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Faecal samples were obtained from 77 first season grazers from 20 Norwegian dairy herds in autumn 2020 for analysis of Cooperia oncophora and Ostertagia ostertagi infection. Strongylid eggs per gram of faeces (EPG) were determined for each sample and the samples underwent larval culture. DNA was extracted from the faeces at different stages of the culture preparation: from faecal slurry (FS), direct extraction before culture (DBC), and direct extraction after culture (DAC). Extracted DNA was subject to molecular assay for both C. oncophora and O. ostertagi using both a published ddPCR assay and a modified qPCR duplex assay targeting the ITS-2 gene. For C. oncophora, DBC samples contained significantly less ITS-2 copies than DAC and FS samples (p ≤ 0.0001 for both) for qPCR, but not between DAC and FS samples (p = 0.339). Droplet digital PCR with DBC samples yielded significantly fewer ITS-2 copies than DAC and FS samples (p ≤ 0.0001). For O. ostertagi, the ITS-2 copies in DBC samples differed significantly from levels in DAC and FS samples on the qPCR platform (p=0.002 and 0.044, respectively) as was the case with ddPCR (p ≤ 0.0001 and 0.0137). Overall, across the various processing techniques, C. oncophora results obtained on both ddPCR and qPCR platforms correlated with EPG better than was found for equivalent results for O. ostertagi and results are strongly indicative of poor correlation when EPG counts are low. Results from this study suggest that DAC faecal processing was of limited practical use., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes., (© 2023. The Author(s).)

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.)

Background: Gastrointestinal nematode (GIN) epidemiology is changing in many regions of the world due to factors such as global warming and emerging anthelmintic resistance. However, the dynamics of these changes in northern continental climate zones are poorly understood due to a lack of empirical data., Methods: We studied the accumulation on pasture of free-living infective third-stage larvae (L3) of different GIN species from fecal pats deposited by naturally infected grazing cattle. The field study was conducted on three organic farms in Alberta, western Canada. Grass samples adjacent to 24 fecal pats were collected from each of three different pastures on each farm. Internal transcribed spacer-2 nemabiome metabarcoding was used to determine the GIN species composition of the harvested larvae. The rotational grazing patterns of the cattle ensured that each pasture was contaminated only once by fecal pat deposition. This design allowed us to monitor the accumulation of L3 of specific GIN species on pastures under natural climatic conditions without the confounding effects of pasture recontamination or anthelmintic treatments., Results: In seven out of the nine pastures, grass L3 counts peaked approximately 9 weeks after fecal deposition and then gradually declined. However, a relatively large number of L3 remained in the fecal pats at the end of the grazing season. Nemabiome metabarcoding revealed that Cooperia oncophora and Ostertagia ostertagi were the two most abundant species on all of the pastures and that the dynamics of larval accumulation on grass were similar for both species. Daily precipitation and temperature across the whole sampling period were similar for most of the pastures, and multiple linear regression showed that accumulated rainfall 1 week prior to sample collection had a significant impact on the pasture L3 population, but accumulated rainfall 3 weeks prior to sample collection did not., Conclusions: The results suggest that the pasture L3 population was altered by short-term microclimatic conditions conducive for horizontal migration onto grass. Overall, the results show the importance of the fecal pat as a refuge and reservoir for L3 of cattle GIN on western Canadian pastures, and provide an evidence base for the risk assessment of rotational grazing management in the region., (© 2021. The Author(s).)

Background: Growing evidence points towards a role of gastrointestinal (GI) helminth parasites of ruminants in modifying the composition of the host gut flora, with likely repercussions on the pathophysiology of worm infection and disease, and on animal growth and productivity. However, a thorough understanding of the mechanisms governing helminth-microbiota interactions and of their impact on host health and welfare relies on reproducibility and replicability of findings. To this aim, in this study, we analysed quantitative and qualitative fluctuations in the faecal microbiota composition of lambs vaccinated against, and experimentally infected with, the parasitic GI nematode Teladorsagia circumcincta over the course of two separate trials performed over two consecutive years., Methods: Two trials were conducted under similar experimental conditions in 2017 and 2018, respectively. In each trial, lambs were randomly assigned to one of the following experimental groups: (i) vaccinated/infected, (ii) unvaccinated/infected and (iii) unvaccinated/uninfected. Faecal samples collected from individual animals were subjected to DNA extraction followed by high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene and bioinformatics and biostatistical analyses of sequence data., Results: Substantial differences in the populations of bacteria affected by immunisation against and infection by T. circumcincta were detected when comparing data from the two trials. Nevertheless, the abundance of Prevotella spp. was significantly linked to helminth infection in both trials., Conclusions: Despite the largely conflicting findings between the two trials, our data revealed that selected gut microbial populations are consistently affected by T. circumcincta infection and/or vaccination. Nevertheless, our study calls for caution when interpreting data generated from in vivo helminth-microbiome interaction studies that may be influenced by several intrinsic and extrinsic host-, parasite- and environment-related factors.

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.)

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 β-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.

Standard diagnostic methods currently in use for the identification of helminth infections in ruminants are based on the morphological analysis of immature and adult stages of parasites. This paper describes a method for the semiquantitative identification of nematodes, mainly Trichostrongyloidea, at species-level resolution. The method is based on amplification and fragment analysis followed by minisequencing of the ITS-2 region (internal transcribed spacer 2) of the ribosomal DNA of parasite eggs or larvae. This method allows for the identification of seven genera (Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus) and 12 species (Chabertia ovina, Cooperia curticei, Cooperia punctata, Cooperia oncophora/Cooperia surnabada, Haemonchus contortus, Haemonchus placei, Haemonchus longistipes, Oesophagostomum asperum, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei, and Trichostrongylus colubriformis) of infectious nematodes of domestic ruminants. The concordance between the morphological and molecular analyses in the detection of genera ranged from 0.84 to 0.99, suggesting the proposed detection method is specific, semiquantitative, less laborious, and highly cost-efficient.

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform-based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites' ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.

The complete mitochondrial (mt) genome of Ostertagia trifurcata , a parasitic nematode of small ruminants, has been sequenced and its phylogenetic relationship with selected members from the superfamily Trichostrongyloidea was investigated on the basis of deduced datasets of mt amino acid sequences. The entire mt genome of Ostertagia trifurcata is circular and 14,151 bp in length. It consists of a total of 36 genes comprising 12 genes coding for proteins (PCGs), 2 genes for ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes and 2 non-coding regions, since all genes are transcribed in the same direction. The phylogenetic analysis based on the concatenated datasets of predicted amino acid sequences of the 12 protein coding genes supported monophylies of the Haemonchidae, Dictyocaulidae and Molineidae families, but rejected monophylies of the Trichostrongylidae family. The complete characterization and provision of the mtDNA sequence of Ostertagia trifurcata provides novel genetic markers for molecular epidemiological investigations, systematics, diagnostics and population genetics of Ostertagia trifurcata and its correspondents.

Background: Gastrointestinal nematodes (GINs) frequently infect South American camelids (alpacas and llamas) and cause economic losses due to reduced production of fiber, meat and/or leather. Our knowledge about the epidemiology and diagnosis of GINs in llamas and alpacas is limited, and reliable keys for the identification of the third-stage larvae (L3s) of some common nematodes (such as Camelostrogylus mentulatus) that infect alpacas and llamas remain undescribed. In this study, we modified two existing semi-quantitative multiplexed-tandem (MT)-PCR assays, originally developed for the GINs of sheep and cattle, to reliably detect and differentiate the common genera/species of GINs in the faeces of alpacas., Results: Following the establishment of the MT-PCR assay using positive and negative control samples, alpaca faecal samples were tested to validate the assay to detect and differentiate nematode genera/species, including C. mentulatus, Cooperia spp., Haemonchus spp., Oesophagostomum spp., Ostertagia ostertagi, Teladorsagia circumcincta and Trichostrongylus spp. Sequencing of the MT-PCR products demonstrated specific (100%) amplification of the target nematode genera/species. Additionally, a comparison of results of the MT-PCR assay and the morphological identification of adult worms collected from the same 35 alpacas revealed that there was a good agreement (37-94%) between the two methods. However, some discrepancies were observed between the results of the MT-PCR assay and the morphological identification of adult worms., Conclusions: The MT-PCR platform is an accurate, sensitive and rapid method for the diagnosis of GINs in alpacas, and it can be used as a substitute to larval culture to identify common nematodes in the faeces of alpacas and llamas.

Background: Many trichostrongylid nematode species are reported to infect bison, some of which are major causes of disase and production loss in North American bison herds. However, there is little information on the species distribution and relative abundance of these parasites in either commercial or conservation herds. This is largely because trichostrongylid nematode species cannot be distinguished by visual microscopic examination of eggs present in feces. Consequently, we have applied ITS2 rDNA nemabiome metabarcoding to describe the trichostrongyle parasite species diversity in 58 bison production groups derived from 38 commercial North American plains bison (Bison bison bison) herds from across western Canada, and two bison conservation herds located in Elk Island National Park (EINP) [plains bison and wood bison (Bison bison athabascae)] and one in Grasslands National Park (GNP) (plains bison)., Results: We report much higher infection intensities and parasite species diversity in commercial bison herds than previously reported in beef cattle herds grazing similar latitudes. Predominant trichostrongyle parasite species in western Canadian commercial bison herds are those commonly associated with Canadian cattle, with Ostertagia ostertagi being the most abundant followed by Cooperia oncophora. Combined with high fecal egg counts in many herds, this is consistent with significant clinical and production-limiting gastrointestinal parasitism in western Canadian bison herds. However, Haemonchus placei was the most abundant species in five of the production groups. This is both surprising and important, as this highly pathogenic blood-feeding parasite has not been reported at such abundance, in any livestock species, at such northerly latitudes. The presence of Trichostrongylus axei as the most abundant parasite in four herds is also unusual, relative to cattle. There were striking differences in parasite communities between the EINP and commercial bison herds. Most notably, Orloffia bisonis was the predominant species in the wood bison herd despite being found at only low levels in all other herds surveyed., Conclusions: This study represents the most comprehensive description of parasite communities in North American bison to date and illustrates the power of deep amplicon sequencing as a tool to study species diversity in gastrointestinal nematode communities.

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 history of European bison Bison bonasus Linnaeus, 1758 has been stormy since its extinction in the wild after the First World War. Due to the fact that the species was restored from just 12 founders, further expansion has suffered from low genetic variability, rendering the bison vulnerable to various pathogens due to inbreeding depression. Parasites are recognised as a key biological threat to bison population. Thus, parasitological examination including monitoring of the level of anthelmintic resistance in a herd should be a routine procedure involved in management and protection of European bison. This study was conducted in a group of 27 bison kept in a European bison breeding centre in Sweden. In April 2015, a faecal egg count reduction test (FECRT) was performed in animals with ≥ 100 gastrointestinal nematode (GIN) eggs per gram faeces, to determine effectiveness of fenbendazole (FBZ) treatment. Additionally, the third stage larvae were cultured for molecular examination by a conventional PCR as well as by real-time quantitative PCR (q-PCR) for detection of the blood-sucking nematode Haemonchus contortus. Faecal sampling was conducted 1 day before and 8 days after deworming each animal. Anthelmintic treatment turned to be entirely efficient toward intestinal nematodes of genera Nematodirus and Trichuris, whereas shedding of strongylid eggs from the subfamily Ostertagiinae was reduced from 81 to 30%. Polymerase chain reaction (PCR) on cultured third-stage larvae (L3) before treatment was positive for H. contortus, Ostertagia ostertagi and Cooperia oncophora, whereas post-treatment examination revealed exclusively the DNA of H. contortus. Thus, only H. contortus was involved in post-treatment faecal egg count (FEC). FECRT showed that the reduction in strongylid FEC to FBZ in the examined bison herd was 87% (95%-confidence intervals [95% CI] = 76-93), suggesting reduced efficacy of FBZ to strongylid GIN including mainly H. contortus.

Background: Benzimidazole (BZ) resistance in gastrointestinal nematodes is associated with a single nucleotide polymorphism (SNP) at codons 167, 198 and 200 in the isotype 1 of beta-tubulin gene although in some species these SNPs have also been associated with resistance to macrocyclic lactones. In the present study we compared the levels of resistance in Teladorsagia circumcincta and Trichostrongylus colubriformis by means of the faecal egg reduction test (FECRT) and the percentage of resistant alleles obtained after pyrosequencing. The study was conducted in 10 naturally infected sheep flocks. Each flock was divided into three groups: i) group treated with albendazole (ABZ); ii) group treated with ivermectin (IVM); iii) untreated group. The number of eggs excreted per gram of faeces was estimated at day 0 and 14 post-treatment., Results: Resistance to ABZ was observed in 12.5% (1/8) of the flocks and to IVM in 44.4% (4/9) of them. One flock was resistant to both drugs according to FECRT. Coprocultures were performed at the same dates to collect L3 for DNA extraction from pooled larvae and to determine the resistant allele frequencies by pyrosequencing analysis. In T. circumcincta, SNPs were not found at any of the three codons before treatment; after the administration of ABZ, SNPs were present only in two different flocks, one of them with a frequency of 23.8% at SNP 167, and the other 13.2% % at SNP 198. In relation to T. colubriformis, we found the SNP200 before treatment in 33.3% (3/9) of the flocks with values between 48.5 and 87.8%. After treatment with ABZ and IVM, the prevalence of this SNP increased to 75 and 100% of the flocks, with a mean frequency of 95.1% and 82.6%, respectively., Conclusion: The frequencies observed for SNP200 in T. colubriformis indicate that the presence of resistance is more common than revealed by the FECRT.

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.)

Macrophage migration inhibitory factor (MIF) of Ostertagia ostertagi, an abomasal parasite of cattle, was characterised in the present study. Phylogenetic analysis identified at least three O. ostertagi MIFs (Oos-MIFs), each encoded by a distinct transcript: Oos-MIF-1.1, Oos-MIF-1.2 and Oos-MIF-2. Oos-MIF-2 is only distantly related to Oos-MIF-1s, but has higher sequence similarity with the Caenorhabditis elegans MIF2. Oos-MIF-1.1 and Oos-MIF-1.2 are similar (93%) and thus collectively referred to as Oos-MIF-1 when characterised with immunoassays. Recombinant Oos-MIF-1.1 (rOos-MIF-1.1) is catalytically active as a tautomerase. A mutation (rOos-MIF-1.1P1G) or duplication of Pro1 residue (rOos-MIF-1.1P1+P) resulted in reduced oligomerisation and loss of tautomerase activity. The tautomerase activity of rOos-MIF-1.1 was only partially inhibited by ISO-1 but was abrogated by a rOos-MIF-1.1-specific antibody. Oos-MIF-1 was detected in all developmental stages of O. ostertagi, with higher levels in the adult stage; it was also detected in adult worm excretory/secretory product. Oos-MIF-1 was localised to the hypodermis/muscle, reproductive tract and intestine, but not to the cuticle. rOos-MIF-1.1, but not rOos-MIF-1.1P1G, was able to specifically bind to human CD74, a MIF cell surface receptor, with an affinity comparable with human MIF. Immunostaining indicated that macrophages were able to internalise rOos-MIF-1.1, further supporting receptor-mediated transportation. Herein we also show that rOos-MIF-1.1 inhibited migration of bovine macrophages and restored glucocorticoid-suppressed, lipopolysaccharide-induced TNF-α and IL-8 in human and/or bovine macrophages. Given its dual role in self-regulation and molecular mimicry, this secreted parasite protein warrants investigation as a vaccine candidate against O. ostertagi infections in cattle., (Published by Elsevier Ltd.)

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 β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-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 β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-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.

Background: Cooperia oncophora and Ostertagia ostertagi are among the most important gastrointestinal nematodes of cattle worldwide. The economic losses caused by these parasites are on the order of hundreds of millions of dollars per year. Conventional treatment of these parasites is through anthelmintic drugs; however, as resistance to anthelmintics increases, overall effectiveness has begun decreasing. New methods of control and alternative drug targets are necessary. In-depth analysis of transcriptomic data can help provide these targets., Results: The assembly of 8.7 million and 11 million sequences from C. oncophora and O. ostertagi, respectively, resulted in 29,900 and 34,792 transcripts. Among these, 69% and 73% of the predicted peptides encoded by C. oncophora and O. ostertagi had homologues in other nematodes. Approximately 21% and 24% were constitutively expressed in both species, respectively; however, the numbers of transcripts that were stage specific were much smaller (~1% of the transcripts expressed in a stage). Approximately 21% of the transcripts in C. oncophora and 22% in O. ostertagi were up-regulated in a particular stage. Functional molecular signatures were detected for 46% and 35% of the transcripts in C. oncophora and O. ostertagi, respectively. More in-depth examinations of the most prevalent domains led to knowledge of gene expression changes between the free-living (egg, L1, L2 and L3 sheathed) and parasitic (L3 exsheathed, L4, and adult) stages. Domains previously implicated in growth and development such as chromo domains and the MADF domain tended to dominate in the free-living stages. In contrast, domains potentially involved in feeding such as the zinc finger and CAP domains dominated in the parasitic stages. Pathway analyses showed significant associations between life-cycle stages and peptides involved in energy metabolism in O. ostertagi whereas metabolism of cofactors and vitamins were specifically up-regulated in the parasitic stages of C. oncophora. Substantial differences were observed also between Gene Ontology terms associated with free-living and parasitic stages., Conclusions: This study characterized transcriptomes from multiple life stages from both C. oncophora and O. ostertagi. These data represent an important resource for studying these parasites. The results of this study show distinct differences in the genes involved in the free-living and parasitic life cycle stages. The data produced will enable better annotation of the upcoming genome sequences and will allow future comparative analyses of the biology, evolution and adaptation to parasitism in nematodes.

Larval inhibition is a common strategy of Trichostrongylidae nematodes that may increase survival of larvae during unfavourable periods and concentrate egg production when conditions are favourable for development and transmission. We investigated the propensity for larval inhibition in a population of Ostertagia gruehneri, the most common gastrointestinal Trichostrongylidae nematode of Rangifer tarandus. Initial experimental infections of 4 reindeer with O. gruehneri sourced from the Bathurst caribou herd in Arctic Canada suggested that the propensity for larval inhibition was 100%. In the summer of 2009 we infected 12 additional reindeer with the F1 and F2 generations of O. gruehneri sourced from the previously infected reindeer to further investigate the propensity of larval inhibition. The reindeer were divided into 2 groups and half were infected before the summer solstice (17 June) and half were infected after the solstice (16 July). Reindeer did not shed eggs until March 2010, i.e. 8 and 9 months post-infection. These results suggest obligate larval inhibition for at least 1 population of O. gruehneri, a phenomenon that has not been conclusively shown for any other trichostrongylid species. Obligate inhibition is likely to be an adaptation to both the Arctic environment and to a migratory host and may influence the ability of O. gruehneri to adapt to climate change.

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.)

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The genetic changes underlying resistance to anthelmintics, particularly ivermectin (IVM), remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of changes in gene expression. In this study, we investigated the expression of putative drug transporter molecules, P-glycoproteins (Pgps), in Teladorsagia circumcincta, the predominant parasitic nematode species of sheep in the UK and the major anthelmintic resistant species. Utilising a degenerate PCR approach, 11 partial Pgp sequences were identified. Constitutive differences in gene expression between an IVM-susceptible (MTci2) and a multidrug-resistant (MTci5) isolate were determined for 10 of the Pgps using the ΔΔCt TaqMan® real-time PCR method. Gene expression differences were particularly marked in one of these genes, namely Tci-pgp-9. In the MTci5 isolate, statistically significant increases in Tci-pgp-9 expression, at the mRNA level, were observed across all life-cycle stages and most notably in eggs (55-fold increase). Comparison of the partial Tci-pgp-9 nucleotide sequences from MTci2 and MTci5 also identified high levels of polymorphism. This work has shown that constitutively increased expression in Tci-pgp-9, coupled with increased sequence polymorphism, could play a role in allowing multidrug-resistant T. circumcincta to survive IVM exposure. The genetic changes underpinning these gene expression changes remain to be elucidated and need to be investigated in other isolates. These changes could form the basis of an IVM resistance marker to monitor the spread of resistance and to evaluate management practices aimed at delaying its spread., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Ivermectin (IVM) resistance is an emerging problem for the control of gastrointestinal nematodes of cattle such as Cooperia oncophora and Ostertagia ostertagi. Although there is still a poor understanding of the molecular basis of macrocyclic lactone (ML)-resistance, it is clear that IVM exerts its activity by binding to glutamate-gated chloride (GluCl) channels within the parasite's neuromuscular system. One of the GluCl genes (avr-14) encodes, via alternative splicing, two subunits, AVR-14A and AVR-14B; the latter is suggested to be the main target for IVM. The genomic DNA (gDNA) sequence of avr-14 in C. oncophora contains 21 exons separated by 20 introns and spans approximately 10 kb of gDNA. Intron 13 contains a sequence with high homology to a mammalian mariner transposase. The L256F polymorphism in the avr-14 gene, which was shown to be associated with IVM resistance in a UK isolate of C. oncophora, was not found in the IVM-resistant C. oncophora and O. ostertagi isolates investigated in this study. However, genetic analyses on C. oncophora indicated a loss in allelic diversity of the avr-14 gene in the resistant isolates compared with the susceptible isolate. This suggests that the avr-14 gene, or another genetically linked locus, is under selection in these Belgian C. oncophora isolates. Comparison of the full-length avr-14B coding sequence in the susceptible and resistant C. oncophora isolates did not show any polymorphisms specifically linked to IVM resistance, although a decrease in the number of avr-14B isoforms was observed in the resistant isolates compared with the susceptible one. Measuring the transcription levels of avr-14B in adult male and female C. oncophora and O. ostertagi worms showed significantly lower levels in resistant worms compared with susceptible ones. Whether the down-regulation of this IVM target actually contributes to the resistance mechanism in these worms remains unclear., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling., (© 2011 Blackwell Publishing Ltd.)

Polymorphisms in the isotype I beta-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276bp fragment immediately surrounding the isotype I beta-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200(Y) mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK., (Copyright (c) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Partial sequences of the second internal transcribed spacer (ITS-2) rDNA were obtained for Ostertagia ostertagi and O. lyrata, which are supposedly constitute two morphologically distinct variants of the species. The 1.4% level of difference between the ITS-2 sequences of O. ostertagi and O. lyrata was reported, whereas the sequences derived from worms of the same morph were completely identical. The data obtained prevent an attribution of O. ostertagi and O. lyrata to the same species.

The second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) is used as a genetic marker to identify trichostrongylid nematodes. However, it is often difficult to amplify by polymerase chain reaction (PCR) the ITS2 rDNA of a single trichostrongylid nematode larva or egg. A nested PCR (nPCR) assay was, therefore, developed to amplify the ITS2 from individual trichostrongylid nematode larvae. The results show that the ITS2 rDNA of a significantly greater proportion of individual larvae was amplified using nPCR compared with a standard PCR. There was also no need to column-purify the genomic DNA before nPCR, which is more time and cost effective for studies involving large sample sizes. The amplicons produced from the secondary phase of the nPCR were subjected to single-strand conformation polymorphism analyses and DNA sequencing to confirm the species identity of the larvae used in the current study as Ostertagia gruehneri. The nPCR assay was also used to amplify the ITS2 from individual trichostrongylid eggs. The ability to amplify the ITS2 rDNA from large numbers of individual nematode eggs and larvae has important implications for diagnostic testing and for conducting epidemiological studies on these parasites of veterinary importance.

Aim: To test the hypothesis that anthelmintic treatment of animals with a well-developed immunity to endoparasites will be more selective for anthelmintic resistance than treatment of animals that are not immunocompetent., Methods: In Experiment 1, five pregnant, mixed-aged ewes, and five newly weaned 3-month-old lambs were housed indoors, and infected three times a week with a mixture of albendazole resistant and -susceptible Teladorsagia (=Ostertagia) circumcincta larvae for 12 weeks. In Experiment 2, six two-tooth ewes, six 9-month-old lambs, and 10 newly weaned 3-month-old lambs were infected three times a week with a mixture of albendazole resistant and -susceptible Trichostrongylus colubriformis larvae for 10 weeks over the winter months of June to August. Egg-hatch assays (EHA) were performed on a weekly basis, to determine the resistance status of eggs passed before and after a treatment with albendazole, that was given at least one week after the infections became patent., Results: Faecal nematode egg counts (FEC) indicated that mature ewes and immunocompetent lambs restricted the establishment of parasite larvae, especially after the anthelmintic treatment. Although EHA were completed for T. circumcincta, the numbers of eggs recovered from the ewes were too few to allow accurate comparisons of anthelmintic resistance status between ewes and lambs. In Experiment 2, viable infections of T. colubriformis only developed in five 9-month-old and five 3-month-old lambs after anthelmintic treatment. Comparison of the concentrations of anthelmintic needed to kill 50% of the eggs (LC(50)) from the lambs showed that where host immunity restricted the establishment of new infection after anthelmintic treatment, the resistance status of eggs passed remained high until the end of the study. In contrast, where new infections established, the resistance status of eggs in the faeces declined to levels close to those prior to anthelmintic treatment., Conclusions: The results support the conclusion that older animals are more refractory to the establishment of ingested parasite larvae, and that this reduces the dilution of resistant worms surviving an anthelmintic treatment. Surviving resistant worms are therefore likely to make a greater proportional contribution to the resulting population of parasites on pasture. The findings support the view that treating mature sheep with an anthelmintic should be considered a higher-risk practice for selecting anthelmintic-resistant parasites, than treating lambs.

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.

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.

The transthyretin-like (ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi (Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes (Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory-secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic-bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing β-sheets, arranged in a β-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.

Activation associated secreted proteins (ASP) are members of a nematode-specific protein family belonging to the SCP/Tpx-1/Ag5/PR-1/Sc7 family. Three different types of molecules have been identified in this family: two-domain ASPs and single-domain ASPs showing homology to either the C-terminal or N-terminal domain of the two-domain ASP. The function of these proteins is still unclear, but a role in transition to parasitism and a role as allergen are often suggested. Here we report that the abomasal cattle parasite Ostertagia ostertagi produces at least 15 ASPs, including two-domain and C- and N-type single-domain ASPs. Ten of these are highly transcribed in the L4 stage, whereas others are highly enriched in adult male worms. The latter was especially the case for the N-type single-domain ASPs Oo-ASP1 and Oo-ASP2 and also for Oo-ASP3, which is homologous with the Haemonchus contortus and Ancylostoma caninum C-type single-domain ASPs. Immunohistochemistry showed that Oo-ASP3 was localised in the oesophagus. Oo-ASP1 and Oo-ASP2 on the other hand were localised in the reproductive tract of both male and female worms, suggesting a role in reproduction or in the development of the reproductive tract.

It has been shown that the bovine abomasal parasite, Ostertagia ostertagi, drastically modulates its microenvironment, causing epithelial cell damage, accumulation of inflammatory cells and pH changes in the stomach. The mechanisms used by the parasite to change the abomasal environment are largely unknown, but an important role has been attributed to excretory-secretory (ES) products from the parasite. In this study we have identified proteins representing a novel ES protein family, characterized by the SCP/Tpx-1/Ag5/PR-1/Sc7 protein motif. These proteins were named Oo-AL1 and Oo-AL2 (O. ostertagi ASP-like protein). Both proteins contain a signal peptide and 1 predicted N-glycosylation site. The transcript for Oo-AL1 was present from the L4 stage onwards in both male and female adult worms, whereas the Oo-AL2 transcript was hardly detectable. Western blots of somatic extracts and ES products from different developmental stages of O. ostertagi, probed with anti-Oo-AL1 antibodies, revealed Oo-AL proteins in the ES products of adult worms. An analysis of the nematode genome and EST databases indicated that these novel ES proteins are unique to O. ostertagi and its relative, Teladorsagia circumcincta, suggesting a key function in these abomasal parasites.

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.

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, beta-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.

In this study, we isolated and analyzed a small heat shock protein (HSP) of Ostertagia ostertagi (Oo-HSP18). Oo-hsp18 is encoded by a single-copy gene and the full-length cDNA represents an 18-kDa protein. The expression of Oo-hsp18 is highly stage specific and restricted to the adult stage. The protein is synthesized in a tissue-specific manner and localized in the body muscle layer. The levels of Oo-hsp18 mRNAs are sharply induced by heat shock but not by other stressors such as levamisole and H2O2. A vaccination trial with recombinant Oo-HSP18 failed to protect calves against a challenge infection.

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.

In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematode Ostertagia ostertagi which were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs of Ostertagia (met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3'- and 5'-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor of Ancylostoma caninum. The full-length cDNA of met-1 consists of an open reading frame (ORF) of 586 amino acids which contains 5 potential N-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence of met-3 contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4 and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.

As an alternative to antihelminthic drugs, we are exploiting vaccination to control infections with the abomasal nematode Ostertagia ostertagi in cattle. Our focus for vaccine targets is excretory-secretory (ES) products of this parasite. One of the most abundant antigens in larval and adult Ostertagia ES products is a protein homologous to nematode polyprotein allergens. We found that the Ostertagia polyprotein allergen (OPA) is encoded by a single-copy gene. OPA comprises three or more repeated units, and only the 15-kDa subunits are found in ES products. The native antigen is localized in the intestinal cells of third-stage larvae and in the hypodermis and cuticle of fourth-stage larvae and adult parasites. Vaccination of cattle with native OPA (nOPA) in combination with QuilA resulted in protection against Ostertagia challenge infections. The geometric mean cumulative fecal egg counts in the nOPA-vaccinated animals were reduced by 60% compared to the counts in the control group during the 2-month course of the experiment. Both male and female adult worms in nOPA-vaccinated animals were significantly shorter than the worms in the control animals. In the abomasal mucus of vaccinated animals the nOPA-specific immunoglobulin G1 (IgG1) and IgG2 levels were significantly elevated compared to the levels in the control animals. Reductions in the Ostertagia egg output and the length of the adult parasites were significantly correlated with IgG1 levels. IgG2 titers were only negatively associated with adult worm length. Protected animals showed no accumulation of effector cells (mast cells, globular leukocytes, and eosinophils) in the mucosa. In contrast to the native antigen, recombinant OPA expressed in Escherichia coli did not stimulate any protection.

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.

Excretory-secretory (ES) products of Ostertagia ostertagi, an abomasal nematode of cattle, are considered to be important for the development and survival of the parasite within the host. To gain insight in the composition of these ES products of both larval (L3, L4) and adult life stages of Ostertagia cDNA libraries of the parasite were immunoscreened with polyclonal rabbit serum raised against these ES products. This approach led to the identification of 41 proteins, amongst which are structural proteins such as actin, kinesin and vitellogenin, housekeeping proteins such as those involved in protein folding, different metabolic pathways or mitochondrial functioning and proteins associated with stress (heat shock protein) or antioxidantia (thioredoxin peroxidase). A large number of the isolated proteins were similar to hypothetical proteins of the model nematode Caenorhabditis elegans. Because somatic proteins can be non-specifically released during in vitro culturing as nematodes deteriorate, it was checked if the isolated proteins are genuinely secreted. The amino acid sequences of the translated cDNAs were investigated for signal peptides and monospecific antibodies against the isolated proteins were purified and used to develop Western blots of ES and somatic extracts. In this manner it could be proven that 15 cDNAs code for genuine secreted proteins. The identification of these ES antigens allows to select proteins with potential protective capacities, which are targets for vaccine development., (Copyright 2003 Elsevier Science B.V.)

The identification of protective helminth antigens remains the most important challenge in the development of parasitic vaccines. To identify protective antigens of Ostertagia ostertagi, an important abomasal parasite of cattle, parasite-specific local antibodies from the abomasal mucus and from the draining lymph nodes were collected from calves immunized with multiple infections and from 'primary infected' animals. With these probes, Western blots of extracts and excretion/ secretion (E/S) material from L3, L4 and adult life-stages as well as cDNA expression libraries were screened to identify antigens that were exclusively recognized by antibodies from 'immunized' calves. In the adult stage, a protein of 32 kDa was specifically detected on Western blot by mucus antibodies from 'immunized' animals. In the L3 and L4 larval stages, proteins situated in the regions of 28-29 kDa were recognized by mucus antibodies and a 59 kDa antigen was specifically recognized by lymph node antibodies from 'immunized' animals. Screening E/S material revealed no specific difference in recognition pattern between 'immunized' and 'primary infected' animals. Screening of the cDNA libraries revealed 26 relevant clones, coding for 15 proteins, among these several with potential protective capacity, immunodominant properties or functional and physiological importance e.g. metalloproteases, an aspartyl protease inhibitor and collagen.

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.

Reverse transcriptase-PCR (RT-PCR) was carried out on total RNA prepared from the third-stage larvae (L3) of Ostertagia ostertagi in order to clone and characterize the major transcripts expressed in this larval stage, as an initial investigation of arrested larval development in the parasite. Distinct bands were visible on an agarose gel and four of these were cloned and sequenced. Three of the bands represented multiple transcripts, while the fourth band encoded the enzyme GTP cyclohydrolase I (GTP-CH), which catalyses the first and rate-limiting step in pteridine biosynthesis. Northern blot analysis and RT-PCR demonstrated that GTP-CH is highly up-regulated in the L3 stage and undetectable in either the L2 or adult stages. Using immunogold electron microscopy, GTP-CH was shown to be predominantly localized to the cell body of the body wall muscles and the cells of the intestine in the L3.

DNA sequences of ITS-1 and ITS-2 of rDNA were determined for 16 individual adult males each of Ostertagia gruehneri and Ostertagia arctica from Svalbard reindeer (Rangifer tarandus platyrhynchus) and Eurasian tundra reindeer (R. t. tarandus). Each ITS was virtually identical in O. gruehneri and O. arctica and the three mixed bases detected were shared by both species. Our results strongly suggest that O. gruehneri and O. arctica are dimorphic males of the same species.