Field evaluation of Duddingtonia flagrans IAH 1297 for the reduction of worm burden in grazing animals: Pasture larval studies in horses, cattle and goats.

A series of placebo-controlled trials were conducted in horses, cattle and goats in different seasons and bioclimatic regions of New South Wales and Queensland, Australia, to evaluate the ability of BioWorma®, a feed supplement containing the spores of Duddingtonia flagrans IAH 1297, to reduce the larval development of parasitic gastrointestinal nematodes (GIN) and their subsequent migration from faeces onto the surrounding pasture. In each trial, faeces were collected from animals harbouring a burden of nematode parasites following a period of supplementation with a placebo and again after supplementation with BioWorma. The faeces were manually placed onto pasture plots at one or two distinct geographical sites and the effect of treatment was determined by subsequent monitoring the numbers of parasitic larvae on the pasture surrounding the faecal pats at two weekly intervals over an eight week period. The results for these studies showed that administration of BioWorma at a minimum daily dose of 3 × 104 spores/kg bodyweight reduced parasite larvae in the pasture surrounding the faeces by 53-99 % over an eight week post treatment period in horses, cattle and goats in a range of bioclimatic zones and in different seasons. Overall, the studies with BioWorma show substantial reductions in GIN infectivity of pasture surrounding faeces of treated horses, cattle and goats (P < 0.05). Results indicate that the use of BioWorma in these host species would lead to decreased levels of GIN infection in animals grazing pasture when this product is used and would provide an alternative means of controlling parasitic nematodes.

Field evaluation of Duddingtonia flagrans IAH 1297 for the reduction of worm burden in grazing animals: Tracer studies in sheep.

The aim of these studies was to determine the reduction in pasture infectivity likely to be achieved by the supplementation of grazing sheep with BioWorma®, a product containing the chlamydospores of the nematophagous fungus Duddingtonia flagrans strain IAH 1297. Four placebo-controlled trials were conducted between 2009 and 2013 in sheep in different climatic regions of New South Wales and Queensland, Australia and across several seasons. The effectiveness of BioWorma was assessed by total worm counts in tracer sheep placed in paddocks grazed by parasitised sheep which were fed a daily supplement with and without BioWorma under group-feeding conditions. Further proof of concept was obtained by assessing the worm burdens and weight gains of the parasitised sheep, as well as the number of anthelmintic ("salvage") treatments required when faecal egg counts exceeded a threshold level. Significant reductions ranging from 57 to 84% (P < 0.05) in worm burdens of the tracer sheep placed in the paddock grazed by BioWorma treated sheep were obtained in all four trials, compared to the Control group. In two of the studies the treatment effect was greater at the end of the trial, indicating that pasture infectivity in the Control paddocks had risen considerably. The main nematodes encountered were Haemonchus spp., Trichostrongylus spp., and Teladorsagia spp. (including multi-resistant strains) and significant reductions were demonstrated for each of these species. Given the results of the four trials it can be concluded that supplementation of pastured sheep with BioWorma was effective in reducing the numbers of parasitic nematode larvae ingested by tracer sheep. It is considered that these levels of reduced pasture larvae would result in productivity increases in grazing sheep and reduce the requirement for intervention with anthelmintic chemicals. Therefore, use of BioWorma will provide an alternative means for control of gastrointestinal nematode (GIN) parasites on pasture.

Web-based self-management support for people with type 2 diabetes (HeLP-Diabetes): randomised controlled trial in English primary care.

OBJECTIVE: To determine the effectiveness of a web-based self-management programme for people with type 2 diabetes in improving glycaemic control and reducing diabetes-related distress. METHODS AND DESIGN: Individually randomised two-arm controlled trial. SETTING: 21 general practices in England. PARTICIPANTS: Adults aged 18 or over with a diagnosis of type 2 diabetes registered with participating general practices. INTERVENTION AND COMPARATOR: Usual care plus either Healthy Living for People with Diabetes (HeLP-Diabetes), an interactive, theoretically informed, web-based self-management programme or a simple, text-based website containing basic information only. OUTCOMES AND DATA COLLECTION: Joint primary outcomes were glycated haemoglobin (HbA1c) and diabetes-related distress, measured by the Problem Areas in Diabetes (PAID) scale, collected at 3 and 12 months after randomisation, with 12 months the primary outcome point. Research nurses, blind to allocation collected clinical data; participants completed self-report questionnaires online. ANALYSIS: The analysis compared groups as randomised (intention to treat) using a linear mixed effects model, adjusted for baseline data with multiple imputation of missing values. RESULTS: Of the 374 participants randomised between September 2013 and December 2014, 185 were allocated to the intervention and 189 to the control. Final (12 month) follow-up data for HbA1c were available for 318 (85%) and for PAID 337 (90%) of participants. Of these, 291 (78%) and 321 (86%) responses were recorded within the predefined window of 10-14 months. Participants in the intervention group had lower HbA1c than those in the control (mean difference -0.24%; 95% CI -0.44 to -0.049; p=0.014). There was no significant overall difference between groups in the mean PAID score (p=0.21), but prespecified subgroup analysis of participants who had been more recently diagnosed with diabetes showed a beneficial impact of the intervention in this group (p = 0.004). There were no reported harms. CONCLUSIONS: Access to HeLP-Diabetes improved glycaemic control over 12 months. TRIAL REGISTRATION NUMBER: ISRCTN02123133.

In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only.

There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and -ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances.

Relative potency of macrocyclic lactones in in vitro assays with larvae of susceptible and drug-resistant Australian isolates of Haemonchus contortus and H. placei.

The present study used in vitro assays to determine the relative potency of commercial macrocyclic lactone (ML) anthelmintics against larvae of drug-susceptible and drug-resistant Australian isolates of important parasites of sheep and cattle, Haemonchus contortus and Haemonchus placei, respectively. Cattle pour-on products containing abamectin, ivermectin, eprinomectin, doramectin or moxidectin were diluted in DMSO and used in larval development assays. Abamectin was the most potent chemical (lowest IC50 value) towards the drug-susceptible H. contortus Kirby isolate. The abamectin IC50 was approximately 2-fold lower than those for ivermectin, moxidectin, eprinomectin and doramectin. Moxidectin and abamectin were the most potent chemicals towards the resistant H. contortus Wallangra isolate. This isolate showed resistance ratios up to 70-fold towards eprinomectin. The resistance ratio for this species was lowest with moxidectin (ratio of 4.0-fold). Abamectin was also the most potent chemical towards both drug-susceptible (Bremner) and drug-resistant (Dayboro) H. placei isolates. The larval development assay only showed low levels of resistance for the drug-resistant H. placei, with resistance ratios ranging from 1.7 to 2.0 fold for moxidectin and abamectin, up to 3.3-fold for eprinomectin. This study examined the readily-accessible larval life stages of these parasites in in vitro assays, and, hence, the relationship between our findings and relative drug efficacies in vivo remains to be determined. Despite this, the study accords with some evidence from the use of these anthelmintics in the field in demonstrating the potency of moxidectin and abamectin against ML-resistant H. contortus. The study also highlights the usefulness of eprinomectin as a readily-available compound which is a more sensitive marker for ML resistance in in vitro larval development assays than the other commercial ML compounds examined.

Phylogenetic characterization of ß-tubulins and development of pyrosequencing assays for benzimidazole resistance in cattle nematodes.

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the ß-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length ß-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be ß-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans ß-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like ß-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in ß-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.

The complexity of the secreted NPA and FAR lipid-binding protein families of Haemonchus contortus revealed by an iterative proteomics-bioinformatics approach.

Two different classes of small nematode specific lipid-binding proteins, the nematode polyprotein allergens/antigens (NPAs) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in Haemonchus contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to reveal the expression and complexity of these proteins in the excretory/secretory products (ESP) of adult H. contortus at the protein and gene levels. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to Ostertagia ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and Caenorhabditis elegans homologues retrieved diverse sequences encoding H. contortus NPA and FAR proteins. H. contortus sequences were then integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the diversity in structures of the encoding genes was revealed by assembling the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least one Hc-NPA gene and six Hc-FAR genes in H. contortus, and life stage gene expression of Hc-FAR-1 to -6 revealed unique transcription patterns for each of these genes.