The cytochrome P450 family in the parasitic nematode <i>Haemonchus contortus</i>

&lt;i&gt;Haemonchus contortus&lt;/i&gt;, a highly pathogenic and economically important parasitic nematode of sheep, is particularly adept at developing resistance to the anthelmintic drugs used in its treatment and control. The basis of anthelmintic resistance is poorly understood for many commonly used drugs with most research being focused on mechanisms involving drug targets or drug efflux. Altered or increased drug metabolism is a possible mechanism that has yet to receive much attention despite the clear role of xenobiotic metabolism in pesticide resistance in insects. The cytochrome P450s (CYPs) are a large family of drug-metabolising enzymes present in almost all living organisms, but for many years thought to be absent from parasitic nematodes. In this paper, we describe the CYP sequences encoded in the &lt;i&gt;H. Contortus&lt;/i&gt; genome and compare their expression in different parasite life-stages, sexes and tissues. We developed a novel real-time PCR approach based on partially assembled CYP sequences “tags” and confirmed findings in the subsequent draft genome with RNA-seq. Constitutive expression was highest in larval stages for the majority of CYPs, although higher expression was detected in the adult male or female for a small subset of genes. Many CYPs were expressed in the worm intestine. A number of &lt;i&gt;H. Contortus&lt;/i&gt; genes share high identity with &lt;i&gt;Caenorhabditis elegans&lt;/i&gt; CYPs and the similarity in their expression profiles supports their classification as putative orthologues. Notably, &lt;i&gt;H. Contortus&lt;/i&gt; appears to lack the dramatic CYP subfamily expansions seen in &lt;i&gt;C. elegans&lt;/i&gt; and other species, which are typical of CYPs with exogenous roles. However, a small group of &lt;i&gt;H. Contortus&lt;/i&gt; genes cluster with the &lt;i&gt;C. elegans&lt;/i&gt; CYP34 and CYP35 subfamilies and may represent candidate xenobiotic metabolising genes in the parasite

A comparative study of the effects of four treatment regimes on ivermectin efficacy, body weight and pasture contamination in lambs naturally infected with gastrointestinal nematodes in Scotland

AbstractRefugia-based drenching regimes have been widely recommended to slow development of anthelmintic resistance but there are few comparisons between different treatment approaches in the UK. The impact of four ivermectin treatment regimes on drug efficacy, lamb body weight and nematode contamination during a 154 day grazing season were evaluated in a consecutive five year field study. Regimes were whole-flock treatment every 4weeks (NST), targeted selective treatment (TST) based on individual performance, strategic whole-flock treatments at pre-determined times (SPT) or whole-flock treatment when clinical signs were apparent (MT). Mean numbers of ivermectin drenches administered per season were 4.0, 1.8, 2.0 and 1.4 for NST, TST, SPT and MT groups, respectively. The mean anthelmintic efficacy (AE) for each treatment group was based on faecal egg count reduction post-treatment employing a bootstrap sampling based algorithm. Mean AE was 95–98% for all groups in 2006 and mean AE (95% confidence limits) for NST declined to 62% (55%, 68%) in 2010. In comparison, AE for TST, SPT and MT in 2010 were 86% (81%, 92%), 86% (83%, 90%) and 83% (78%, 88%), respectively. Body weight in TST and SPT was similar to NST in all years (p>0.05), however MT lambs were lighter than NST in 2006–2008 (p⩽0.04). Tracer lamb worm burdens was lowest in NST but was not significantly different between other groups. Overall, both the TST and SPT regimes appeared to maintain animal performance and conserve anthelmintic efficacy compared with a neo-suppressive anthelmintic treatment regime

Oral dosing with papaya latex is an effective anthelmintic treatment for sheep infected with Haemonchus contortus

Background The cysteine proteinases in papaya latex have been shown to have potent anthelmintic properties in monogastric hosts such as rodents, pigs and humans, but this has not been demonstrated in ruminants. Methods In two experiments, sheep were infected concurrently with 5,000 infective larvae of Haemonchus contortus and 10,000 infective larvae of Trichostrongylus colubriformis and were then treated with the supernatant from a suspension of papaya latex from day 28 to day 32 post-infection. Faecal egg counts were monitored from a week before treatment until the end of the experiment and worm burdens were assessed on day 35 post-infection. Results We found that the soluble fraction of papaya latex had a potent in vivo effect on the abomasal nematode H. contortus, but not on the small intestinal nematode T. colubriformis. This effect was dose-dependent and at tolerated levels of gavage with papaya latex (117 μmol of active papaya latex supernatant for 4 days), the H. contortus worm burdens were reduced by 98%. Repeated treatment, daily for 4 days, was more effective than a single dose, but efficacy was not enhanced by concurrent treatment with the antacid cimetidine. Conclusions Our results provide support for the idea that cysteine proteinases derived from papaya latex may be developed into novel anthelmintics for the treatment of lumenal stages of gastro-intestinal nematode infections in sheep, particularly those parasitizing the abomasum

A quantitative analysis of attitudes and behaviours concerning sustainable parasite control practices from Scottish sheep farmers

Nematode control in sheep, by strategic use of anthelmintics, is threatened by the emergence of roundworms populations that are resistant to one or more of the currently available drugs. In response to growing concerns of Anthelmintic Resistance (AR) development in UK sheep flocks, the Sustainable Control of Parasites in Sheep (SCOPS) initiative was set up in 2003 in order to promote practical guidelines for producers and advisors. To facilitate the uptake of ‘best practice’ approaches to nematode management, a comprehensive understanding of the various factors influencing sheep farmers’ adoption of the SCOPS principles is required. A telephone survey of 400 Scottish sheep farmers was conducted to elicit attitudes regarding roundworm control, AR and ‘best practice’ recommendations. A quantitative statistical analysis approach using structural equation modelling was chosen to test the relationships between both observed and latent variables relating to general roundworm control beliefs. A model framework was developed to test the influence of socio-psychological factors on the uptake of sustainable (SCOPS) and known unsustainable (AR selective) roundworm control practices. The analysis identified eleven factors with significant influences on the adoption of SCOPS recommended practices and AR selective practices. Two models established a good fit with the observed data with each model explaining 54% and 47% of the variance in SCOPS and AR selective behaviours, respectively. The key influences toward the adoption of best practice parasite management, as well as demonstrating negative influences on employing AR selective practices were farmer’s base line understanding about roundworm control and confirmation about lack of anthelmintic efficacy in a flock. The findings suggest that improving farmers’ acceptance and uptake of diagnostic testing and improving underlying knowledge and awareness about nematode control may influence adoption of best practice behaviour

Transfer hydrogenation of methyl levulinate with methanol to gamma valerolactone over Cu-ZrO₂:A sustainable approach to liquid fuels

Cu-ZrO2 is demonstrated to be a highly effective catalyst for the transfer hydrogenation of methyl levulinate to γ-valerolactone, using methanol as the hydrogen donor. The emergence of several new strategies for synthesising green methanol, underlines its potential as a sustainable hydrogen source for such transformations. Transfer hydrogenation of methyl levulinate over Cu-ZrO2 was determined to proceed through a two-step ‘hydrogen borrowing’ process. The first step involves methanol dehydrogenation (rate limiting) and the second, levulinate reduction. This proof-of-concept study demonstrates that methanol can be used effectively as a hydrogen source for such transformations when a suitable catalyst is employed

Hidden in plain sight - Multiple resistant species within a strongyle community

Ovine parasitic gastroenteritis is a complex disease routinely treated using anthelmintics. Although many different strongyle species may contribute to parasitic gastroenteritis, not all are equally pathogenic: in temperate regions, the primary pathogen is Teladorsagia circumcincta. In this study we investigated benzimidazole and ivermectin resistance on a commercial sheep farm in southeast Scotland. We assessed the impact of species diversity on the diagnosis of resistance using the faecal egg count reduction test and in vitro bioassays, and correlated the results with the frequency of benzimidazole resistance-associated genotypes measured in the T. circumcincta population by pyrosequencing of the β-tubulin isotype-1 gene. Faecal egg count reduction test results showed efficacies of 65% for albendazole and 77% for ivermectin, indicating moderate resistance levels on the farm. However, PCR speciation of the same populations pre- and post-treatment revealed that removal of susceptible species had masked the presence of a highly resistant population of T. circumcincta. Less than 25% of individuals in the pre-treatment populations were T. circumcincta, the remainder consisting of Cooperia curticei, Chabertia ovina, Oesophagostomum venulosum and Trichostrongylus spp. In contrast, post-treatment with albendazole or ivermectin, the majority (88% and 100% respectively) of the populations consisted of T. circumcincta. The egg hatch test for benzimidazole resistance and the larval development test for ivermectin resistance were carried out using eggs obtained from the same populations and the results were broadly consistent with the faecal egg count reduction test. Thirty individual T. circumcincta from each sampling time point were assessed for benzimidazole resistance by pyrosequencing, revealing a high frequency and diversity of resistance-associated mutations, including within the population sampled post-ivermectin treatment. These results highlight the potential diversity of parasite species present on UK farms, and their importance in the diagnosis of anthelmintic resistance. On this particular farm, we demonstrate the presence of a highly dual-resistant population of T. circumcincta, which was strongly selected by treatment with either benzimidazoles or ivermectin, while other potentially less pathogenic species were removed

Increased expression of a microRNA correlates with anthelmintic resistance in parasitic nematodes

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3′ UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3′ UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species

Serum and acute phase protein changes in laying hens, infested with poultry red mite

The poultry red mite (PRM) is one of the most economically important ectoparasites of laying hens globally. This mite can have significant deleterious effects on its fowl host including distress, anemia, reduced egg production, and reduced egg quality. This study was conducted to evaluate the influence of PRM on the serum protein profile in laying hens and its effect on the acute phase proteins (APPs) to assess their potential as biomarkers for mite infestation. Three APPs: alpha-1 acid glycoprotein (AGP), serum amyloid-A (SAA), and ceruloplasmin (CP) were measured in serum samples collected from laying hens at 12 and 17 wk of age, and then for up to 4 mo after a challenge with PRM (starting at 18.5 wk of age). The serum protein profile (SDS-PAGE/nanoflow HPLC electrospray tandem mass spectrometry) and concentration of individual serum proteins (SDS-PAGE-band densitometry) were also compared. Post challenge there was a positive correlation (r = 0.489; P &lt; 0.004) between the levels of SAA and the PRM numbers. The levels of SAA steadily increased after the PRM challenge and were significantly different than the pre-challenge levels at 28, 32, and 36 wk of age (P &lt; 0.01). The PRM numbers also peaked around 31-33 wk of age. The results for AGP and CP in comparison were inconsistent. Proteomics revealed the presence of 2 high molecular weight proteins in the serum between 12 and 17 wk of age. These were identified as Apolipoprotein-B and Vitellogenin-2, and their increase was commensurate with the onset of lay. No other major differences were detected in the protein profiles of blood sera collected pre and post challenge. We conclude that SAA could be used as a useful biomarker to monitor PRM infestation in commercial poultry flocks and that PRM infestation does not disrupt the production of the major proteins in the serum that are associated with egg formation

Determining the influence of socio-psychological factors on the adoption of individual 'best practice' parasite control behaviours from Scottish sheep farmers

Since 2003, the Sustainable Control Of Parasites in Sheep (SCOPS) group have provided the UK sheep farming industry with guidance on ways to mitigate the development and dissemination of anthelmintic resistance (AR). However our empirical understanding of sheep farmers’ influences towards such ‘best practice’ parasite control approaches is limited, and therefore requires further assessment and evaluation to identify the potential factors influencing their implementation. In 2015, a telephone questionnaire was conducted in order to elicit Scottish sheep farmers’ attitudes and behaviours regarding the SCOPS recommended practices, as well as gauging farmers’ general attitudes to gastrointestinal nematodes (GIN; term roundworm used in questionnaire) control. A quantitative structural equation modelling (SEM) approach was employed to determine the influences of socio-psychological factors and the uptake of individual anthelmintic resistance mitigating practices including: the implementation of a quarantine strategy for parasite control and the use of parasite diagnostic testing for monitoring faecal egg counts (FEC) and detecting AR. The proposed models established a good fit with the observed data and explained 61%, 54% and 27% of the variance in the adoption of AR testing, FEC monitoring, and quarantine behaviours respectively. The results presented highlight a number of consistent and distinct factors significantly influencing the implementation of selected SCOPS recommended practices. The negative influences of topography and farmer experience was frequently demonstrated in relation to multiple GIN control practices, as well as the positive influences of social norms, worm control knowledge, AR risk perception and positive attitudes to the services provided by the veterinary profession. Factors that were shown to have the greatest relative effects on individual parasite control practices included: the perceived expectation of others (i.e. Social norms) for implementing a quarantine strategy, farmer’s suspicions to the presence of AR on the holding for instigating AR testing and the confirmation of AR for adopting FEC monitoring. Determining the influences of behaviour-specific factors on farmers’ decision making processes will help to identify and address positive and negative influences concerning implementation of AR mitigating practices, as well as contribute to the development of more evidence based intervention strategies in the future