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Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Haemonchus contortus is one of the most pathogenic nematodes affecting small ruminants globally and is responsible for large economic losses in the sheep and goat industry. Anthelmintic resistance is rampant in this parasite and thus parasite control programs must account for drug efficacy on individual farms and, sometimes, whether H. contortus is the most prevalent trichostrongylid. Historically, coproculture has been the main way to determine the prevalence of H. contortus in faecal samples due to the inability to morphologically differentiate between trichostrongylid egg types, but this process requires a skilled technician and takes multiple days to complete. Fluoresceinated peanut agglutinin (PNA) has been shown to specifically bind H. contortus and thus differentiate eggs based on whether they fluoresce, but this method has not been widely adopted. The Parasight TM System (PS) fluorescently stains helminth eggs in order to identify and quantify them, and the H. contortus PNA staining method was therefore adapted to this platform using methodology requiring only 20 min to obtain results. In this study, 74 fecal samples were collected from sheep and analyzed for PNA-stained H. contortus, using both PS and manual fluorescence microscopy. The percentage of H. contortus was determined based on standard total strongylid counts with PS or brightfield microscopy. Additionally, 15 samples were processed for coproculture with larval identification, and analyzed with both manual and automated PNA methods. All methods were compared using the coefficient of determination (R 2 ) and the Lin's concordance correlation coefficient (ρc). Parasight TM and manual PNA percent H. contortus results were highly correlated with R 2  = 0.8436 and ρc = 0.9100 for all 74 fecal samples. Coproculture versus PS percent H. contortus were also highly correlated with R 2  = 0.8245 and ρc = 0.8605. Overall, this system provides a rapid and convenient method for determining the percentage of H. contortus in sheep and goat fecal samples without requiring specialized training., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Santa Ines (SI) and Ile de France (IF) sheep are known to be resistant and susceptible to Haemonchus contortus infection, respectively. Several studies have shown some genes as potential biological markers for sheep resistance against gastrointestinal nematodes using molecular tools, including transcriptomic analysis. In this study, we sequenced the polyadenylated RNA of the abomasal tissue of SI and IF suckling lambs to identify mucosa-specific transcript alterations between breeds artificially infected with H. contortus. Naïve SI (n = 4) and IF (n = 4) lambs were artificially infected every other day, over a period of 52 days, from 14 to 66 days old, with a total of 5,400 H. contortus infective larvae. Fundic abomasal tissue samples were collected at 68 days old, and submitted to high-throughput RNA sequencing (RNA-seq). Differential expression analysis (P value < 0.001 and False Discovery Rate (FDR) < 0.05) between SI and IF samples identified 292 genes, most of which showed greater expression in SI lambs. To help annotate and assign possible function to differentially expressed genes (DEGs), we used previously available single-cell RNA-seq (scRNA-seq) data from ovine abomasal mucosa to putatively identify cell types and possible mechanisms involved in resistance to H. contortus. In particular, genes associated with endothelial and tuft cells showed the greatest increases in expression in SI relative to IF lambs. SI lambs had higher percentages of tuft cells than IF lambs in the fundic abomasal mucosa. Although we found innate immunity (cell-mediated in mucosa) acting as a protagonist in impairing H. contortus infection, a stronger acquired immune response was being modulated at an earlier stage by SI lambs. We suggest that the complex connection between innate and adaptive immunity is via cellular antigen processing and presentation (APP). Based on comparison with scRNA-seq data, SI lambs showed a robust APP mechanism characterized mainly by greater T cell APP, macrophage differentiation, and cytokine signalling. We identified potential mechanisms and markers to advance knowledge for selection of H. contortus resistance at a very early age, in SI as well as in other commercial sheep breeds., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Improvement in the use of existing anthelmintics is a high priority need for the pharmaco-parasitology research field, considering the magnitude and severity of anthelmintic resistance as an important issue in livestock production. In the work described here, monepantel (MNP) was given alone or co-administered with either macrocyclic lactone (ML) or benzimidazole (BZ) anthelmintics to calves naturally infected with ML- and BZ-resistant gastrointestinal (GI) nematodes on two different commercial cattle farms. Both pharmacokinetic (PK) and efficacy assessments were performed. On Farm A, male calves (n = 15 per group) were treated with either MNP orally (2.5 mg/kg), IVM s.c. (0.2 mg/kg), ricobendazole (RBZ) s.c. (3.75 mg/kg) or remained untreated. On Farm B, eight groups (n = 15) of male calves received treatment with either: MNP, abamectin (ABM, oral, 0.2 mg/kg), RBZ (s.c., 3.75 mg/kg), albendazole (ABZ, oral, 5 mg/kg), MNP+ABM, MNP+RBZ, MNP+ABZ (all at the above-mentioned routes and doses) or remained untreated. Seven animals from each treated group (Farm B) were randomly selected to perform the PK study. MNP and its metabolite monepantel sulphone (MNPSO 2 ) were the main analytes recovered in plasma after HPLC analysis. The combined treatments resulted in decreased systemic exposures to MNP parent drug compared with that observed after treatment with MNP alone (P < 0.05). However, the systemic availability of the main MNP metabolite (MNPSO 2 ) was unaffected by co-administration with either ABM, RBZ or ABZ. Efficacies of 98% (Farm A) and 99% (Farm B) demonstrated the high efficacy of MNP given alone (P < 0.05) against GI nematodes resistant to ML and BZ in cattle. While the ML (IVM, ABM) failed to control Haemonchus spp., Cooperia spp. and Ostertagia spp., MNP achieved 99% to 100% efficacy against those nematode species on both commercial farms. However, MNP alone failed to control Oesophagostomum spp. (60% efficacy) on Farm A. The co-administered treatments MNP+ABZ and MNP+RBZ reached a 100% reduction against all GI nematode genera. In conclusion, the oral treatment with MNP should be considered to deal with resistant nematode parasites in cattle. The use of MNP in combination with BZ compounds could be a valid strategy to extend its lifespan for use in cattle as well as to reverse its poor activity against Oesophagostomum spp., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Infections caused by Haemonchus spp. and Trichostrongylus spp. are major health problems for sheep and cattle. The objective of this study was to determine the efficacy of copper chloride (CuCl 2 ), and copper sulphate (CuSO 4 ) at 2.0, 7.0, 30.0, 125.0, 500.0, and 2000.0 µM formulations, and nitroxynil 34% (NTX) at 0.235 mM against gastrointestinal nematodes (GINs) of ruminants. Hence, the in vitro egg hatch test (EHT), the larval development test (LDT), and the larval migration inhibition test (LMIT) were used. Haemonchus spp. (52%) and Trichostrongylus spp. (38%) were the most frequently found parasites. The data fitted a concentration-dependent shape with the highest efficacies of CuCl 2 and CuSO 4 at 95.2 and 97.3% for parasites collected from sheep, and 95.8 and 93.4% from cattle, respectively. The combination of the 50% inhibitory concentration (IC 50 ) of CuCl 2 and CuSO 4 and the IC 10 of NTX showed up to a 52% increase in efficacy above the expected additive results, demonstrating a synergic/drug enhancer interaction. NTX may retain Cu-II ions by complexation, in a hitchhiking mechanism carrying the salts across the parasite cell wall, causing oxidative stress as a consequence of free radical production and cell damage. Synergy data between NTX and CuCl 2 , and CuSO 4 represent a viable opportunity to develop new formulations for combating parasites of ruminants (i.e., Fasciola hepatica, Haemonchus spp., and Oesophagostomum spp.)., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Gastrointestinal nematode (GIN) infections are ubiquitous and often cause morbidity and reduced performance in livestock. Emerging anthelmintic resistance and increasing change in climate patterns require evaluation of alternatives to traditional treatment and management practices. Mathematical models of parasite transmission between hosts and the environment have contributed towards the design of appropriate control strategies in ruminants, but have yet to account for relationships between climate, infection pressure, immunity, resources, and growth. Here, we develop a new epidemiological model of GIN transmission in a herd of grazing cattle, including host tolerance (body weight and feed intake), parasite burden and acquisition of immunity, together with weather-dependent development of parasite free-living stages, and the influence of grass availability on parasite transmission. Dynamic host, parasite and environmental factors drive a variable rate of transmission. Using literature sources, the model was parametrised for Ostertagia ostertagi, the prevailing pathogenic GIN in grazing cattle populations in temperate climates. Model outputs were validated on published empirical studies from first season grazing cattle in northern Europe. These results show satisfactory qualitative and quantitative performance of the model; they also indicate the model may approximate the dynamics of grazing systems under co-infection by O. ostertagi and Cooperia oncophora, a second GIN species common in cattle. In addition, model behaviour was explored under illustrative anthelmintic treatment strategies, considering impacts on parasitological and performance variables. The model has potential for extension to explore altered infection dynamics as a result of management and climate change, and to optimise treatment strategies accordingly. As the first known mechanistic model to combine parasitic and free-living stages of GIN with host feed-intake and growth, it is well suited to predict complex system responses under non-stationary conditions. We discuss the implications, limitations and extensions of the model, and its potential to assist in the development of sustainable parasite control strategies., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Haemonchus contortus is the most prevalent and important gastrointestinal nematode (GIN) in small ruminants. Since it reduces the packed cell volume (PCV), causing anemia, early diagnosis can be used for targeted selective treatment (TST) of sheep, reducing antiparasitic drug use and anthelmintic resistance. This study aimed to predict PCV values through near-infrared spectroscopy (NIRS) and to develop a classification and diagnosis model of H. contortus infection using PCV values, eggs per gram of feces (EPG) counts and mean daily weight gain (DWG). A total of 1728 spectra were collected from blood samples of 216 lambs with a portable NIR spectroscope. In parallel, other parameters indicative of infection were measured: PCV by hematocrit, FAffa MAlan CHArt (FAMACHA) scores, EPG and DWG. To evaluate the relationship between NIRS spectra and the evaluated parameters, principal component analysis (PCA) was used for an exploratory analysis, regression by the partial least squares method (PLS) for the prediction of PCV values via NIRS, and PCA linear discriminant analysis (PCA-LDA) as a classification model for diagnosis. The absorption peaks in the NIRS region associated with the excitation of overtones of nitrogen-hydrogen (N-H) functional groups of proteins had a strong impact on the principal components (PCs), indicating that blood proteins, especially hemoglobin, can be estimated by the NIRS technique. The model for predicting PCV by PLS presented a standard error of prediction of 2.53, root-mean-square error of 2.48, and coefficient of determination of 0.84, indicating good correlation between the PCV values predicted by the model and the PCV obtained by hematocrit. The PCA-LDA model presented 93.33% sensitivity and 82.18% accuracy, both higher than those of the FAMACHA method, as was expected for resilient Morada Nova lambs. The multivariate models associated with the NIRS technique reported here can be used in the future as a quick and versatile tool for H. contortus infection diagnosis and TST application in lambs., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Gastrointestinal nematodes (GINs) are damaging parasites of global sheep populations. The key weapons in fighting GINs have been anthelmintic drugs, but the emergence of drug-resistant parasites has meant that alternative control methods are needed. One of these alternatives is to breed for enhanced host resistance to GINs, and decades of research have estimated the genetic contribution to different measures of resistance to GINs and their genetic correlations with other desirable performance traits. It is clear that parasite resistance is a heritable trait that can be selected for. Despite this consensus, estimates of both heritability of resistance and genetic correlations with other traits vary widely between studies, and the reasons for this variation have not been examined. This study provides a comprehensive and quantitative meta-analysis of genetic parameters for resistance to GINs in sheep, including measures of worm burden (faecal egg counts, FECs), anti-parasite immunity (GIN-specific antibodies), and parasite-induced pathology (FAMACHA© scores). Analysis of 591 heritability estimates from 121 studies revealed a global heritability estimate for resistance to GINs of 0.25 (95% confidence interval (CI) = 0.22-0.27) that was stable across breeds, ages, geographical location and analytical methods. Meanwhile, analysis of 559 genetic correlations from 54 studies revealed that resistance to GINs overall has a positive genetic correlation of +0.10 (95% CI = 0.02-0.19) with performance traits, and that this was consistent across breeds, ages, sexes and analytical methods. Importantly, the direction of the genetic correlation varied with the resistance trait measured: while FECs and FAMACHA© scores were favourably correlated with performance traits, adaptive immune markers were unfavourably correlated, suggesting that selection for enhanced immune responses to GINs could reduce animal performance. Overall, the results suggest that breeding for resistance to GINs should continue to form part of integrated management programs to reduce the impact of parasites on health and performance, but that selection for enhanced immune responses should be avoided., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Macrocyclic lactones have been the most widely used drugs for equine parasite control during the past four decades. Unlike ivermectin, moxidectin exhibits efficacy against encysted cyathostomin larvae, and is reported to have persistent efficacy with substantially longer egg reappearance periods. However, shortened egg reappearance periods have been reported recently for both macrocyclic lactones, and these findings have raised several questions: (i) are egg reappearance period patterns different after ivermectin or moxidectin treatment? (ii) Are shortened egg reappearance periods associated with certain cyathostomin species or stages? (iii) How does moxidectin's larvicidal efficacy affect egg reappearance period? To address these questions, 36 horses at pasture, aged 2-5 years old, were randomly allocated to three treatment groups: 1, moxidectin; 2, ivermectin; and 3, untreated control. Strongylid fecal egg counts were measured on a weekly basis, and the egg reappearance period was 5 weeks for both compounds. Strongylid worm counts were determined for all horses: 18 were necropsied at 2 weeks post-treatment (PT), and the remaining 18 at 5 weeks PT. Worms were identified to species morphologically and by internal transcribed spacer-2 (ITS-2) rDNA metabarcoding. Moxidectin and ivermectin were 99.9% and 99.7% efficacious against adults at 2 weeks post treatment, whereas the respective efficacies against luminal L4s were 84.3% and 69.7%. At 5 weeks PT, adulticidal efficacy was 88.3% and 57.6% for moxidectin and ivermectin, respectively, while the efficacy against luminal L4s was 0% for both drugs. Moxidectin reduced early L3 counts by 18.1% and 8.0% at 2 or 5 weeks, while the efficacies against late L3s and mucosal L4s were 60.4% and 21.2% at the same intervals, respectively. The luminal L4s surviving ivermectin treatment were predominantly Cylicocyclus (Cyc.) insigne. The ITS-2 rDNA metabarcoding was in good agreement with morphologic species estimates but suggested differential activity between moxidectin and ivermectin for several species, most notably Cyc. insigne and Cylicocyclus nassatus. This study was a comprehensive investigation of current macrocyclic lactone efficacy patterns and provided important insight into potential mechanisms behind shortened egg reappearance periods., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Gastrointestinal nematode (GIN) parasites in grazing cattle are a major cause of production loss and their control is increasingly difficult due to anthelmintic resistance and climate change. Rotational grazing can support control and decrease reliance on chemical intervention, but is often complex due to the need to track grazing periods and infection levels, and the effect of weather on larval availability. In this paper, a simulation model was developed to predict the availability of infective larvae of the bovine GIN, Ostertagia ostertagi, at the level of individual pastures. The model was applied within a complex rotational grazing system and successfully reproduced observed variation in larval density between fields and over time. Four groups of cattle in their second grazing season (n = 44) were followed throughout the temperate grazing season with regular assessment of GIN faecal egg counts, which were dominated by O. ostertagi, animal weight and recording of field rotations. Each group of cattle was rotationally grazed on six group-specific fields throughout the 2019 grazing season. Maps and calendars were produced to illustrate the change in pasture infectivity (density of L3 on herbage) across the 24 separate grazing fields. Simulations predicted differences in pasture contamination levels in relation to the timing of grazing and the return period. A proportion of L3 was predicted to persist on herbage over winter, declining to similar intensities across fields before the start of the following grazing season, irrespective of contamination levels in the previous year. Model predictions showed good agreement with pasture larval counts. The model also simulated differences in seasonal pasture infectivity under rotational grazing in systems that differed in temperature and rainfall profiles. Further application could support individual farm decisions on evasive grazing and refugia management, and improved regional evaluation of optimal grazing strategies for parasite control. The integration of weather and livestock movement is inherent to the model, and facilitates consideration of climate change adaptation through improved disease control., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Anthelmintic resistance threatens the sustainability of sheep production globally. Advice regarding strategies to reduce the development of anthelmintic resistance incorporates the outcomes of modelling exercises. Further understanding of gastrointestinal nematode species diversity, and population dynamics and genetics (which may vary between species) is required to refine these models; and field studies combining faecal egg outputs, species composition and resistance genetics are needed to calibrate them. In this study, faecal samples were taken from ewes and lambs on a commercial farm in south-eastern Scotland at approximately 3 t-4 week intervals between spring and autumn over a period of 4 years. Faecal egg counts were performed on these samples, and L 3 were collected from pooled coprocultures. Deep amplicon sequencing was used to determine both the species composition of these L 3 and the proportions of benzimidazole-resistant single nucleotide polymorphisms in the isotype-1 β-tubulin locus of the predominant species, Teladorsagia circumcincta L 3 . Despite consistent management throughout the study, the results show variation in gastrointestinal nematode species composition with time and between age groups, that was potentially associated with weather conditions. The F200Y benzimidazole resistance mutation is close to genetic fixation in the T. circumcincta population on this farm. There was no evidence of variation in isotype-1 β-tubulin single nucleotide polymorphisms frequency between age groups, and no genetic evidence of reversion to benzimidazole susceptibility, despite targeted benzimidazole usage. This study highlights the need to include speciation when investigating gastrointestinal nematode epidemiology and anthelmintic resistance, and serves as an example of how genetic data may be analysed alongside species diversity and faecal egg counts, when markers for other anthelmintic classes are identified., (Copyright © 2021 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections., (Copyright © 2020 Australian Society for Parasitology. All rights reserved.)

Gastrointestinal nematodes are a significant threat to the economic and environmental sustainability of keeping livestock, as adequate control becomes increasingly difficult due to the development of anthelmintic resistance in some systems and climate-driven changes to infection dynamics. To mitigate any negative impacts of climate on gastrointestinal nematode epidemiology and slow anthelmintic resistance development, there is a need to develop effective, targeted control strategies that minimise the unnecessary use of anthelmintic drugs and incorporate alternative strategies such as vaccination and evasive grazing. However, the impacts climate and gastrointestinal nematode epidemiology may have on the optimal control strategy are generally not considered, due to lack of available evidence to drive recommendations. Parasite transmission models can support control strategy evaluation to target field trials, thus reducing the resources and lead-time required to develop evidence-based control recommendations incorporating climate stochasticity. Gastrointestinal nematode population dynamics arising from natural infections have been difficult to replicate and model applications have often focussed on the free-living stages. A flexible framework is presented for the parasitic phase of gastrointestinal nematodes, GLOWORM-PARA, which complements an existing model of the free-living stages, GLOWORM-FL. Longitudinal parasitological data for two species that are of major economic importance in cattle, Ostertagia ostertagi and Cooperia oncophora, were obtained from seven cattle farms in Belgium for model validation. The framework replicated the observed seasonal dynamics of infection in cattle on these farms and overall, there was no evidence of systematic under- or over-prediction of faecal egg counts. However, the model under-predicted the faecal egg counts observed on one farm with very young calves, highlighting potential areas of uncertainty that may need further investigation if the model is to be applied to young livestock. The model could be used to drive further research into alternative parasite control strategies such as vaccine development and novel treatment approaches, and to understand gastrointestinal nematode epidemiology under changing climate and host management., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Horses are infected by a wide range of parasite species that form complex communities. Parasite control imposes significant constraints on parasite communities whose monitoring remains, however, difficult to track through time. Postmortem examination is a reliable method to quantify parasite communities. Here, we compiled 1,673 necropsy reports accumulated over 29 years, in the reference necropsy centre from Normandy (France). The burden of non-strongylid species was quantified and the presence of strongylid species was noted. Details of horse deworming history and the cause of death were registered. Building on these data, we investigated the temporal trend in non-strongylid epidemiology and we determined the contribution of parasites to the deaths of horses throughout the study period. Data analyses revealed the seasonal variations of non-strongylid parasite abundance and reduced worm burden in race horses. Beyond these observations, we found a shift in the species responsible for fatal parasitic infection from the year 2000 onward, whereby fatal cyathostominosis and Parascaris spp. infection have replaced cases of death caused by Strongylus vulgaris and tapeworms. A concomitant break in the temporal trend of parasite species prevalence was also found within a 10 year window (1998-2007) that has seen the rise of Parascaris spp. and the decline of both Gasterophilus spp. and tapeworms. A few cases of parasite persistence following deworming were identified, which all occurred after 2000. Altogether, these findings provide insights into major shifts in non-strongylid parasite prevalence and abundance over the last 29 years. They also underscore the critical importance of Parascaris spp. in young equids., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Lambs with the Major Histocompatibility Complex DRB1*1101 allele have been shown to produce fewer nematode eggs following natural and deliberate infection. These sheep also possess fewer adult Teladorsagia circumcincta than sheep with alternative alleles at the DRB1 locus. However, it is unclear if this allele is responsible for the reduced egg counts or merely acts as a marker for a linked gene. This study defined the MHC haplotypes in a population of naturally infected Scottish Blackface sheep by PCR amplification and sequencing, and examined the associations between MHC haplotypes and faecal egg counts by generalised linear mixed modelling. The DRB1*1101 allele occurred predominately on one haplotype and a comparison of haplotypes indicated that the causal mutation or mutations occurred in or around this locus. Additional comparisons with another resistant haplotype indicated that mutations in or around the DQB2*GU191460 allele were also responsible for resistance to nematode infections. Further analyses identified six amino acid substitutions in the antigen binding site of DRB1*1101 that were significantly associated with reductions in the numbers of adult T. circumcincta., (Copyright © 2019. Published by Elsevier Ltd.)

Of anthropogenic methane emissions, 40% can be attributed to agriculture, the majority of which are from enteric fermentation in livestock. With international commitments to tackle drivers of climate change, there is a need to lower global methane emissions from livestock production. Gastrointestinal helminths (parasitic worms) are globally ubiquitous and represent one of the most pervasive challenges to the health and productivity of grazing livestock. These parasites influence a number of factors affecting methane emissions including feed efficiency, nutrient use, and production traits. However, their effects on methane emissions are unknown. This is to our knowledge the first study that empirically demonstrates disease-driven increases in methane (CH 4 ) yield in livestock (grams of CH 4 per kg of dry matter intake). We do this by measuring methane emissions (in respiration chambers), dry matter intake, and production parameters for parasitised and parasite-free lambs. This study shows that parasite infections in lambs can lead to a 33% increase in methane yield (g CH 4 /kg DMI). This knowledge will facilitate more accurate calculations of the true environmental costs of parasitism in livestock, and reveals the potential benefits of mitigating emission through controlling parasite burdens., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Worm expulsion is known to occur in mammalian hosts exposed to mono-species helminth infections, whilst this phenomenon is poorly described in avian hosts. Mono-species infections, however, are rather rare under natural circumstances. Therefore, we quantified the extent and duration of worm expulsion by chickens experimentally infected with both Ascaridia galli and Heterakis gallinarum, and investigated the accompanying humoral and cell-mediated host immune responses in association with population dynamics of the worms. Results demonstrated the strong co-expulsion of the two ascarid species in three phases. The expulsion patterns were characterized by non-linear alterations separated by species-specific time thresholds. Ascaridia galli burden decreased at a daily expulsion rate (e) of 4.3 worms up to a threshold of 30.5 days p.i., followed by a much lower second expulsion rate (e = 0.46), which resulted in almost, but not entirely, complete expulsion. Heterakis gallinarum was able to induce reinfection within the experimental period (9 weeks). First generation H. gallinarum worms were expelled at a daily rate of e = 0.8 worms until 36.4 days p.i., and thereafter almost no expulsion occurred. Data on both humoral and tissue-specific cellular immune responses collectively indicated that antibody production in chickens with multispecies ascarid infections is triggered by Th2 polarisation. Local Th2 immune responses and mucin-regulating genes are associated with the regulation of worm expulsion. In conclusion, the chicken host is able to eliminate the vast majority of both A. galli and H. gallinarum in three distinct phases. Worm expulsion was strongly associated with the developmental stages of the worms, where the elimination of juvenile stages was specifically targeted. A very small percentage of worms was nevertheless able to survive, reach maturity and induce reinfection if given sufficient time to complete their life cycle. Both humoral and local immune responses were associated with worm expulsion., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

A growing body of evidence, particularly in humans and rodents, supports the existence of a complex network of interactions occurring between gastrointestinal (GI) helminth parasites and the gut commensal bacteria, with substantial effects on both host immunity and metabolic potential. However, little is known of the fundamental biology of such interactions in other animal species; nonetheless, given the considerable economic losses associated with GI parasites, particularly in livestock and equines, as well as the global threat of emerging anthelmintic resistance, further explorations of the complexities of host-helminth-microbiota interactions in these species are needed. This study characterises the composition of the equine gut commensal flora associated with the presence, in faecal samples, of low (Clow) and high (Chigh) numbers of eggs of an important group of GI parasites (i.e. the cyathostomins), prior to and following anthelmintic treatment. High-throughput sequencing of bacterial 16S rRNA amplicons and associated bioinformatics and statistical analyses of sequence data revealed strong clustering according to faecal egg counts (P = 0.003). A trend towards increased populations of Methanomicrobia (class) and Dehalobacterium (genus) was observed in Clow in comparison with Chigh. Anthelmintic treatment in Chigh was associated with a significant reduction of the bacterial Phylum TM7 14 days post-ivermectin administration, as well as a transient expansion of Adlercreutzia spp. at 2 days post-treatment. This study provides a first known insight into the discovery of the intimate mechanisms governing host-parasite-microbiota interactions in equines, and sets a basis for the development of novel, biology-based intervention strategies against equine GI helminths based on the manipulation of the commensal gut flora., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Next-generation deep amplicon sequencing, or metabarcoding, has revolutionized the study of microbial communities in humans, animals and the environment. However, such approaches have yet to be applied to parasitic helminth communities. We recently described the first example of such a method - nemabiome sequencing - based on deep-amplicon sequencing of internal transcribed spacer 2 (ITS-2) rDNA, and validated its ability to quantitatively assess the species composition of cattle gastro-intestinal nematode (GIN) communities. Here, we present the first application of this approach to explore GIN species diversity and the impact of anthelmintic drug treatments. First, we investigated GIN species diversity in cow-calf beef cattle herds in several different regions, using coproculture derived L3s. A screen of 50 Canadian beef herds revealed parasite species diversity to be low overall. The majority of parasite communities were comprised of just two species; Ostertagia ostertagi and Cooperia oncophora. Cooperia punctata was present at much lower levels overall, but nevertheless comprised a substantive part of the parasite community of several herds in eastern Canada. In contrast, nemabiome sequencing revealed higher GIN species diversity in beef calves sampled from central/south-eastern USA and Sao Paulo State, Brazil. In these regions C. punctata predominated in most herds with Haemonchus placei predominating in a few cases. Ostertagia ostertagi and C. oncophora were relatively minor species in these regions in contrast to the Canadian herds. We also examined the impact of routine macrocyclic lactone pour-on treatments on GIN communities in the Canadian beef herds. Low treatment effectiveness was observed in many cases, and nemabiome sequencing revealed an overall increase in the proportion of Cooperia spp. relative to O. ostertagi post-treatment. This work demonstrates the power of nemabiome metabarcoding to provide a detailed picture of GIN parasite community structure in large sample sets and illustrates its potential use in research, diagnostics and surveillance., (Copyright © 2017. Published by Elsevier Ltd.)

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50μg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50μg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R(2)⩾0.999) and faecal samples (R(2)⩾0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5-6 months old Assaf lambs using Al(OH)(3) or Escherichia coli lipopolysaccharide+inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%-85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

We evaluated a combined microscopic-molecular approach for the diagnosis of key strongylid infections in sheep using panels of well-defined control and test samples. The method established is based on the separation of nematode eggs from faecal samples using a salt flotation procedure, the extraction and column-purification of genomic DNA, followed by real-time PCR and melting-curve analysis. Specific and semi-quantitative amplification from (a minimum of 0.1-2.0pg) genomic DNA of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia oncophora, Oesophagostomum columbianum, Oesophagostomum venulosum or Chabertia ovina is achieved using a specific, forward oligonucleotide primer located in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) together with a conserved reverse primer in the large subunit of rDNA. Using a panel of well-defined genomic DNA samples from eggs from sheep monospecifically infected with H. contortus or Te. circumcincta, there was a correlation between cycle threshold (Ct) values in the PCR and numbers of egg per gram of faeces, thus allowing the semi-quantitation of parasite DNA in faeces. The findings of the present study indicate that a microscopic-molecular approach provides a useful tool for diagnosis, for epidemiological and ecological surveys as well as for integration into parasite monitoring, drug resistance (i.e. 'egg count reduction') testing or control programmes, particularly following semi- or full-automation.

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P>0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.

This paper describes a series of five pilot trials to test the feasibility of inducing a protective mucosal immune response against a non-blood-feeding intestinal nematode by delivery of antigens across the mucosal epithelium. A number of antigen preparations from Trichostrongylus colubriformis (viable larvae, larval homogenate and recombinant 17kDa excretory-secretory protein) were delivered to the luminal surface of the mucosal epithelium overlying jejunal or rectal lymphoid tissue in cellulose or chitosan formulations. Significant protection was induced following delivery of viable larvae, larval homogenate or recombinant protein to the epithelium overlying rectal Peyer's patches, and recombinant protein to the epithelium overlying jejunal Peyer's patches. Viable larvae were associated with a jejunal IgE/IgG1 response, while the 17kDa antigen was associated with a jejunal IgA response. The results demonstrate that delivery of Trichostrongylus native and recombinant antigens across the epithelium overlying rectal lymphoid patches can result in significant protective immunity even in the absence of adjuvant. They warrant the further investigation of appropriate mucosal delivery methods and adjuvants for induction of protective mucosal responses to stages and species of gastrointestinal helminths which do not ingest serum antibodies.

Monepantel (AAD 1566) is the first compound from the recently discovered amino-acetonitrile derivative (AAD) class of anthelmintics to be developed for use in sheep. Three dose determination studies were conducted in Australia and Europe to identify the therapeutic dose of monepantel, when formulated for the oral treatment of sheep, to control adult gastro-intestinal nematodes. In each study, sheep infected with various nematode species were treated with either 1.25, 2.5 or 5.0mg monepantel/kg bodyweight. Following euthanasia and worm counting, their worm burdens were compared with those from untreated control groups. At a dose rate of 1.25mg/kg, monepantel showed efficacy above 91.9% against all major nematode species, with the exception of Chabertia ovina and Oesophagostomum venulosum. Efficacy against these two species was 93.6% and 94.0%, respectively, at a dose of 2.5mg/kg. At this dose, efficacy was above 99.2% against nine other nematode species including Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp. and Nematodirus spp. It was concluded that 2.5mg/kg would be a suitable dose rate for a commercial product.

Background: Schistosomiasis is the second most common parasitic disease after malaria. Imported cases of schistosomiasis are reported in nonendemic regions due to an increased number of travelers to affected geographical areas. The present case report aimed to emphasize the importance of a correct history of a patient, including information regarding travel history and its consequences, and to overview the evolution of a tropical disease diagnosed rarely in Romania., Materials and Methods: Retrospective analysis of a single case of imported schistosomiasis diagnosed in Timisoara, Romania., Result: A 25-year old male patient had traveled and worked abroad for a long period of time in tropical areas in Asia and South America (Brazil). The clinical pattern at admission to hospital included fever and diarrhea with multiple liquid stools with mucus. Physical examination was normal. Routine laboratory tests revealed an increased number of leucocytes., Conclusions: Final diagnosis was established with difficulty in this case. Failure to associate a febrile prolonged syndrome or chronic diarrhea with a history of travel can cause a delayed diagnosis.

Fasciola hepatica infection causes significant clinical disease in ruminants. Current control methods, based on flukicidal drugs, are becoming less useful because of resistance in fluke populations. Vaccination would be a viable alternative, but as yet no vaccine to protect ruminants against liver fluke infection has been commercialised. Adjuvants can be used to enhance and promote protective immune responses by vaccines. In previous vaccination trials, we have observed a distinct adjuvant effect, or a degree of protection, in animals administered adjuvant alone in the absence of any specific F. hepatica antigen. Understanding this effect will be important for continuing efforts to develop vaccines effective against fasciolosis. This study investigated the effects of three adjuvants (Quil A, Freund's Incomplete and TiterMax Gold) on the course of experimental F. hepatica infection in 6-month-old sheep (n=33). At completion of the trial, all animals were necropsied to determine fluke burden and fluke weight. Quil A administration led to a significant reduction in faecal egg count (P<0.0001) and significantly higher parasite-specific serum antibody activity for all isotypes measured (P<0.01). This suggests that Quil A, which promotes a Th1 response, may be useful as an adjuvant in anti-Fasciola vaccines. Furthermore, it reinforces the results of our previous studies indicating that enhanced Th1 responsiveness to vaccine antigens is required to achieve protection against challenge by F. hepatica.

The nutritional cost of, and the sequential cellular changes associated with the developing immune response to the abomasal parasite Teladorsagia circumcincta were investigated using corticosteroid-induced immune-suppression. Six-month-old lambs with minimal nematode experience were either infected with 4000 L3 T. circumcincta per day (group IF), similarly infected and concurrently immune-suppressed with methylprednisolone acetate (group ISIF), immune-suppressed only (group IS) or remained as controls (group C). Food intake, faecal egg count (FEC) and antibody titres in plasma were recorded weekly, worm burden at necropsy on day 63 p.i. and body composition by X-ray computed tomography on days -2 and 62 p.i. Furthermore, sequential immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from additional animals that were fitted with an abomasal cannula and either infected with the same regime as IF animals above (group CnIF) or concurrently infected and immune-suppressed as above (group CnISIF). Corticosteroid treatment resulted in greater FECs (P<0.01) and worm burdens (P<0.01) in both ISIF and CnISIF compared with IF and CnIF sheep, respectively. Infection reduced feed intake by 17% between 14 and 28 days p.i. (P<0.05) and efficiency of energy utilisation by 20% (P=0.07) in IF animals but not in ISIF animals. Mast cells, globule leukocytes and IgA in tissue biopsy samples were elevated in CnIF from 42 days p.i., all of which were abrogated by corticosteroid treatment. The ability to regulate the worm population appeared to be associated with a rise in tissue IgA concentration and numbers of globule leucocytes (GL). The results support the hypothesis that a majority of the production losses that occur during infection of lambs with T. circumcincta in lambs are a consequence of the host immune response. These findings may have implications for regimes that promote the development of a strong host immune reaction to gastrointestinal parasites in lambs.

The inheritance of resistance to louse infestation and the related allergic skin disease, cockle, was examined in Romney lambs. The lambs used in the study were the 2001- and 2004-born progeny of four experimental breeding lines ("Resistant", "Susceptible", "Resilient" and "Control") developed as part of a long-term study of the genetics of host resistance (maintenance of low faecal egg count (FEC) under nematode challenge) or resilience (maintenance of health and productivity under nematode challenge irrespective of FEC) to nematode parasites in sheep. Between 13 and 22 progeny (equally distributed between males and females, where possible) from each of five sires in each line were selected each year for this trial. All lambs (n=701) were examined for lice (Bovicola ovis) before artificial infestation; in 2001 the lambs were free of natural infestation, whilst in 2004 naturally acquired infestation was evident. In November 2001 and May 2002, approximately 60 B. ovis were transferred to each lamb, followed by monitoring at approximately 2-monthly intervals until August 2002. Similar procedures, but with fewer monitoring times, were repeated on the 2004 lambs. Overall, lambs in the Control line were significantly more susceptible to louse infestation and cockle compared with those in the other three lines (P<0.001). Least squares-means (SEM) of log-transformed louse score for the control, resistant, susceptible and resilient lines, respectively, were 2.178 (0.045), 1.499 (0.050), 1.618 (0.050) and 1.587 (0.044), and for cockle score were 1.36 (0.05), 0.76 (0.05), 0.95 (0.05) and 0.78 (0.05). From all progeny together, the heritability of log-transformed louse score was 0.22 (Standard Error (SE) 0.06) in autumn and 0.34 (SE 0.08) in winter, with a value of 0.44 (SE 0.09) when these data were combined. These estimates were similar to those obtained for resistance to gastro-intestinal nematodes in these breeding lines, using log-transformed FECs. Heritability estimates for cockle score in autumn, winter and when combined were 0.06 (SE 0.04), 0.45 (SE 0.09) and 0.40 (SE 0.09), respectively. The genetic correlations of mean log-transformed louse score with mean cockle score and levels of two different louse antigens in wool were, respectively, 0.97 (SE 0.04), 0.96 (SE 0.08) and 0.95 (SE 0.09). However, there was no significant genetic correlation between louse scores and FEC. These results suggest that selective breeding would be effective in reducing louse infestation and cockle in sheep, but that differences in louse burdens were not related to differences in nematode burdens as indicated by FECs.

Ivermectin and moxidectin are closely related avermectin/milbemycin anthelmintics and available data suggest that side resistance occurs with these two drugs. However, moxidectin remains effective against many species of ivermectin-resistant worms due to its higher potency. The larval development assay (LDA) is routinely used to diagnose ivermectin resistance in Haemonchus contortus but laboratory diagnosis of moxidectin resistance is hampered by the lack of any validated in vitro tests. The objective of this study was to measure the relative susceptibility/resistance of H. contortus to moxidectin on goat farms in Georgia, and to validate the DrenchRite LDA for detecting resistance to moxidectin. Fecal egg count reduction tests (FECRT) were performed at five different moxidectin dose levels and DrenchRite LDAs were performed in duplicate on nine meat goat farms in Georgia, USA. To improve our ability to make inferences on the relative levels of resistance between farms, FECRT data were first analysed using a linear mixed model, and then Tukey's sequential trend test was used to evaluate the trend in response across dose levels. LDA data were analysed using log-dose logit-response and probit models. Using these statistical results, we were able to rank the nine farms from the least to the most resistant, and to develop a set of criteria for interpreting DrenchRite LDA results so that this assay can be used to diagnose both clinically apparent moxidectin resistance, as well as sub-clinical emerging resistance. These results suggest that our novel approach for examining these types of data provides a method for obtaining an increased amount of information, thus permitting a more sensitive detection of resistance. Based on results of the LDA, moxidectin-resistant farms had resistance ratios, compared with an ivermectin-sensitive farm, ranging from 32 to 128, and had resistance ratios of 6-24 compared with an ivermectin-resistant/moxidectin naive farm. Moxidectin resistance was diagnosed both in Haemonchus and Trichostrongylus on almost half of the farms tested, despite this drug only being used on these farms for 2-3 years.

Bovine abortions due to Neospora caninum infection are a major cattle-production problem worldwide. The parasite is readily maintained in cattle populations by vertical transmission. The domestic dog excretes oocysts in its feces and, after sporulation, these oocysts are infectious to cattle. Current control measures are aimed at culling infected cows and limiting the access of cattle to infective oocysts. The recent revelations that coyotes (Canis latrans) can excrete N. caninum oocysts in their feces and that white-tailed deer (Odocoileus virginianus) are natural intermediate hosts of the parasite demonstrate the existence of a sylvatic cycle of neosporosis in North America. This complicates parasite-prevention programs but opens many new and exciting avenues of research. Similar canid-ruminant sylvatic cycles might exist in other countries and, if so, need to be investigated.

The gastrointestinal nematode Ostertagia gruehneri is a parasite of reindeer that can have a significant impact on host population dynamics. To gain a better understanding of the population dynamics of O. gruehneri, we parameterise a model for its fecundity that describes the observed seasonal and intensity dependent pattern of faecal egg counts well. The faecal egg count model is combined with a model for the seasonal faecal production rate of Svalbard reindeer to obtain quantitative estimates of the fecundity of O. gruehneri. The model is used to evaluate the relative contribution to pasture contamination of variation in the abundance of O. gruehneri and variation in reindeer densities. It is concluded that due to the intensity dependence in nematode fecundity, variation in reindeer population densities is likely to be the most important of these factors for pasture contamination.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding São Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.

Within-host mathematical models of Eimeria maxima and Eimeria praecox infections of the chicken are presented and used to investigate the role of host cell availability as a possible determinant of the so-called 'crowding effect'; whereby the fecundity of the parasites decreases as infectious dose increases. Assumptions about the number of available host cells, the average lifespan of these cells and the age structure within the host-cell population were made and mathematical models were constructed and combined with experimental data to test whether these conditions could reproduce the crowding effect in the two species. Experimental data demonstrated that crowding during in vivo infections was apparent following very low infectious doses, but none of the models could adequately reproduce crowding at the same doses while maintaining realistic estimates of the dynamics of the enterocyte pool. However, both the size and lifespan of the enterocyte pool were demonstrated to have substantial effects on the fecundity of the infections, particularly at higher doses. These data indicate that host cell availability cannot be solely responsible for the crowding effect. Alternative factors such as the influence of the primary immune response to the parasite may also be explored using within-host models and other applications of these models are discussed.

The 'crowding effect' in avian coccidia, following administration of graded numbers of sporulated oocysts to naïve hosts, is recognisable by two characteristics. First, increasing doses of oocysts give rise to progressively higher oocyst yields, until a level of infection is reached (the 'maximally producing dose') above which further dose increases result in progressive decreases in oocyst yields. Second, the number of oocysts produced per oocyst administered (the 'reproductive potential') tends to decrease as the oocyst dose is increased. The dose that gives the maximal reproductive potential is the 'crowding threshold' and doses exceeding this are 'crowded doses'. Graded doses of Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox or Eimeria tenella were given to chickens of the same breed, sex and age, reared on the same diet, under identical management. The two characteristics of the crowding effect were demonstrated graphically and, by interpolation, the estimated crowding thresholds were 903, < or =16, 39, < or =14, < or =16, < or =16 or 72 sporulated oocysts, respectively, for the seven Eimeria species enumerated above. This is apparently the first report of definitive experiments to quantify a crowding effect in E. brunetti, E. maxima, E. mitis, E. necatrix and E. praecox. Maximum experimental reproductive potentials were considerably lower than the theoretical reproductive potentials for all seven species. The interaction between availability of host intestinal cells and immunity contributing to the crowding effect is discussed. Standard curves obtained under specified conditions should be used to estimate appropriate infective doses for experimental designs or in vivo production of oocyst stocks. For experiments on effects of chemotherapy or immunisation on oocyst production, an infective dose lower than the crowding threshold should be used. For efficient production of laboratory or factory oocyst stocks, the maximally producing dose (which is greater than the crowding threshold), should be used.

Following the unexpected activity of the excipient beta-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of beta-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with beta-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. beta-cyclodextrin was well tolerated by all of the treated animals.

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.

Naive wethers were used to investigate the long-term effects of dietary condensed tannins from Quebracho extract, during an intestinal parasitic infection in sheep. Sheep were allocated to eight groups; seven groups were daily infected with 3000 L(3) Trichostrongylus colubriformis for 10 weeks and the eighth group was the uninfected control. The 10-week experiment was divided into two periods; Period 1 (P(1), week 1-5) corresponded to high worm establishment and acquisition of immunity, whereas Period 2 (P(2), week 6-10) to the established worm population and expression of host immunity. Three experimental foods with similar composition were formulated: Q0, Q3 and Q6. Their difference was in the content of Quebracho extract which was 0, 30 and 60 g per kg fresh matter, respectively. All foods were offered at an allowance of 3.5% of sheep liveweight. During P(1), parasitised sheep were offered one of the three experimental foods and during P(2) they either remained on the same food or changed food according to the design (P(1)-P(2)): Q0-Q0, Q0-Q3, Q0-Q6, Q3-Q0, Q3-Q3, Q6-Q0, Q6-Q6. Control sheep were offered the allowance of Q0 throughout. Sheep that consumed Q3 and Q6 reduced their faecal egg counts (FEC) compared to sheep offered Q0, during both periods (P<0.05). No differences were observed in the FEC between sheep offered Q3 and Q6. The changeover from Q0 in P(1) to either Q3 or Q6 during P(2), was accompanied by a reduction in FEC (P<0.05), whereas an increase in FEC was observed when food changed from Q3 or Q6 to Q0 (P<0.05). Worm burdens and fecundity at the end of the experiment were reduced in sheep offered foods Q3 and Q6 compared to sheep offered Q0. A significant decrease in liveweight gain and in food conversion efficiency of parasitised sheep offered Q3 and Q6 compared to sheep offered Q0, was observed in P(1) (P<0.05) but not in P(2). By the end of the experiment control sheep had achieved higher liveweight and converted food more efficiently than parasitised sheep (P<0.05). In conclusion, evidence for a long-term effect of Quebracho extract, during both the initial establishment and on the established T. colubriformis population in sheep, was provided by the present study. It is suggested that the effect observed was a direct anthelmintic effect of the condensed tannins included in sheep diets.

The study reported here investigated acquired resistance of mice and pigs to challenge-infections with Schistosoma japonicum. Two morphologically indistinguishable isolates of the parasite (from the Anhui and Zhejiang provinces of China), which could be typed by polymerase chain reaction-linked restriction fragment length polymorphism analysis (PCR-RFLP), were used for the infections. In two parallel infection studies, 60 female outbred NMRI mice and 29 Danish Landrace/Yorkshire/Duroc crossbred pigs were used. Two of the groups received a primary infection with either the Anhui or the Zhejiang isolate, respectively. The remaining groups received a primary infection with the Zhejiang isolate and challenge-infections with the Anhui isolate at either week 2, 3, 4 or 6 post primary infection. The results of the study indicated that both mice and pigs are partially resistant to challenge-infection from week 4 post primary infection. Resistance appeared to decrease in pigs 6 weeks after primary infection, while it remained effective in mice. These results suggest that the mechanism responsible for acquired resistance in mice and pigs may not be the same and support the theory that worm burdens in pigs receiving repeated infection are in a balance between acquisition and loss of worms.

The aim of this research was to determine whether metabolisable protein supply during the early period of infection with Trichostrongylus colubriformis influenced resilience and the later stages in the development and magnitude of host resistance in previously nematode-naïve lambs. Eighty TexelxGreyface lambs were fed pelleted feeds calculated to provide grossly different amounts of metabolisable protein. Sixty of the lambs received a trickle infection of T. colubriformis and 20 lambs were kept as uninfected controls. There were four initial groups, namely infected or uninfected and fed either a moderate or a high protein feed. After 5weeks of infection, a further four groups were established by changing the feed of half of the animals fed the moderate protein feed to the high protein feed and of half of the animals fed high protein to the moderate protein feed. Live weight gain and feed conversion ratio were greatest for lambs fed the high protein feed and were reduced by infection. Faecal egg counts, worm burdens and per capita fecundity of adult female nematodes were unaffected by changes to metabolisable protein supply. Decreasing metabolisable protein supply following 5weeks of infection reduced live weight gain without any effect on resistance to T. colubriformis. Haematological variables, indicative of improved resistance, were also largely unaffected by metabolisable protein supply. It is concluded that the requirements of immune function probably had priority over those of growth and that the metabolisable protein supply provided by the moderate protein feed was sufficient to account for the requirements for the expression of immunity. It is probable that the potential for metabolisable protein supply to enhance resistance to infection from T. colubriformis is dependent on the levels and magnitude (i.e. in relation to maintenance requirements) of metabolisable protein supply being compared and the demand of other competing physiological functions.

Serum gastrin and pepsinogen concentrations, food intake, abomasal pH and abomasal aerotolerant and anaerobic bacterial populations were measured in sheep infected with Ostertagia circumcincta to search for links between hypergastrinaemia, food intake and changes in the abomasal environment. Abomasal pH and serum gastrin and pepsinogen concentrations were elevated in each of five sheep infected via abomasal cannulae with 150000 exsheathed larval stage three, followed 11 days later by 100000 sheathed larvae given intraruminally. Unparasitised abomasa contained aerotolerant bacterial population densities of between 10(3) and 10(6) cells ml(-1) and these did not change significantly following parasitism. In contrast, anaerobic bacterial population densities increased markedly by about 10(4)-fold following parasitism. Anaerobic numbers changed rapidly when abomasal pH increased from 2.5 to 3.5. At pH 4 and above, anaerobic bacterial numbers approached levels expected in rumen contents but parameters other than pH did not relate to bacterial numbers. Brief periods when serum gastrin was lower than expected, coinciding with raised abomasal pH, were not explicable by increased bacterial numbers. Food intake, which decreased for a variable period from around Day 5 p.i., correlated poorly with serum gastrin concentration, suggesting hypergastrinaemia is not the sole cause of anorexia in parasitised animals. The survival of substantial numbers of rumen bacteria in the abomasum at only slightly raised pH may significantly lower the bacterial protein available to the sheep.

Two morphologically marked strains of Haemonchus contortus, CAVRS (smooth-macrocyclic lactone resistant) and McMaster (linguiform-macrocyclic lactone susceptible), were used to investigate the selection for anthelmintic resistance following exposure to ivermectin (IVM), a non-persistent anthelmintic. and a more persistent anthelmintic, oral moxidectin (MOX). Three types of selection were investigated: (1) selection of resident worms at the time of treatment (Head selection); (2) selection of incoming-larvae post-treatment (Tail selection); and (3) selection of both resident population and incoming larvae (Head + Tail selection). The experimental animals were adult sheep and lambs. In the controls where there was no anthelmintic selection, the proportion of CAVRS in the adult worm population was the same as the proportion in larvae given to both adults and lambs indicating that CAVRS and McMaster H. contortus were equally infective. There was a significant effect of anthelmintic on total worm numbers in adult sheep with MOX treated adults having less worms, but selection type was non-significant. Anthelmintic type had a significant effect on numbers of resistant worms in adult sheep with less resistant worms in the MOX treated groups, but selection type had no effect. Analysis of variance of arcsine-transformed proportions of resistant worms found that the type of anthelmintic had a highly significant effect, with MOX treated adults having a higher proportion of resistant worms, while type of selection was not significant. In the lambs, nil treated controls and IVM Head + Tail and Tail selected groups had similar geometric mean total worm burdens while Head selected had less total worms. In the MOX treated lamb groups the worm burdens were similar within selection type but less than the IVM treated groups. In the lambs, the types of selection that resulted in more resistant worms were IVM Tail, MOX Head + Tail and MOX Tail. Resistant worm numbers were similar in both adult and lamb groups with Head selection by either MOX or IVM. Moxidectin selected out higher proportions of resistant worms than did IVM in the lambs, with Tail and Head + Tail being stronger selectors than Head. Computer simulations were used to estimate the rate at which resistance developed in the field using the information generated in the present study. The anthelmintic treatments used in the simulation followed a strategic parasite control program for H. contortus in which all sheep receive three Closantel (CLS) treatments in summer. all sheep receive a broad-spectrum (BS) drench or capsule at weaning and lambs receive an additional two BS drenches insummer or no further treatment in the case of the capsule. Moxidectin, IVM-capsule and IVM were the broad spectrum anthelmintics simulated. All simulations were run four times assuming high or low efficacy against resident resistant worms and in the presence or absence of CLS resistance. The simulations indicated that the presence of CLS resistance hastened selection for macrocyclic lactone (ML) resistance. While the IVM-capsule will select most rapidly for ML resistance, IVM oral is expected to be least selective. Moxidectin treatment is intermediate, except in simulations with no CLS resistance and when MOX is assumed to be highly effective against resident ML-resistant worms, in which case MOX can be expected to select more slowly than IVM oral treatments.

Experimental vaccine trials against hydatid disease have been undertaken in sheep using the EG95 recombinant vaccine. Challenge infection was with viable Echinococcus granulosus eggs obtained from a New Zealand isolate (dog/sheep cycle), an Australian isolate (dingo/wallaby cycle) and an Argentine isolate (dog/sheep cycle). Vaccination with EG95 conferred a high degree of protection against challenge with all three parasite isolates (protection range 96-100%). Taken together, the trials demonstrated that 86% of vaccinated sheep were completely free of viable hydatid cysts when examined approximately 1 year after challenge infection. Vaccination reduced the number of viable cysts by 99.3% compared with unvaccinated controls. These results suggest that the EG95 vaccine could have wide applicability as a new tool for use in hydatid control campaigns.

The mammary glands of 103 pasture-reared non-lactating, non-pregnant Merino ewes were infused via the teat canal with antigens prepared from the nematode Haemonchus contortus, and the inflammatory response to infusion assessed by washing the gland of its contents after 24 h and 14 days. The ewes were of two genotypes: one with proven high levels of resistance to infection with the nematode H. contortus, the other random-bred animals with relative susceptibility to infection. On day 0 of a H. contortus infection, one gland of the subgroups of both genotypes was infused with the antigen preparation. At the same time, the other gland of the random-bred ewes was infused with sterile physiological saline. A third group of infected random-bred ewes was infused with only sterile physiological saline. Similar infusions were performed on other subgroups on days 12, 21 and 35 of infection, which was then terminated with anthelmintic. A fourth group of uninfected random-bred control ewes was given both infusions 35 days after the other groups were infected. Sheep of the resistant genotype had lower worm egg counts and smaller reductions in blood packed cell volumes from day 21 of infection. Infusion of antigen had no effect on the course of infection and no effect on the response of the other gland, which had been infused with saline alone. The dominant leukocyte response from the antigen-infused gland was eosinophilia. On all days of infusion, and after both 24 h and 14 days, eosinophil counts from the resistant genotype were higher than those from their random-bred counterparts. The sheep mammary gland provides a source of eosinophils whose number is related to host genotype and stage of infection and may provide a model for the investigation of cellular responses in mucosal immunity to nematode infections.

Weaner sheep that had been hand-fed on diets containing increasing concentrations of protein for a 9-week period (when uninfected, or infected with Haemonchus contortus) were studied during the next 69 weeks when put on to pasture as a single, unsupplemented flock. During the 9-week period, groups of 12 sheep (six infected, six uninfected) were offered one of five iso-energetic (9.0 MJ kg(-1)) diets containing 10, 13, 16, 19 or 22% crude protein. All sheep were treated with anthelmintic at the end of the 9 weeks and then put out to pasture for 69 weeks, where they were all subject to the same environmental variables including nematode larval challenge. During the grazing period, animals that had previously received the higher protein diets consistently had higher live-weight gain and wool production, higher antibody responses to both H. contortus and Trichostrongylus colubriformis antigenic challenge in vitro, and lower faecal nematode egg counts than did the lambs previously offered the lower protein diets. Faecal egg counts of the grazing sheep that had been artificially infected with H. contortus while being hand-fed were similar to those of the uninfected sheep and there was no interaction between previous infection and dietary protein concentration. We conclude that short periods of enhanced post-weaning nutrition can have long-term and perhaps life-long effects on production.

Variation between hosts of different ages and genotypes in the developmental success of trichostrongylid parasite eggs from sheep was investigated in two trials. The percentage development to infective third-stage larvae of eggs collected from lambs and adult ewes infected with Ostertagia circumcincta was compared in an indoor trial. In addition, sheep previously bred for either high or low faecal egg count and grazed outdoors on parasite contaminated pasture were sampled; egg development, faecal egg count, generic profile and faecal dry matter were measured. In both trials, development to L3 was significantly lower in eggs derived from adult ewes than from lambs and, in the field trial, from animals selected for low faecal egg count. The observed differences could not be accounted for by variations in faecal egg count, faecal dry matter content nor by differences in the generic composition of worm egg output. Although not shown conclusively, the results are consistent with an immune mechanism influencing the development of free-living stages of gastrointestinal nematodes outside the host. These results have significant implications for our understanding of parasite epidemiology, particularly as it relates to sources of pasture contamination.

The objectives of this work were to clone and express Chinese strain Schistosoma japonicum antigens and evaluate their immunogenicity and protective efficacy in the natural ovine host in China. Recombinant antigens selected for testing were: isoforms of glutathione S-transferase Sj28GST and Sj26GST; the large hydrophilic domain of Sj23, the homologue of the protective S. mansoni membrane antigen Sm23; and a 3' fragment of S. japonicum paramyosin. In addition, Chinese strain S. japonicum native paramyosin and GST were purified and used for vaccination. Antigens were co-administered with Freund's adjuvants or BCG. We also examined the effects of co-administration of native unfractionated GSTs with keyhole limpet haemocyanin (KLH), which shares a cross-reactive protective epitope with schistosomes. These are the first side-by-side comparisons of candidate defined-antigen schistosomiasis vaccines in a natural host. Significant partial protection was obtained with each of the antigens tested. Less protection was obtained with a recombinant fragment of S. japonicum paramyosin compared with native paramyosin. Co-administration of native GST and KLH was no more effective than vaccination with either antigen alone. Although encouraging levels of protection against S. japonicum were demonstrated using each of these antigens, further work is needed to optimise vaccine delivery and vaccination schedules.

The ability of young Merino lambs to achieve protective immunity following vaccination via viable nematode infections was assessed. Lambs were infected from 1 month of age by repeated continuous low dose (trickle) administration of Trichostrongylus colubriformis or Haemonchus contortus infective larvae (L3), or by truncated infections with high doses of viable T. colubriformis L3. After 7 weeks all groups were drenched with anthelmintic and at 3 months of age they were re-infected with the homologous species. Protection was assessed by faecal egg counts at 3, 4, 5, 6 and 7 weeks after challenge, and worm count at 7 weeks after challenge. Young lambs were partially protected by 3 months of age against Trichostrongylus by trickle infection. This protection correlated with local mast cell and T-cell priming, increased numbers of local antigen-presenting cells and T-cells and increased worm-specific antibody titres in the intestine. However, there was no evidence that young lambs were capable of immunologically recognising H. contortus antigens following trickle infection, nor did trickle infection significantly protect young lambs against Haemonchus challenge.