Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

Preliminary results of the recombinase polymerase amplification technique for the detection of Haemonchus contortus from Hungarian field samples.

Haemonchus contortus is a parasitic nematode of small ruminants responsible for significant economic losses and animal health concerns globally. Detection of gastrointestinal nematode (GIN) infection in veterinary practice typically relies on microscopy-based methods such as the faecal egg count and morphological identification of larval culture. However, mixed co-infections are common and species-specific identification is typically time-consuming and expertise-intensive. Compounded by increasing anthelmintic resistance, there is an urgent need to implement the molecular diagnosis of GIN in the livestock industry, preferably in field settings. Advances in isothermal amplification techniques including recombinase polymerase amplification (RPA) assays could improve this. Yet, constraints in RPA kit availability and amplicon detection systems limit the use of this technology in point of care settings. In this study, we present an early-stage, proof-of-concept demonstration of RPA targeting the internal transcribed spacer (ITS2) region of H. contortus. Having tested against eight closely related nematodes and also against five farm isolates in Eastern Hungary, preliminary results derived from a comparative analysis of 3 primer sets showed the assay detects H. contortus DNA and has a limit of detection of 10-5 ng/µl. We also tested an end-result naked eye detection system using various DNA binding dyes, of which EvaGreen® dye was successful for a qualitative RPA detection that could be adaptable at farm sites.

An evaluation of newborn hearing screening brochures and parental understanding of screening result terminology.

OBJECTIVE: To assess the suitability of newborn hearing screening brochures by evaluating current state-level brochures and pregnant people's understanding of screening result terminology. DESIGN: In Study 1, state-level brochures were evaluated based on readability, design, picture appropriateness, and use of the word "refer." In Study 2, pregnant people completed a questionnaire that queried their understanding of and expected anxiety about three newborn hearing screening outcomes ("refer," "did not pass," and "pass"). STUDY SAMPLE: In Study 1, 59 newborn hearing screening brochures were analysed. In Study 2, 43 pregnant people completed surveys during a prenatal appointment. RESULTS: Most of the brochures were found deficient on at least one element. Thirty percent of brochures used the word "refer" to indicate a hearing screening failure; yet, fewer than half of participants understood its meaning. Ratings of expected anxiety were highest in response to the term "did not pass." CONCLUSIONS: Based on four study criteria of brochure suitability, 88% of available state-level newborn hearing screening brochures should be modified to make them readily understandable by a broad educational demographic. Discretion in use of the term "refer" should be made when indicating screening results, because the term is not readily understood.

Microfluidic-Based Formulation of Essential Oils-Loaded Chitosan Coated PLGA Particles Enhances Their Bioavailability and Nematocidal Activity.

In this study, poly (lactic-co-glycolic) acid (PLGA) particles were synthesized and coated with chitosan. Three essential oil (EO) components (eugenol, linalool, and geraniol) were entrapped inside these PLGA particles by using the continuous flow-focusing microfluidic method and a partially water-miscible solvent mixture (dichloromethane: acetone mixture (1:10)). Encapsulation of EO components in PLGA particles was confirmed by Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction, with encapsulation efficiencies 95.14%, 79.68%, and 71.34% and loading capacities 8.88%, 8.38%, and 5.65% in particles entrapped with eugenol, linalool, and geraniol, respectively. The EO components' dissociation from the loaded particles exhibited an initial burst release in the first 8 h followed by a sustained release phase at significantly slower rates from the coated particles, extending beyond 5 days. The EO components encapsulated in chitosan coated particles up to 5 µg/mL were not cytotoxic to bovine gut cell line (FFKD-1-R) and had no adverse effect on cell growth and membrane integrity compared with free EO components or uncoated particles. Chitosan coated PLGA particles loaded with combined EO components (10 µg/mL) significantly inhibited the motility of the larval stage of Haemonchus contortus and Trichostrongylus axei by 76.9%, and completely inhibited the motility of adult worms (p < 0.05). This nematocidal effect was accompanied by considerable cuticular damage in the treated worms, reflecting a synergistic effect of the combined EO components and an additive effect of chitosan. These results show that encapsulation of EO components, with a potent anthelmintic activity, in chitosan coated PLGA particles improve the bioavailability and efficacy of EO components against ovine gastrointestinal nematodes.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus.

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths.

Integrating applied parasitological and molecular epidemiological methodologies to investigate the capacity of Haemonchus contortus to over-winter on pasture in Scotland.

BACKGROUND: The Barber's Pole worm, Haemonchus contortus is of major concern to sheep producers, particularly in the southern hemisphere. This nematode is also commonly found in many sheep flocks in Northern hemisphere countries but is generally not associated with acute clinical pathology. As with other nematode species, the pattern of disease is changing in the United Kingdom. Changes in management practices, climate, anthelmintic resistance prevalence and parasite adaptation are possible factors thought to be responsible for this. METHODS: In the present study, a combination of traditional applied parasitological and molecular species identification techniques were used to assess the capability of H. contortus infective larvae to over-winter on pasture and infect lambs in early spring. RESULTS: Adult and inhibited H. contortus worms were identified in previously worm-free tracer lambs that had grazed contaminated pasture in late winter/early spring (February/March). CONCLUSION: The study illustrated the benefit of using classical applied parasitology techniques in conjunction with molecular species identification methods to explore the epidemiology of gastro-intestinal nematodes of livestock. This study also demonstrated that larvae were able to survive over-winter, albeit in small numbers, and potentially contaminate pastures earlier than previously considered in northern regions of the UK.

The impact of Baby Friendly Initiative accreditation: An overview of systematic reviews.

Despite its reported benefits, breastfeeding rates are low globally, and support systems such as the Baby Friendly Initiative (BFI) have been established to support healthy infant feeding practices and infant bonding. Increasingly reviews are being undertaken to assess the overall impact of BFI accreditation. A systematic synthesis of current reviews has therefore been carried out to examine the state of literature on the effects of BFI accreditation. A systematic search of CINAHL, MEDLINE, Maternal and Infant Health, Scopus, the Cochrane Library and PROSPERO was undertaken. Study selection, data extraction and critical appraisal of included reviews using the AMSTAR-2 tool were undertaken by two authors, with disagreements resolved through discussion with the third author. Due to heterogeneity, a narrative synthesis of findings was applied. Fourteen reviews met the inclusion criteria. Overall confidence in the results of the review was rated as high for three reviews, low for two reviews and critically low for nine reviews. Most evidence suggests some increase in breastfeeding initiation, exclusivity and duration of breastfeeding, and one main trial suggests decreased gastrointestinal infection and allergic dermatitis in infants. However, overall certainty in the evidence was rated as very low across all outcomes due to concerns over risk of bias within and heterogeneity between the original studies. More contemporary, good-quality randomised controlled trials or well-controlled prospective comparative cohorts are required to better evaluate the impact of full BFI accreditation, with particular attention paid to the context of the research and to long-term maternal and infant health outcomes.

Molecular and phenotypic characterisation of fenbendazole resistance in a field-derived isolate of Ostertagia ostertagi.

The prevalence of anthelmintic resistance in the bovine nematode Cooperia oncophora has been well documented globally but lack of efficacy against the more pathogenic nematode species Ostertagia ostertagi is less common. The sensitivity of an O. ostertagi isolate to the benzimidazole class of anthelmintic was investigated using classical parasitological techniques following apparent clinical failure of controlled release fenbendazole capsule administration in first season grazers at pasture. A controlled efficacy test (CET) was conducted in conjunction with sequencing of the ß-tubulin isotype 1 gene of larvae pre- and post-fenbendazole administration. Twelve helminth-naïve calves were infected experimentally with 20,000 third stage larvae; six received oral fenbendazole (7.5 mg/kg bodyweight) 28 days post infection. Total abomasal nematode burdens were compared between treatment and control groups to determine efficacy. Fenbendazole resistance in O. ostertagi was confirmed with a total treatment failure in reducing worm burden: efficacy of 0%. Sequence analysis of the ß-tubulin isotype-1 gene from forty-five infective larvae from both control and treated groups was performed. The three commonest single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance, namely F167Y, E198A and F200Y, were examined. The predominant resistance-associated SNPs were F200Y (78 % control and 79 % treated groups) and F167Y (remaining genotypes) and emphasises the importance of these SNPs in clinical disease in this isolate. The development of diagnostic molecular tools based on a characterised field-derived isolate of benzimidazole-resistant Ostertagia will enable future prevalence surveys to be undertaken to assess the possible risk posed by resistance in this economically important species.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance.

BACKGROUND: Benzimidazole resistance is associated with isotype-1 ß-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 ß-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Nematocidal Effects of a Coriander Essential Oil and Five Pure Principles on the Infective Larvae of Major Ovine Gastrointestinal Nematodes In Vitro.

The anthelmintic effects of extracted coriander oil and five pure essential oil constituents (geraniol, geranyl acetate, eugenol, methyl iso-eugenol, and linalool) were tested, using larval motility assay, on the third-stage larvae (L3s) of Haemonchus contortus, Trichostrongylus axei, Teladorsagia circumcincta, Trichostrongylus colubriformis, Trichostrongylus vitrinus and Cooperia oncophora. Coriander oil and linalool, a major component of tested coriander oil, showed a strong inhibitory efficacy against all species, except C. oncophora with a half maximal inhibitory concentration (IC50) that ranged from 0.56 to 1.41% for the coriander oil and 0.51 to 1.76% for linalool. The coriander oil and linalool combinations conferred a synergistic anthelmintic effect (combination index [CI] <1) on larval motility comparable to positive control (20 mg/mL levamisole) within 24 h (p < 0.05), reduced IC50 values to 0.11-0.49% and induced a considerable structural damage to L3s. Results of the combined treatment were validated by quantitative fluorometric microplate-based assays using Sytox green, propidium iodide and C12-resazurin, which successfully discriminated live/dead larvae. Only Sytox green staining achieved IC50 values comparable to that of the larval motility assay. The cytotoxicity of the combined coriander oil and linalool on Madin-Darby Canine Kidney cells was evaluated using sulforhodamine-B (SRB) assay and showed no significant cytotoxic effect at concentrations < 1%. These results indicate that testing essential oils and their main components may help to find new potential anthelmintic compounds, while at the same time reducing the reliance on synthetic anthelmintics.

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematodes, resistance in N. battus is likely to be conferred by single nucleotide polymorphisms (SNP) within the ß-tubulin isotype 1 locus at codons 167, 198 and 200. Pyrosequencing and deep amplicon sequencing assays were designed to identify the F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) SNPs. Nematodirus battus populations from 253 independent farms were analysed by pyrosequencing; 174 farm populations were included in deep amplicon sequencing and 170 were analysed using both technologies. F200Y was the most prevalent SNP identified throughout the UK, in 12-27% of the populations tested depending on assay, at a low overall individual frequency of 2.2 ±â€¯0.6% (mean ±â€¯SEM, based on pyrosequencing results). Four out of the five populations with high frequencies (>20%) of the F200Y mutation were located in NW England. The F167Y SNP was identified, for the first time in this species, in four of the populations tested at a low frequency (1.2% ±â€¯0.01), indicating the early emergence of the mutation. E198A or E198L were not identified in any of the isolates. Results obtained were comparable between both techniques for F200Y (Lins' CCC, rc = 0.96) with discrepancies being limited to populations with low frequencies. The recent emergence of resistance in this species will provide a unique opportunity to study the early stages of anthelmintic resistance within a natural setting and track its progress in the future.

Correlation of air-bone gap and size of Enlarged Vestibular Aqueduct in children.

OBJECTIVE: Enlarged vestibular aqueduct (EVA) is an inner ear malformation that represents an important cause of pediatric hearing loss. While certain elements in the history or audiogram may suggest EVA, it is most often diagnosed using computed tomography (CT). The present investigation was conducted to determine if the size of the audiometric air-bone gap (ABG) is correlated with the size of the vestibular aqueduct in the pediatric population using three vestibular aqueduct measurements. These included the fundus, midpoint, and porous widths of the vestibular aqueduct. STUDY DESIGN: This is a retrospective cohort study. SETTING: This study took place at a tertiary care referral center. PATIENTS: Fifty-five children (33 female; 22 male) with a confirmed diagnosis of unilateral or bilateral EVA as determined by prior imaging of the inner ear were included in the study. MAIN OUTCOME MEASURES: Associations of EVA measurements with ABGs at 0.5 and 1 kHz were evaluated using Pearson correlation coefficients. RESULTS: All of the correlation coefficients were positive, indicating that as EVA measurements increased so did the ABG. Only the correlation between fundus width and ABG at 1 kHz was not statistically significant. CONCLUSIONS: ABGs measured during audiometric testing correlate with the size of the EVA and ABGs can be clinical predictors of the severity of the bony abnormality. These data support the third window theory of conductive hearing loss in pediatric EVA.

Genotypic characterisation of monepantel resistance in historical and newly derived field strains of Teladorsagia circumcincta.

Recent reports of monepantel (MPTL) resistance in UK field isolates of Teladorsagia circumcincta has highlighted the need for a better understanding of the mechanism of MPTL-resistance in order to preserve its anthelmintic efficacy in this economically important species. Nine discrete populations of T. circumcincta were genotypically characterised; three MPTL-susceptible isolates, three experimentally selected MPTL-resistant strains and three field derived populations. Full-length Tci-mptl-1 gene sequences were generated and comparisons between the MPTL-susceptible isolates, MPTL-resistant strains and one field isolate, showed that different putative MPTL-resistance conferring mutations were present in different resistant isolates. Truncated forms of the Tci-mptl-1 gene were also observed. The genetic variability of individual larvae, within and between populations, was examined using microsatellite analyses at 10 'neutral' loci (presumed to be unaffected by MPTL). Results confirmed that there was little background genetic variation between the populations, global FST <0.038. Polymorphisms present in exons 7 and 8 of Tci-mptl-1 enabled genotyping of individual larvae. A reduction in the number of genotypes was observed in all MPTL-resistant strains compared to the MPTL-susceptible strains that they were derived from, suggesting there was purifying selection at Tci-mptl-1 as a result of MPTL-treatment. The potential link between benzimidazole (BZ)-resistance and MPTL-resistance was examined by screening individual larvae for the presence of three SNPs associated with BZ-resistance in the ß-tubulin isotype-1 gene. The majority of larvae were BZ-susceptible homozygotes at positions 167 and 198. Increased heterozygosity at position 200 was observed in the MPTL-resistant strains compared to their respective MPTL-susceptible population. There was no decrease in the occurrence of BZ-resistant genotypes in larvae from each population. These differences, in light of the purifying selection at this locus in all MPTL-resistant isolates, suggests that Tci-mptl-1 confers MPTL-resistance in T. circumcincta, as in Haemonchus contortus, but that different mutations in Tci-mptl-1 can confer resistance in different populations.

The dynamics of ovine gastrointestinal nematode infections within ewe and lamb cohorts on three Scottish sheep farms.

Gastrointestinal nematodes (GIN) are a serious concern for sheep producers worldwide. However, there is a paucity of evidence describing the epidemiology of GIN on modern UK sheep farms. The aim of this paper was to understand whether expected seasonal variations of infection are still found in ewes and lambs under varying management strategies in temperate climates. Faecal egg counts (FEC) were conducted on freshly voided samples collected from groups of ewes and lambs every third week for twelve months on three farms in southeast Scotland. The patterns of egg output have been described here in relation to management practices undertaken on the farms. Despite changes in farming practice and climatic conditions, the findings complement historical studies detailing the epidemiology of GIN. Findings include a periparturient rise in ewe FEC on two of the farms, while lambing time treatment appeared to suppress this on the third farm. On the same two farms lamb FEC increased during the summer, reaching a peak in the autumn. The work also highlights how the ad hoc use of anthelmintics does little to impact these patterns.

Development of amplicon sequencing for the analysis of benzimidazole resistance allele frequencies in field populations of gastrointestinal nematodes.

Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 ß-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 ß-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

Microbiome analysis as a platform R&D tool for parasitic nematode disease management.

The relationship between bacterial communities and their host is being extensively investigated for the potential to improve the host's health. Little is known about the interplay between the microbiota of parasites and the health of the infected host. Using nematode co-infection of lambs as a proof-of-concept model, the aim of this study was to characterise the microbiomes of nematodes and that of their host, enabling identification of candidate nematode-specific microbiota member(s) that could be exploited as drug development tools or for targeted therapy. Deep sequencing techniques were used to elucidate the microbiomes of different life stages of two parasitic nematodes of ruminants, Haemonchus contortus and Teladorsagia circumcincta, as well as that of the co-infected ovine hosts, pre- and post infection. Bioinformatic analyses demonstrated significant differences between the composition of the nematode and ovine microbiomes. The two nematode species also differed significantly. The data indicated a shift in the constitution of the larval nematode microbiome after exposure to the ovine microbiome, and in the ovine intestinal microbial community over time as a result of helminth co-infection. Several bacterial species were identified in nematodes that were absent from their surrounding abomasal environment, the most significant of which included Escherichia coli/Shigella. The ability to purposefully infect nematode species with engineered E. coli was demonstrated in vitro, validating the concept of using this bacterium as a nematode-specific drug development tool and/or drug delivery vehicle. To our knowledge, this is the first description of the concept of exploiting a parasite's microbiome for drug development and treatment purposes.

First detection of ivermectin resistance in oesophagostomum dentatum in pigs.

In 2016 suspected reduced ivermectin (IVM) efficacy in Oesophagostomum species in pigs was reported in England. Following this initial report, APHA raised awareness amongst private pig veterinary practitioners of the need to monitor the efficacy of the worm control on pig units. In 2017 another veterinary practitioner highlighted a potential in-field lack of IVM efficacy in treating Oesophagostomum species in sows on another breeder-finisher unit. In this trial, the efficacy of IVM against Oesophagostomum species worms has been investigated to determine whether suspected reduced efficacy (52% reduction in mean faecal egg count 14 days post ivermectin administration) on a mixed indoor and outdoor breeder-finisher pig farm in England reflected true IVM resistance under controlled experimental conditions. On days 0 and 40 of the trial, twenty helminth-naive pigs were artificially infected per os with 5000 Oesophagostomum L3 obtained from the farm under investigation. The pigs were allocated to treatment or control groups (n = 10 per group). Treatment group pigs received IVM (0.3 mg kg body weight) by sub-cutaneous injection as per manufacturer's instructions on day 44. Control group animals were left untreated. Faecal worm egg counts were monitored throughout the trial from day 15 post infection to determine time to patency. On day 50 all pigs were euthanased to assess the worm burdens. Resistance to IVM was confirmed in Oesophagostomum dentatum based on the results of a faecal egg count reduction test (FECRT) and a controlled efficacy test (CET). Efficacy based on mean reduction in faecal egg count of IVM-treated pigs compared to untreated control pigs was 86%. Mean reduction in IVM-treated pig worm burdens was 5% against an adult worm population and 94% against an L3/L4 population. The apparent discrepancy between FECRT and CET efficacy results appears to be due to egg development and/or oviposition suppression in IVM-treated female worms. The detection of IVM resistance in Oesophagostomum species worms for the first time in UK pigs is particularly important considering the global situation where resistance to pyrantel, levamisole and benzimidazole anthelmintics in Oesophagostomum species in pigs have already been reported. The results also provide an opportunity to discuss the wider issue of anthelmintic usage and efficacy on pig farms and highlight the need for wider surveillance for the occurrence of anthelmintic resistance in pigs.

Mating barriers between genetically divergent strains of the parasitic nematode Haemonchus contortus suggest incipient speciation.

Haemonchus contortus, in common with many nematode species, has extremely high levels of genetic variation within and between field populations derived from distant geographical locations. MHco10(CAVR), MHco3(ISE) and MHco4(WRS) are genetically divergent H. contortus strains, originally derived from Australia, Kenya and South Africa, respectively, that have been maintained by numerous rounds of in vivo experimental infection of sheep. In order to explore potential pre-zygotic competition or post-zygotic incompatibility between the strains, we have investigated the ability of MHco10(CAVR) to interbreed with either MHco3(ISE) or MHco4(WRS) during dual strain co-infections. Sheep were experimentally co-infected with 4000 infective larvae (L3) per os of the MHco10(CAVR) strain and an equal number of either the MHco3(ISE) or the MHco4(WRS) strain L3. The adult worm establishement rates and the proportions of F1 progeny resulting from intra- and inter-strain mating events were determined by admixture analysis of microsatellite multi-locus genotypes. Although there was no difference in adult worm establishment rates, the proportions of F1 progeny of both the MHco10(CAVR) × MHco3(ISE) and MHco10(CAVR) × MHco4(WRS) dual strain co-infections departed from Mendelian expectations. The proportions of inter-strain hybrid F1 progeny were lower than the expected 50%, suggesting either pre-zygotic competition or post-zygotic incompatibility between the co-infecting strains. To investigate this further, both eggs and hatched L1 of broods from single adult female worms recovered from each dual co-infection were genotyped. Unhatched eggs from the broods revealed no inter-strain hybrid genotype deficit, suggesting there is no pre-zygotic competition between the strains. In contrast, there was a deficit in L1 inter-strain hybrid genotypes in the broods derived from MHco3(ISE) or MHco4(WRS) maternal parents, but not from MHco10(CAVR) maternal parents. This suggests that hybrid progeny of MHco10(CAVR) paternal parents have reduced post-zygotic development and/or viability consistent with incipient speciation of the MHco10(CAVR) strain. The presence of mating barriers between allopatric H. contortus strains has important implications for parasite ecology, including the ability of newly introduced anthelmintic-resistant parasite populations to compete and interbreed with populations already established in a region.