Investigating the perceived versus actual gastrointestinal nematode challenge on extensive sheep farms.

Extensive farming systems form an integral part of sheep production systems across Europe. However, with innate production handicaps, declining sheep numbers and narrow economic margins, production is becoming increasingly challenging threatening the future sustainability of the industry. Gastrointestinal nematodes (GINs) are a significant cause of production losses to the global sheep industry, with well-established resistance to the major anthelmintic groups. Traditionally, extensive farming systems are not thought to have a significant parasite challenge compared with intensive farms, but there is a need to identify the scale and importance of GINs on extensive farms to inform the need for sustainable control strategies. In this study, a questionnaire of extensive farmers (n=34) was conducted and parasitological data were collected from nine study farms to investigate the perceived versus actual GIN and anthelmintic resistance challenge faced by extensive farms. The results showed a production-limiting challenge on most farms, with a higher GIN challenge observed on improved pastures. Furthermore, over half of the extensive farmers perceived anthelmintic resistance to be a greater problem for intensive farmers, with only 20% of respondents reporting known anthelmintic resistance. However, all study farms had evidence of resistance to at least one group of anthelmintics. Consequently, this study has demonstrated that despite the traditional perception of parasitism on extensive farms, there is a need to increasingly consider its impact and take a proactive approach to sustainable control, with solutions tailored to their unique management.

Long-term temporal trends in gastrointestinal parasite infection in wild Soay sheep.

Monitoring the prevalence and abundance of parasites over time is important for addressing their potential impact on host life histories, immunological profiles and their influence as a selective force. Only long-term ecological studies have the potential to shed light on both the temporal trends in infection prevalence and abundance and the drivers of such trends, because of their ability to dissect drivers that may be confounded over shorter time scales. Despite this, only a relatively small number of such studies exist. Here, we analysed changes in the prevalence and abundance of gastrointestinal parasites in the wild Soay sheep population of St. Kilda across 31 years. The host population density (PD) has increased across the study, and PD is known to increase parasite transmission, but we found that PD and year explained temporal variation in parasite prevalence and abundance independently. Prevalence of both strongyle nematodes and coccidian microparasites increased during the study, and this effect varied between lambs, yearlings and adults. Meanwhile, abundance of strongyles was more strongly linked to host PD than to temporal (yearly) dynamics, while abundance of coccidia showed a strong temporal trend without any influence of PD. Strikingly, coccidian abundance increased 3-fold across the course of the study in lambs, while increases in yearlings and adults were negligible. Our decades-long, intensive, individual-based study will enable the role of environmental change and selection pressures in driving these dynamics to be determined, potentially providing unparalleled insight into the drivers of temporal variation in parasite dynamics in the wild.

Helminth parasites are associated with reduced survival probability in young red deer.

Helminths are common parasites of wild ungulates that can have substantial costs for growth, mortality and reproduction. Whilst these costs are relatively well documented for mature animals, knowledge of helminths' impacts on juveniles is more limited. Identifying these effects is important because young individuals are often heavily infected, and juvenile mortality is a key process regulating wild populations. Here, we investigated associations between helminth infection and overwinter survival in juvenile wild red deer (Cervus elaphus) on the Isle of Rum, Scotland. We collected fecal samples non-invasively from known individuals and used them to count propagules of 3 helminth taxa (strongyle nematodes, Fasciola hepatica and Elaphostrongylus cervi). Using generalized linear models, we investigated associations between parasite counts and overwinter survival for calves and yearlings. Strongyles were associated with reduced survival in both age classes, and F. hepatica was associated with reduced survival in yearlings, whilst E. cervi infection showed no association with survival in either age class. This study provides observational evidence for fitness costs of helminth infection in juveniles of a wild mammal, and suggests that these parasites could play a role in regulating population dynamics.

Longitudinal dynamics of co-infecting gastrointestinal parasites in a wild sheep population.

Within-year variation in infection is a ubiquitous feature of natural populations, but is determined by a complex interplay of environmental, parasitological and host factors. At the same time, co-infection is the norm in the wild. Longitudinal dynamics of co-infecting parasites may therefore be further complicated by covariation across multiple parasites. Here, we used fecal parasite egg and oocyst counts collected repeatedly from individually marked wild Soay sheep to investigate seasonal dynamics of six gastrointestinal parasite groups. Prevalence and abundance tended to be higher in spring and summer, and abundance was higher in lambs compared to adults. We found that within-year variation in highly prevalent strongyle nematode counts was dependent on adult reproductive status, where reproductive ewes had distinct dynamics compared to males and barren ewes. For similarly prevalent coccidia we found an overall peak in oocyst counts in spring but no differences among males, barren and pregnant ewes. Using multivariate mixed-effects models, we further show that apparent positive correlation between strongyle and coccidia counts was driven by short-term within-individual changes in both counts rather than long-term among-individual covariation. Overall, these results demonstrate that seasonality varies across demographic and parasite groups and highlight the value of investigating co-infection dynamics over time.

Functionally distinct T-helper cell phenotypes predict resistance to different types of parasites in a wild mammal.

The adaptive immune system is critical to an effective response to infection in vertebrates, with T-helper (Th) cells pivotal in orchestrating these responses. In natural populations where co-infections are the norm, different Th responses are likely to play an important role in maintaining host health and fitness, a relationship which remains poorly understood in wild animals. In this study, we characterised variation in functionally distinct Th responses in a wild population of Soay sheep by enumerating cells expressing Th-subset specific transcription factors and quantifying Th-associated cytokines. We tested the prediction that raised Th1 and Th2 responses should predict reduced apicomplexan and helminth parasite burdens, respectively. All measures of Th-associated cytokine production increased with age, while Th17- and regulatory Th-associated cytokine production increased more rapidly with age in males than females. Independent of age, sex, and each other, IL-4 and Gata3 negatively predicted gastro-intestinal nematode faecal egg count, while IFN-γ negatively predicted coccidian faecal oocyst count. Our results provide important support from outside the laboratory that Th1 and Th2 responses predict resistance to different kinds of parasites, and illustrate how harnessing specific reagents and tools from laboratory immunology will illuminate our understanding of host-parasite interactions in the wild.

Fitness Costs of Parasites Explain Multiple Life-History Trade-Offs in a Wild Mammal.

AbstractReproduction in wild animals can divert limited resources away from immune defense, resulting in increased parasite burdens. A long-standing prediction of life-history theory states that these parasites can harm the reproductive individual, reducing its subsequent survival and fecundity, producing reproduction-fitness trade-offs. Here, we examined associations among reproductive allocation, immunity, parasitism, and subsequent survival and fecundity in a wild population of individually identified red deer (Cervus elaphus). Using path analysis, we investigated whether costs of lactation in terms of downstream survival and fecundity were mediated by changes in strongyle nematode count and mucosal antibody levels. Lactating females exhibited increased parasite counts, which were in turn associated with substantially decreased fitness in the following year in terms of overwinter survival, fecundity, subsequent calf weight, and parturition date. This study offers observational evidence for parasite regulation of multiple life-history trade-offs, supporting the role of parasites as an important mediating factor in wild mammal populations.

Refugia-Based Strategies for Parasite Control in Livestock.

Refugia-based strategies are intended to help slow the development of anthelmintic resistance by providing a population of parasites that are not exposed to the treatment. Evidence from field studies is lacking. There is no single way to incorporate refugia into a parasite control program. There are many options available varying greatly in complexity and practicality, and none are suitable for all situations. Incorporating refugia into production systems typically requires a change in farmer mindset and greater input of time, labor, and/or technology, but is necessary to help preserve anthelmintic efficacy and promote sustainable animal production systems.

Refugia and anthelmintic resistance: Concepts and challenges.

Anthelmintic resistance is a threat to global food security. In order to alleviate the selection pressure for resistance and maintain drug efficacy, management strategies increasingly aim to preserve a proportion of the parasite population in 'refugia', unexposed to treatment. While persuasive in its logic, and widely advocated as best practice, evidence for the ability of refugia-based approaches to slow the development of drug resistance in parasitic helminths is currently limited. Moreover, the conditions needed for refugia to work, or how transferable those are between parasite-host systems, are not known. This review, born of an international workshop, seeks to deconstruct the concept of refugia and examine its assumptions and applicability in different situations. We conclude that factors potentially important to refugia, such as the fitness cost of drug resistance, the degree of mixing between parasite sub-populations selected through treatment or not, and the impact of parasite life-history, genetics and environment on the population dynamics of resistance, vary widely between systems. The success of attempts to generate refugia to limit anthelmintic drug resistance are therefore likely to be highly dependent on the system in hand. Additional research is needed on the concept of refugia and the underlying principles for its application across systems, as well as empirical studies within systems that prove and optimise its usefulness.

The Fine-Scale Landscape of Immunity and Parasitism in a Wild Ungulate Population.

Spatial heterogeneity in susceptibility and exposure to parasites is a common source of confounding variation in disease ecology studies. However, it is not known whether spatial autocorrelation acts on immunity at small scales, within wild animal populations, and whether this predicts spatial patterns in infection. Here we used a well-mixed wild population of individually recognized red deer (Cervus elaphus) inhabiting a heterogeneous landscape to investigate fine-scale spatial patterns of immunity and parasitism. We noninvasively collected 842 fecal samples from 141 females with known ranging behavior over 2 years. We quantified total and helminth-specific mucosal antibodies and counted propagules of three gastrointestinal helminth taxa. These data were analyzed with linear mixed models using the Integrated Nested Laplace Approximation, using a Stochastic Partial Differentiation Equation approach to control for and quantify spatial autocorrelation. We also investigated whether spatial patterns of immunity and parasitism changed seasonally. We discovered substantial spatial heterogeneity in general and helminth-specific antibody levels and parasitism with two helminth taxa, all of which exhibited contrasting seasonal variation in their spatial patterns. Notably, Fasciola hepatica intensity appeared to be strongly influenced by the presence of wet grazing areas, and antibody hotspots did not correlate with distributions of any parasites. Our results suggest that spatial heterogeneity may be an important factor affecting immunity and parasitism in a wide range of study systems. We discuss these findings with regards to the design of sampling regimes and public health interventions, and suggest that disease ecology studies investigate spatial heterogeneity more regularly to enhance their results, even when examining small geographic areas.

100 Questions in Livestock Helminthology Research.

An elicitation exercise was conducted to collect and identify pressing questions concerning the study of helminths in livestock, to help guide research priorities. Questions were invited from the research community in an inclusive way. Of 385 questions submitted, 100 were chosen by online vote, with priority given to open questions in important areas that are specific enough to permit investigation within a focused project or programme of research. The final list of questions was divided into ten themes. We present the questions and set them briefly in the context of the current state of knowledge. Although subjective, the results provide a snapshot of current concerns and perceived priorities in the field of livestock helminthology, and we hope that they will stimulate ongoing or new research efforts.

Seasonality of helminth infection in wild red deer varies between individuals and between parasite taxa.

Parasitism in wild mammals can vary according to myriad intrinsic and extrinsic factors, many of which vary seasonally. However, seasonal variation in parasitism is rarely studied using repeated samples from known individuals. Here we used a wild population of individually recognized red deer (Cervus elaphus) on the Isle of Rum to quantify seasonality and intrinsic factors affecting gastrointestinal helminth parasitism over the course of a year. We collected 1020 non-invasive faecal samples from 328 known individuals which we then analysed for propagules of three helminth taxa: strongyle nematodes, the common liver fluke Fasciola hepatica and the tissue nematode Elaphostrongylus cervi. Zero-inflated Poisson models were used to investigate how season, age and sex were associated with parasite prevalence and count intensity, while Poisson models were used to quantify individual repeatability within and between sampling seasons. Parasite intensity and prevalence varied according to all investigated factors, with opposing seasonality, age profiles and sex biases between parasite taxa. Repeatability was moderate, decreased between seasons and varied between parasites; both F. hepatica and E. cervi showed significant between-season repeatability, while strongyle nematode counts were only repeatable within-season and showed no repeatability within individuals across the year.

P-glycoprotein-9 and macrocyclic lactone resistance status in selected strains of the ovine gastrointestinal nematode, Teladorsagia circumcincta.

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.

Worm Control in Livestock: Bringing Science to the Field.

Parasitic roundworm infections are ubiquitous in grazing livestock. Chemical control through the frequent 'blanket' administration of anthelmintics (wormers) has been, and remains, the cornerstone in controlling these infections, but this practice is unsustainable. Alternative strategies are available but, even with the plethora of best practice advice available, have yet to be integrated into routine farming practice. This is probably due to a range of factors, including contradictory advice from different sources, changes to advice following increased scientific understanding, and top-down knowledge exchange patterns. In this article, we discuss the worm control options available, the translation of new best practice advice from science bench to field, and ideas for future work and directions.

Faecal egg counts and immune markers in a line of Scottish Cashmere goats selected for resistance to gastrointestinal nematode parasite infection.

This study aimed to investigate the effect of selection for low faecal egg count (FEC) in Scottish Cashmere goats in comparison to a control line of unselected goats grazing the same pasture. Goats from generations F2 through to F9 were monitored for FEC, bodyweight, peripheral eosinophilia and IgG, IgA and IgE response against Teladorsagia circumcincta from the end of their first grazing season, through winter housing (during which a single artificial challenge dose of 10,000 drug susceptible T. circumcincta was given) and the following full grazing season. The study demonstrated that selected line animals excreted a significantly lower number of parasite eggs (P<0.01) in the majority of generations examined. Liveweight productivity was unaffected by selection. Although selected line animals had greater numbers of circulating eosinophils in many of the generations (four generations of males and six generations of females, P<0.05), there was no direct link between eosinophilia and reduced FEC. Immunoglobulin levels showed no consistent difference between selected and control lines. IgG, IgA and IgE levels were not different between lines over the whole dataset (P>0.05), although the selected line had significantly elevated or reduced levels (P<0.05) for all three within individual generations. There were significant associations between increased IgG and reduced FEC under artificial infection conditions (P=0.02). Increased IgA was also significantly associated with elevated FEC during the second grazing season (P<0.001). The study demonstrates that selection produced a line of goats with consistently reduced FEC compared with control animals, but did not identify a clear relationship between any of the immune markers measured and faecal egg output.

Gastrointestinal nematode species diversity in Soay sheep kept in a natural environment without active parasite control.

Molecular methods based on ITS2 sequence analysis were used to identify strongylid parasites and describe their diversity in a management intervention and anthelmintic drug treatment-free sheep flock. Fourteen different nematode parasite species were identified in the flock and the results showed a greater level of nematode species diversity than is normally reported in managed farmed flocks, with the presence of parasites such as Bunostomum trigonocephalum, Ostertagia leptospicularis, Spiculopteragia houdemeri and Trichostrongylus retortaeformis that are considered to be absent or rare in sheep kept in comparable localities. The implied prevalences of Haemonchus contortus in lambs, and of Trichostrongylus axei in lambs, ewes and rams, were higher than those in farmed sheep kept in similar regions, while those of Teladorsagia circumcincta and Trichostrongylus vitrinus were lower. Comparison of the patterns of nematode parasite infection between the summer and autumn sampling periods showed differences from the scenarios that are commonplace in comparable managed flocks; with T. vitrinus burdens of the lambs being higher in the summer than in the winter, and Oesophagostomum venulosum being the predominant nematode species in the adult sheep during the summer, while more-or-less absent from these groups during the winter. Rams played an important role in the epidemiology of certain parasitic nematode species. The relatively non-pathogenic O. venulosum was the only parasitic nematode species to predominate in any group during the study. This preliminary characterisation of the nematode parasite burdens of sheep extensively grazed on diverse unimproved pastures will aid in the understanding of the parasitological consequences of intensive grazing management and of the manner in which modern agriculture upsets the equilibrium between parasites and their hosts. These factors must be accounted for when defining the concept of sustainable parasite control and informing sustainability with reference to commercially efficient sheep farming.

Pooling sheep faecal samples for the assessment of anthelmintic drug efficacy using McMaster and Mini-FLOTAC in gastrointestinal strongyle and Nematodirus infection.

In small ruminants, faecal egg counts (FECs) and reduction in FECs (FECR) are the most common methods for the assessment of intensity of gastrointestinal (GI) nematodes infections and anthelmintic drug efficacy, respectively. The main limitation of these methods is the time and cost to conduct FECs on a representative number of individual animals. A cost-saving alternative would be to examine pooled faecal samples, however little is known regarding whether pooling can give representative results. In the present study, we compared the FECR results obtained by both an individual and a pooled examination strategy across different pool sizes and analytical sensitivity of the FEC techniques. A survey was conducted on 5 sheep farms in Scotland, where anthelmintic resistance is known to be widespread. Lambs were treated with fenbendazole (4 groups), levamisole (3 groups), ivermectin (3 groups) or moxidectin (1 group). For each group, individual faecal samples were collected from 20 animals, at baseline (D0) and 14 days after (D14) anthelmintic administration. Faecal samples were analyzed as pools of 3-5, 6-10, and 14-20 individual samples. Both individual and pooled samples were screened for GI strongyle and Nematodirus eggs using two FEC techniques with three different levels of analytical sensitivity, including Mini-FLOTAC (analytical sensitivity of 10 eggs per gram of faeces (EPG)) and McMaster (analytical sensitivity of 15 or 50 EPG).For both Mini-FLOTAC and McMaster (analytical sensitivity of 15 EPG), there was a perfect agreement in classifying the efficacy of the anthelmintic as 'normal', 'doubtful' or 'reduced' regardless of pool size. When using the McMaster method (analytical sensitivity of 50 EPG) anthelmintic efficacy was often falsely classified as 'normal' or assessment was not possible due to zero FECs at D0, and this became more pronounced when the pool size increased. In conclusion, pooling ovine faecal samples holds promise as a cost-saving and efficient strategy for assessing GI nematode FECR. However, for the assessment FECR one will need to consider the baseline FEC, pool size and analytical sensitivity of the method.

Effect of anthelmintic treatment strategy on strongylid nematode species composition in grazing lambs in Scotland.

BACKGROUND: Refugia based anthelmintic protocols aim to reduce the rate of development of anthelmintic resistance in gastrointestinal nematodes (GIN). Previous studies have illustrated the impact of different drenching regimes on drug efficacy and animal growth; however, the impact on nematode populations has yet to be characterised within natural infections. This study investigated the changes in species composition of GIN throughout the grazing season, following implementation of four different ivermectin drenching regimes over six years: neo-suppressive monthly treatment (NST), targeted selective treatment (TST), strategic prophylactic treatment (SPT) and treatment upon observation of clinical signs (MT). METHODS: Lambs were grazed on one of eight replicate paddocks each grazing season following treatment regimes assigned in year 1. Faecal samples were collected fortnightly from all animals and hatched to first stage larvae (L1). DNA was extracted from individual L1 and a multiplex PCR assay targeting the internal transcribed spacer 2 (ITS2) region of Teladorsagia circumcincta, Trichostrongylus spp. and Haemonchus contortus conducted. Other species were identified using species-specific PCR. Worm-naïve tracer lambs were grazed on the paddocks at the start and end of each grazing season and adult worms recovered at post mortem to investigate the parasite population on pasture. RESULTS: Results showed an overall decrease in species diversity in egg output from the NST group which occurred within a single grazing season and was consistent throughout the experiment. Species diversity was protected over six years in groups implementing TST, SPT and MT treatment regimes, designed to offer refugia. The expected shift in species prevalence throughout the season from Teladorsagia to Trichostrongylus was observed in all but the NST group where only Teladorsagia spp. were recovered from trial lambs by the end of the experiment. Worm burdens indicated the presence of several species at relatively low abundance on pasture in the NST group in 2011. However, these species were not represented in egg output from trial lambs, probably due to the frequent anthelmintic treatment administered throughout the grazing season. CONCLUSION: The molecular methods utilised here worked well. The comparable results of the three refugia-based treatment regimes suggest that nematode diversity can be maintained using part or whole group treatments if a rich supra-population of parasites are available to re-infect animals post treatment.

Viability of the Happy Factor™ targeted selective treatment approach on several sheep farms in Scotland.

The aim of this study was to examine the use of Happy Factor™ weight based targeted selective treatment (TST) on several commercial farms in Scotland in combination with findings from a long term trial on a research farm to assess the potential for TST use in varying farming operations as an alternative to the current regimen of whole flock treatment. Lambs on each farm were regularly weighed and climatic conditions and pasture availability measured for inclusion into the Happy Factor™ model to calculate weight targets. Half of the lambs were allocated to TST treatment and any failing to reach the weight target was treated with the anthelmintic of choice on that farm, while the remaining half of each flock was treated with anthelmintic as per normal practice on that farm (routine treatment, RT). The research farm (farm 1) hosted a long term trial using four anthelmintic treatment regimes over 6 years, and data from two regimes are presented here, alongside findings from three further farms: two commercial enterprises (farms 2 and 3) and a research farm operating as a commercial analogue with two breeds (farms 4a and 4b). The effect of TST strategy on lamb productivity and the number of anthelmintic treatments was investigated. There was no evidence (p>0.300) that mean bodyweight or growth rate was different between TST and RT groups on any of the farms and 95% confidence intervals of TST and RT groups generally suggested that TST had negligible unfavourable effects on the average growth of lambs for most of the farms. Growth rates ranged from 97.39 to 189.16g/day reflecting the varied nature of the farms. All commercial farms used significantly less (1.34 RT versus 1.14 TST treatments per animal, p<0.05) anthelmintic in lambs following TST, with a reduction from 1, 1, 1.03 and 1.14 to 0.77, 0.57, 0.82 and 0.81 in the number of treatments per animal for farms 2, 3, 4a and 4b respectively. This study suggests that TST is a viable means of controlling parasitic disease without incurring production losses.

Development of a loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Haemonchus contortus eggs in ovine faecal samples.

A major constraint on the effective control and management of helminth parasites in livestock is the lack of rapid and reliable diagnostic tests to identify the parasite species responsible for disease and to allow informed treatment decisions to be made. In the present study, we have developed a novel DNA-based assay for the detection of Haemonchus contortus eggs in ovine faecal samples, using loop-mediated isothermal amplification (or LAMP). LAMP allows for rapid detection of H. contortus DNA under isothermal incubation conditions. The robust nature of this assay negates the need for extensive DNA extraction, allowing amplification from relatively crude samples. Preliminary results suggest that LAMP is highly specific, and does not cross-react with DNA from other common co-infecting parasites. The Haemonchus LAMP assay is also highly sensitive, exhibiting a 10 times lower detection limit than the equivalent PCR; 10(-5) ng/µl and 10(-4) ng/µl DNA, respectively, allowing detection in a faecal samples containing two Haemonchus eggs per gram. Translation of this assay onto a real-time platform provided rapid results and highlighted its potential as a quantitative assay which could inform treatment decisions in the future.