatment to evaluate the comparative efficacy against TCBZ-resistant F. hepatica. RESULTS: The presence of IVM and MTZ did not affect the plasma disposition kinetics of TCBZ metabolites after the i.r. administration of TCBZ. The AUC value of TCBZ.SO obtained after TCBZ administration (653.9 +/- 140.6 microgxh/ml) was similar to that obtained after TCBZ co-administered with IVM and MTZ (650.7 +/- 122.8 microgxh/ml). Efficacy values of 56 and 38% were observed for TCBZ alone and for the combined treatment, respectively. No statistical differences (P > 0.05) were observed in fluke counts between treated groups and untreated control, which confirm the resistant status of the Sligo isolate. CONCLUSIONS: The presence of IVM and MTZ did not affect the disposition kinetics of TCBZ and its metabolites. Thus, the combined drug treatment did not reverse the poor efficacy of TCBZ against TCBZ-resistant F. hepatica.

Fasciola hepatica: disruption of spermatogenesis by the fasciolicide compound alpha.

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48, and 72 h post-treatment (p.t.). Changes to the spermatogenic cells in the testis were examined by histology and transmission electron microscopy. Disruption to the testes became increasingly severe over time. The testis tubules shrank in size, became vacuolated, and contained fewer cells. Identification of cell types became difficult, and apoptotic eosinophilic bodies were the predominant feature at 72 h p.t. Changes to the spermatogonia were evident at 24 h p.t., the cells containing swollen and electron-lucent mitochondria. The proportion of tertiary spermatogonia increased at 48 h p.t., and they showed signs of autophagy. Multinucleate spermatogonia were a feature of drug treatment at this time point, and they contained autophagic vacuoles. By 72 h p.t., it was difficult to identify primary and secondary spermatogonia, and there were no recognisable clusters of tertiary spermatogonia. Most spermatogonial cells were multinucleate and in the process of breaking down. With regard to the primary spermatocytes, fragmentation of the cytophore was observed at 24 h p.t. Intact rosettes were rare after 48 h treatment; collections of cells were seen, but were not organised into clusters. By 72 h p.t., no spermatocyte cells could be recognised. The results indicate that spermatogenesis was severely affected by compound alpha.

Fasciola hepatica expresses multiple alpha- and beta-tubulin isotypes.

We have identified five alpha-tubulin and six beta-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke alpha-tubulin and between 65 and 97% for beta-tubulin isotypes. Nucleotide sequence identity ranged between 68-77% and 62-80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the alpha-tubulins and two of the beta-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on beta-tubulin, three of the fluke isotypes had tyrosine at position 200 of beta-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.

No difference in 1RM strength and muscle activation during the barbell chest press on a stable and unstable surface.

Exercise or Swiss balls are increasingly being used with conventional resistance exercises. There is little evidence supporting the efficacy of this approach compared to traditional resistance training on a stable surface. Previous studies have shown that force output may be reduced with no change in muscle electromyography (EMG) activity while others have shown increased muscle EMG activity when performing resistance exercises on an unstable surface. This study compared 1RM strength, and upper body and trunk muscle EMG activity during the barbell chest press exercise on a stable (flat bench) and unstable surface (exercise ball). After familiarization, 13 subjects underwent testing for 1RM strength for the barbell chest press on both a stable bench and an exercise ball, each separated by at least 7 days. Surface EMG was recorded for 5 upper body muscles and one trunk muscle from which average root mean square of the muscle activity was calculated for the whole 1RM lift and the concentric and eccentric phases. Elbow angle during each lift was recorded to examine any range-of-motion differences between the two surfaces. The results show that there was no difference in 1RM strength or muscle EMG activity for the stable and unstable surfaces. In addition, there was no difference in elbow range-of-motion between the two surfaces. Taken together, these results indicate that there is no reduction in 1RM strength or any differences in muscle EMG activity for the barbell chest press exercise on an unstable exercise ball when compared to a stable flat surface. Moreover, these results do not support the notion that resistance exercises performed on an exercise ball are more efficacious than traditional stable exercises.