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A large-scale Fecal Egg Count Reduction Test (FECRT) was integrated with ITS-2 rDNA nemabiome metabarcoding to investigate anthelmintic resistance in gastrointestinal nematode (GIN) parasites in western Canadian beef cattle. The study was designed to detect anthelmintic resistance with the low fecal egg counts that typically occur in cattle in northern temperate regions. Two hundred and thirty-four auction market-derived, fall-weaned steer calves coming off pasture were randomized into three groups in feedlot pens: an untreated control group, an injectable ivermectin treatment group, and an injectable ivermectin/oral fenbendazole combination treatment group. Each group was divided into six replicate pens with 13 calves per pen. Individual fecal samples were taken pre-treatment, day 14 post-treatment, and at monthly intervals for six months for strongyle egg counting and metabarcoding. Ivermectin treatment resulted in an 82.4% mean strongyle-type fecal egg count reduction (95% CI 67.8-90.4) at 14 days post-treatment, while the combination treatment was 100% effective, confirming the existence of ivermectin-resistant GIN. Nemabiome metabarcoding of third-stage larvae from coprocultures revealed an increase in the relative abundance of Cooperia oncophora, Cooperia punctata, and Haemonchus placei at 14 days post-ivermectin treatment indicating ivermectin resistance in adult worms. In contrast, Ostertagia ostertagi third-stage larvae were almost completely absent from day 14 coprocultures, indicating that adult worms of this species were not ivermectin resistant. However, there was a recrudescence of O. ostertagi third stage larvae in coprocultures at three to six months post-ivermectin treatment, which indicated ivermectin resistance in hypobiotic larvae. The calves were recruited from the auction market and, therefore, derived from multiple sources in western Canada, suggesting that ivermectin-resistant parasites, including hypobiotic O. ostertagi larvae, are likely widespread in western Canadian beef herds. This work demonstrates the value of integrating ITS-2 rDNA metabarcoding with the FECRT to enhance anthelmintic resistance detection and provide GIN species- and stage-specific information., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2023 Merck Sharp & Dohme LLC., a subsidiary Merck & Co., Inc., The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Gastrointestinal nematodes (GIN) are amongst the most important pathogens of grazing ruminants worldwide, resulting in negative impacts on cattle health and production. The dynamics of infection are driven in large part by the influence of climate and weather on free-living stages on pasture, and computer models have been developed to predict infective larval abundance and guide management strategies. Significant uncertainties around key model parameters limits effective application of these models to GIN in cattle, however, and these parameters are difficult to estimate in natural populations of mixed GIN species. In this paper, recent advances in molecular biology, specifically ITS-2 rDNA 'nemabiome' metabarcoding, are synthesised with a modern population dynamic model, GLOWORM-FL, to overcome this limitation. Experiments under controlled conditions were used to estimate rainfall constraints on migration of infective L3 larvae out of faeces, and their survival in faeces and soil across a temperature gradient, with nemabiome metabarcoding data permitting species-specific estimates for Ostertagia ostertagi and Cooperia oncophora in mixed natural populations. Results showed that L3 of both species survived well in faeces and soil between 0 and 30 °C, and required at least 5 mm of rainfall daily to migrate out of faeces, with the proportion migrating increasing with the amount of rainfall. These estimates were applied within the model using weather and grazing data and use to predict patterns of larval availability on pasture on three commercial beef farms in western Canada. The model performed well overall in predicting the observed seasonal patterns but some discrepancies were evident which should guide further iterative improvements in model development and field methods. The model was also applied to illustrate its use in exploring differences in predicted seasonal transmission patterns in different regions. Such predictive modelling can help inform evidence-based parasite control strategies which are increasingly needed due climate change and drug resistance. The work presented here also illustrates the added value of combining molecular biology and population dynamics to advance predictive understanding of parasite infections., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: John Gilleard reports financial support was provided by Beef Cattle Research Council. Eric Morgan reports financial support was provided by UK Research and Innovation. Hannah Rose Vineer reports financial support was provided by UK Research and Innovation. John Gilleard reports a relationship with Beef Cattle Research Council that includes: funding grants., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Anthelmintic resistance (AR) is an ever increasing problem for the sheep industry. Several studies worldwide have investigated reversing the trend of increasing AR and documented evidence for reversion toward susceptibility has been found. The hypothesis that resistance mutations compromise parasite fitness was drawn from this evidence. The aim of this study was to assess whether there were measurable differences in the fitness of Teladorsagia circumcincta isolates depending on their AR status. Four isolates were selected for the trial based on their known resistance status; D and M were multi-drug resistant, and T and W were susceptible to the benzimidazole, levamisole, and macrocyclic lactone anthelmintic classes. A secondary aim was to develop a series of in vitro bioassays for assessing fitness characteristics of parasites. The in vitro assays included; the cold stress test measured the number of third stage larvae (L3) developing from eggs stored at 4 °C for different lengths of time. Larval aging measured the locomotory activity of L3 after storage at 30 °C for different lengths of time. The exsheathment assay measured the exsheathment percentage of L3. Larval Length used length as a proxy for fecundity. The egg hatch assay evaluated egg hatch rate in water at room temperature. All isolates exhibited a decrease in the number of L3 recovered after storage of eggs at 4 °C (p < 0.001). Storage of L3 at 30 °C significantly influenced the ability of L3 to migrate through a 20 µm sieve (p < 0.001), however, there were no differences between isolates (p > 0.05). Exsheathment rate was higher for isolate D in comparison to isolates M and W, and for isolate T compared to isolate W. Isolate W was significantly longer than all other isolates (p < 0.05), whilst isolate M was significantly longer than isolate D (p < 0.05). No significant differences were found between isolates in egg hatch (p > 0.05). Overall, the results do not support differences in fitness associated with anthelmintic resistance status, even though differences were seen between the isolates for some assays. This suggests there is considerable variation in fitness parameters between isolates, making it difficult to determine whether resistance genotypes come with lower fitness., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 β-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 β-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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Teladorsagia circumcincta P-glycoprotein-9 (Tci-pgp-9) gene has previously been implicated in multiple-anthelmintic resistance in this parasite. Here we further characterise genetic diversity in Tci-pgp-9 and its possible role in ivermectin (IVM) and multi-drug resistance using two UK field isolates of T. circumcincta, one susceptible to anthelmintics (MTci2) and the other resistant to most available anthelmintics including IVM (MTci5). A comparison of full-length Tci-pgp-9 cDNA transcripts from the MTci2 and MTci5 isolates (∼3.8 kb in both cases) indicated that they shared 95.6% and 99.5% identity at the nucleotide and amino acid levels, respectively. Nine non-synonymous SNPs were found in the MTci5 sequences relative to their MTci2 counterparts. Twelve genomic sequence variants of the first internucleotide binding domain of Tci-pgp-9 were identified and up to 10 of these were present in some individual worms, strongly supporting previous evidence that amplification of this gene has occurred in T. circumcincta. On average, fewer distinct sequence variants of Tci-pgp-9 were present in individual worms of the MTci5 isolate than in those of the MTci2 isolate. A further reduction in the number of sequence variants was observed in individuals derived from an IVM-treated sub-population of MTci5. These findings suggest that Tci-pgp-9 was under purifying selection in the face of IVM treatment in T. circumcincta, with some sequence variants being selected against., (Copyright © 2018. Published by Elsevier Ltd.)

The efficacy of ivermectin (IVM) against gastrointestinal nematodes in Danish cattle was assessed by faecal egg count reduction test (FECRT). Six cattle farms with history of clinical parasitism and avermectin use were included. On the day of treatment (Day 0), 20 naturally infected calves per farm (total n = 120) were stratified by initial faecal egg counts (FEC) and randomly allocated to a treatment group dosed with 0.2 mg IVM kg -1 body weight s.c. (IVM; n = 10) or an untreated control group (CTL; n = 10). Individual FEC were obtained at Day 0 and Day 14 post-treatment and pooled faeces by group were cultured to isolate L3 for detection of Ostertagia ostertagi and Cooperia oncophora by qPCR. Treatment efficacies were analysed using the recommended WAAVP method and two open-source statistical procedures based on Bayesian modelling: 'eggCounts' and 'Bayescount'. A simulation study evaluated the performance of the different procedures to correctly identify FEC reduction percentages of simulated bovine FEC data representing the observed real data. In the FECRT, reduced IVM efficacy was detected in three farms by all procedures using data from treated animals only, and in one farm according to the procedures including data from treated and untreated cattle. Post-treatment, O. ostertagi and C. oncophora L3 were detected by qPCR in faeces of treated animals from one and three herds with declared reduced IVM efficacy, respectively. Based on the simulation study, all methods showed a reduced performance when FEC aggregation increased post-treatment and suggested that a treatment group of 10 animals is insufficient for the FECRT in cattle. This is the first report of reduced anthelmintic efficacy in Danish cattle and warrants the implementation of larger surveys. Advantages and caveats regarding the use of Bayesian modelling and the relevance of including untreated cattle in the FECRT are discussed., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Resistance to benzimidazoles (BZs) is widespread in sheep nematodes and increasing in those of cattle. Several reasons including the predominant use of pour-on anthelmintics and lack of scales in field conditions lead to under-dosing of cattle and therefore to increased selection pressure. In an field experiment the frequency of BZ-resistance associated allele (TAC) in codon 200 in the β-tubulin isotype 1 gene of Ostertagia ostertagi was monitored over one grazing season (approximately 30 weeks). Group 1, consisting of four calves, was experimentally infected with a pure O. ostertagi population displaying ∼50% of the TAC allele. The subsequently following groups of calves (four groups of two calves each) acquired natural infections by grazing contaminated pastures. Each group was treated with increasing percentages of sub-therapeutic dosages of albendazole (35-65%). Larvae obtained from faecal cultures pre and post treatment were subjected to species/genus-specific PCR as well as pyrosequencing to determine allele frequencies. PCR revealed the presence of Ostertagia, Trichostrongylus, Haemonchus and Cooperia in pre-treatment samples and predominantly Ostertagia as well as some Trichostrongylus in post treatment samples. Faecal egg count reduction was always less than 90% 7-10 days post treatment. In naturally infected calves TAC allele frequencies were significantly increased (p<0.05) after treatment and they also rapidly increased during the grazing season (pre: 15-63%; post: 55-89%). The more than 4-fold increase in resistant genotypes before treatment indicates how fast selection for BZ resistance can occur when sub-therapeutic dosages are combined with a high treatment frequency, even under moderated climatic conditions and in the presence of a refugium., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Anthelmintic resistance of gastrointestinal nematodes is considered as one of the main limiting factors causing significant economic losses to the small ruminant industry. The anthelmintic properties of some plants are among the suggested alternative solutions to control these parasitic worms. The present study investigated the anthelmintic activity of neem (Azadirachta indica) and cassava (Manihot esculenta) leaf extracts against the susceptible and resistant strains of one of the most important nematodes in small ruminants, Teladorsagia (Ostertagia) circumcincta. Three different in vitro tests: egg hatch test, larval development assay, and larval paralysis assay were used to determine the efficiency of neem and cassava extracts on three pre-parasitic stages of T. circumcincta. The LC(50) was determined for the most potent extract in each plant as well as the phytochemical tests, total tannin quantification and cytotoxicity on peripheral blood mononuclear cells of goats. The results revealed a high anthelmintic activity of neem methanol extract (NME) and cassava methanol extract (CME) on both strains of T. circumcincta without significant differences between the strains. The first stage larvae were more sensitive with the lowest LC(50) at 7.15 mg/ml and 10.72 mg/ml for NME and CME, respectively, compared with 44.20mg/ml and 56.68 mg/ml on eggs and 24.91 mg/ml and 71.96 mg/ml on infective stage larvae., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ostertagia ostertagi is a gastrointestinal parasitic nematode that affects cattle and leads to a loss of production. In this study, we present the first large-scale genomic survey of O. ostertagi by the analysis of expressed transcripts from three stages of the parasite: third-stage larvae, fourth-stage larvae and adult worms. Using an in silico approach, 2284 genes were identified from over 7000 expressed sequence tags and abundant transcripts were analyzed and characterized by their functional profile. Of the 2284 genes, 66% had similarity to other known or predicted genes while the rest were novel and potentially represent genes specific to the species and/or stages. Furthermore, a subset of the novel proteins were structurally annotated and assigned putative function based on orthologs in Caenorhabditis elegans and corresponding RNA interference phenotypes. Hence, over 70% of the genes were annotated using protein sequences, domains and pathway databases. Differentially expressed transcripts from the two larval stages and their functional profiles were also studied leading to a more detailed understanding of the parasite's life-cycle. The identified transcripts are a valuable resource for genomic studies of O. ostertagi and can facilitate the design of control strategies and vaccine programs.

Recent reports of suspected ivermectin (IVM) resistance in Ostertagia ostertagi have highlighted the need for research into the mechanisms of IVM resistance. However, there are no reports of resistant field isolates of O. ostertagi, which have been characterized for molecular research. Therefore, an anthelmintic susceptible O. ostertagi population was selected for IVM resistance by repeatedly exposing the population to subtherapeutic and therapeutic levels of IVM over 10 generations. In each selection round, a group of calves was infected with the progeny of the previous IVM-selected O. ostertagi population. In the last selection round a therapeutic IVM dose (0.2 mg/kg BW) only reduced the faecal egg counts by 57% and 65% on days 7 and 14 after treatment, respectively. In contrast, the therapeutic IVM dose was 100% effective at eliminating the parental IVM-susceptible isolate.

A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.

A region within the first internal transcribed spacer (ITS-1) of the ribosomal DNA repeat of Ostertagia ostertagi has been identified that is 408 base pairs (bp) in length and is comprised of a 2 x 204 bp repeat. Universal polymerase chain reaction (PCR) primers which span this region, as well as a portion of the 5.8S rDNA, generate a 1011 bp fragment using genomic DNA from O. ostertagi. However, these same primers generate only a 600 bp (approximate) fragment using DNA from Haemonchus contortus, Cooperia oncophora and Oesophagostomum radiatum, as well as other species within the genus Ostertagia. When DNA samples derived from adult parasites of the different genera were mixed and simultaneously amplified, the O. ostertagi component could be identified within the mixed DNA populations. Furthermore, a correlation was observed between relative fluorescence intensities of the 1011 bp and the 600 bp PCR fragments and the percentage of O. ostertagi DNA within a mixture of parasite DNAs. A similar high correlation was obtained between the percentage of O. ostertagi DNA and percent O. ostertagi eggs in feces containing eggs of other nematode genera. This resulted in the generation of a protocol that can determine the percentage of O. ostertagi eggs within a mixed population of gastrointestinal nematode eggs. Results indicate a detection equivalent to 0.05 eggs.

Most genetic work to distinguish strains of parasitic helminths focuses on searching for genetic markers to correlate with phenotypes of interest, but in this study genetic diversity among individual Ostertagia ostertagi adults is partitioned into components within and between populations. Restriction fragment polymorphism data on mitochondrial DNA from ten individual worms from each of five different parasite populations are analyzed. Three of these populations are characterized by arrested larval development (hypobiosis) over the summer months, and the other two by hypobiosis over the winter months. Sequence divergence is scored by the presence or absence of 37 different restriction sites. Although the populations are genetically differentiated with respect to the timing of hypobiosis, greater than 98% of the total mitochondrial DNA sequence diversity is partitioned within a single population, and the geographic distribution of individual mitochondrial DNA haplotypes suggests high gene flow among populations. Further, estimates of within-population mitochondrial DNA diversity are five to ten times greater in O. ostertagi than typical estimates reported for species in other taxa.