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Simple Summary There is increasing evidence for the importance of gut bacteria in animal health and disease. This is particularly relevant for gastrointestinal infections, such as parasitic worms, which share a niche with gut bacteria. Parasitic worms are highly prevalent in domestic horses and are a significant cause of disease in this population. This commentary explores the complex relationships between the most common parasitic worm in horses (cyathostomins) and gut bacteria, based on recent studies in horses and other species. We propose novel theories and avenues for research that harness these relationships and have the potential to improve control of parasitic worms, and overall equine health, in the future. Abstract The importance of the gut microbiome for host health has been the subject of intense research over the last decade. In particular, there is overwhelming evidence for the influence of resident microbiota on gut mucosal and systemic immunity; with significant implications for the outcome of gastrointestinal (GI) infections, such as parasitic helminths. The horse is a species that relies heavily on its gut microbiota for GI and overall health, and disturbances in this complex ecosystem are often associated with life-threatening disease. In turn, nearly all horses harbour parasitic helminths from a young age, the most prevalent of which are the small strongyles, or cyathostomins. Research describing the relationship between gut microbiota and cyathostomin infection is in its infancy, however, to date there is evidence of meaningful interactions between these two groups of organisms which not only influence the outcome of cyathostomin infection but have long term consequences for equine host health. Here, we describe these interactions alongside supportive evidence from other species and suggest novel theories and avenues for research which have the potential to revolutionize our approach to cyathostomin prevention and control in the future.

Anthelmintic resistance is a global problem that threatens sustainable control of the equine gastrointestinal cyathostomins (Phylum Nematoda; Superfamily Strongyloidea). Of the three novel anthelmintic classes that have reached the veterinary market in the last decade, none are currently licenced in horses, hence current control regimens focus on prolonging the useful lifespan of licenced anthelmintics. This approach would be facilitated by knowledge of the resistance mechanisms to the most widely used anthelmintics, the macrocyclic lactones (ML). There are no data regarding resistance mechanisms to MLs in cyathostomins, although in other parasitic nematodes, the ABC transporters, P-glycoproteins (P-gps), have been implicated in playing an important role. Here, we tested the hypothesis that P-gps are, at least in part, responsible for reduced sensitivity to the ML ivermectin (IVM) in cyathostomins; first, by measuring transcript levels of pgp-9 in IVM resistant versus IVM sensitive third stage larvae (L3) pre-and post-IVM exposure in vitro. We then tested the effect of a range of P-gp inhibitors on the effect of IVM against the same populations of L3 using the in vitro larval development test (LDT) and larval migration inhibition test (LMIT). We demonstrated that, not only was pgp-9 transcription significantly increased in IVM resistant compared to IVM sensitive L3 after anthelmintic exposure (p, Graphical abstract Image 1, Highlights • Pgp-9 transcript levels were higher in ivermectin resistant versus susceptible cyathostomin populations. • P-gp inhibitors increased ivermectin effect against cyathostomins in vitro. • P-gp activity may play a role in ivermectin resistance in cyathostomins.

Gastrointestinal helminth parasites share their habitat with a myriad of other organisms, that is, the commensal microbiota. Increasing evidence, particularly in humans and rodent models of helminth infection, points towards a multitude of interactions occurring between parasites and the gut microbiota, with a profound impact on both host immunity and metabolic potential. Despite this information, the exploration of the effects that parasite infections exert on populations of commensal gut microbes of veterinary species is a field of research in its infancy. In this article, we summarise studies that have contributed to current knowledge of helminth-microbiota interactions in species of veterinary interest, and identify possible avenues for future research in this area, which could include the exploitation of such relationships to improve parasite control and delay or prevent the development of anthelmintic resistance.

A plethora of data points towards a role of the gastrointestinal (GI) microbiota of neonatal and young vertebrates in supporting the development and regulation of the host immune system. However, knowledge of the impact that infections by GI helminths exert on the developing microbiota of juvenile hosts is, thus far, limited. This study investigates, for the first time, the associations between acute infections by GI helminths and the faecal microbial and metabolic profiles of a cohort of equine youngstock, prior to and following treatment with parasiticides (ivermectin). We observed that high versus low parasite burdens (measured via parasite egg counts in faecal samples) were associated with specific compositional alterations of the developing microbiome; in particular, the faecal microbiota of animals with heavy worm infection burdens was characterised by lower microbial richness, and alterations to the relative abundances of bacterial taxa with immune-modulatory functions. Amino acids and glucose were increased in faecal samples from the same cohort, which indicated the likely occurrence of intestinal malabsorption. These data support the hypothesis that GI helminth infections in young livestock are associated with significant alterations to the GI microbiota, which may impact on both metabolism and development of acquired immunity. This knowledge will direct future studies aimed to identify the long-term impact of infection-induced alterations of the GI microbiota in young livestock.

The multifaceted interactions occurring between gastrointestinal (GI) parasitic helminths and the host gut microbiota are emerging as a key area of study within the broader research domain of host-pathogen relationships. Over the past few years, a wealth of investigations has demonstrated that GI helminths interact with the host gut flora, and that such interactions result in modifications of the host immune and metabolic statuses. Nevertheless, whilst selected changes in gut microbial composition are consistently observed in response to GI helminth infections across several host-parasite systems, research in this area to date is largely characterised by inconsistent findings. These discrepancies are particularly evident when data from studies of GI helminth-microbiota interactions conducted in humans from parasite-endemic regions are compared. In this review, we provide an overview of the main sources of variance that affect investigations on helminth-gut microbiota interactions in humans, and propose a series of methodological approaches that, whilst accounting for the inevitable constraints of fieldwork, are aimed at minimising confounding factors and draw biologically meaningful interpretations from highly variable datasets.

The control of equid gastrointestinal nematodes in developed countries, in particular the cyathostomins, is threatened by high levels of anthelmintic resistance. In recent years, there has been increasing interest in the evaluation of traditional ‘ethnoveterinary’ medicines as alternatives to chemical anthelmintics. The cysteine proteinases (CPs), a group of enzymes derived from fruits such as papaya (Carica papaya), pineapple (Ananas comosus) and figs (Ficus spp.), have shown good efficacy against adult stages of a range of parasitic nematodes, in vitro and in vivo. The efficacy of CPs against cyathostomins remains to be explored. In this study, the efficacy of a crude preparation of CPs, papaya latex supernatant (PLS), against the free-living stages of cyathostomins was evaluated using two in vitro tests, the egg hatch test (EHT) and the larval migration inhibition test (LMIT). It was demonstrated that PLS had a potent effect in the EHT, with EC-50 values in the range of 0.12–0.22 μM. At concentrations above 6.25 μM the eggs did not develop, below this concentration the L1 developed but they lost integrity of the cuticle upon hatching. These effects were inhibited by pre-incubation of PLS with the CP inhibitor L-trans-epoxysuccinyl- l -leucylamido-(4-guanidino butane) (E64), indicating that CPs were responsible for the anti-parasitic activity. A dose-dependent inhibition of migration of third stage larvae (L3) in the LMIT was demonstrated at higher concentrations of PLS, with EC-50 values in the range of 67.35–106.31 μM. Incubation of PLS with E64 prior to use in the LMIT did not reverse the anti-migratory effect, suggesting that CPs were not responsible for the reduced migration of cyathostomin L3 and that PLS also contains an additional active compound. This is the first report of PLS and/or CPs showing activity against the free-living stages of a parasitic helminth. In addition, it suggests that cyathostomins are highly sensitive to the effects of CPs and further evaluation of their efficacy against parasitic stages and in vivo are strongly indicated.

In spite of the extensive contribution of intestinal pathology to the pathophysiology of schistosomiasis, little is known of the impact of schistosome infection on the composition of the gut microbiota of its mammalian host. Here, we characterised the fluctuations in the composition of the gut microbial flora of the small and large intestine, as well as the changes in abundance of individual microbial species, of mice experimentally infected with Schistosoma mansoni with the goal of identifying microbial taxa with potential roles in the pathophysiology of infection and disease. Bioinformatic analyses of bacterial 16S rRNA gene data revealed an overall reduction in gut microbial alpha diversity, alongside a significant increase in microbial beta diversity characterised by expanded populations of Akkermansia muciniphila (phylum Verrucomicrobia) and lactobacilli, in the gut microbiota of S. mansoni-infected mice when compared to uninfected control animals. These data support a role of the mammalian gut microbiota in the pathogenesis of hepato-intestinal schistosomiasis and serves as a foundation for the design of mechanistic studies to unravel the complex relationships amongst parasitic helminths, gut microbiota, pathophysiology of infection and host immunity.

Background In spite of the emergence of populations of drug-resistant cyathostomines worldwide, little is known of parasite species responsible for ‘early egg shedding’ in cohorts of horses subjected to treatment with widely used anthelmintics, e.g. ivermectin (IVM). In this study, we determined the cyathostomine egg reappearance period (ERP) after IVM treatment in a cohort of yearlings from a large Thoroughbred (TB) stud farm in the United Kingdom, and identified species of cyathostomines with reduced ERP using a combination of fundamental parasitology techniques coupled with advanced molecular tools. Methods Individual faecal samples were collected from TB yearlings with cyathostomine infection prior to IVM treatment, as well as at 14, 21, 28, 35, 42 and 49 days post-treatment. Faecal egg counts (FEC) were performed for each individual sample for determination of ERPs. In addition, individual larval cultures were performed and representative numbers of third-stage larvae (L3s) harvested from each culture were subjected to molecular species identification via PCR-Reverse Line Blot (RLB). Results Prior to IVM treatment, 11 cyathostomine species were detected in faecal samples from TB horses enrolled in this study, i.e. Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicostephanus goldi, Cylicocyclus nassatus, Cylicostephanus calicatus, Cyathostomum pateratum, Cylicocyclus radiatus, Paraposteriostomum mettami, Coronocyclus labratus, Cylicocyclus insigne and Cylicocyclus radiatus variant A. Of these, eggs of Cya. catinatum, Cys. longibursatus, Cyc. nassatus and Cyc. radiatus could be detected at 28 days post-treatment, while from day 42 onwards, cyathostomine species composition reflected data obtained pre-IVM treatment, with the exception of eggs of Cor. labratus and Cyc. insigne which could no longer be detected post-IVM administration. Conclusions This study provides valuable data on the occurrence of IVM-resistance in cyathostomines in the UK. Nevertheless, further investigations are needed to shed light on the prevalence and incidence of drug-resistance in this country, as well as other areas of the world where equine trade is substantial. Electronic supplementary material The online version of this article (10.1186/s13071-018-2638-6) contains supplementary material, which is available to authorized users.

Cyathostomins are the most important gastrointestinal nematode infecting equids. Their effective control is currently under threat due to widespread resistance to the broad spectrum anthelmintics licenced for use in equids. In response to similar resistance issues in other helminths, there has been increasing interest in alternative control strategies, such as bioactive plant compounds derived from traditional ethnoveterinary treatments. This study used an evidence-based approach to evaluate the potential use of plant extracts from the UK and Ethiopia to treat cyathostomins. Plants were shortlisted based on findings from a literature review and additionally, in Ethiopia, the results of a participatory rural appraisal (PRA) in the Oromia region of the country. Systematic selection criteria were applied to both groups to identify five Ethiopian and four UK plants for in vitro screening. These included Acacia nilotica (L.) Delile, Cucumis prophetarum L., Rumex abyssinicus Jacq., Vernonia amygdalina Delile. and Withania somnifera (L.) Dunal from Ethiopia and Allium sativum L. (garlic), Artemisia absinthium L., Chenopodium album L. and Zingiber officinale Roscoe. (ginger) from the UK. Plant material was collected, dried and milled prior to hydro-alcoholic extraction. Crude extracts were dissolved in distilled water (dH2O) and dimethyl sulfoxide (DMSO), serially diluted and screened for anthelmintic activity in the larval migration inhibition test (LMIT) and the egg hatch test (EHT). Repeated measures ANOVA was used to identify extracts that had a significant effect on larval migration and/or egg hatch, compared to non-treated controls. The median effective concentration (EC-50) for each extract was calculated using PROBIT analysis. Of the Ethiopian extracts A. nilotica, R. abyssinicus and C. prophetarum showed significant anthelmintic activity. Their lowest EC-50 values were 0.18 (confidence interval (CI): 0.1-0.3), 1.1 (CI 0.2-2.2) and 1.1 (CI 0.9-1.4)mg/ml, respectively. All four UK extracts, A. sativum, C. album, Z. officinale and A. absinthium, showed significant anthelmintic activity. Their lowest EC-50 values were 1.1 (CI 0.9-1.3), 2.3 (CI 1.9-2.7) and 0.3 (CI 0.2-0.4)mg/ml, respectively. Extract of A. absinthium had a relatively low efficacy and the data did not accurately fit a PROBIT model for the dose response relationship, thus an EC-50 value was not calculated. Differences in efficacy for each extract were noted, dependent on the assay and solvent used, highlighting the need for a systematic approach to the evaluation of bioactive plant compounds. This study has identified bioactive plant extracts from the UK and Ethiopia which have potential as anthelmintic forages or feed supplements in equids.

The gastrointestinal (GI) tract of vertebrates is inhabited by a vast array of organisms, i.e., the microbiota and macrobiota. The former is composed largely of commensal microorganisms, which play vital roles in host nutrition and maintenance of energy balance, in addition to supporting the development and function of the vertebrate immune system. By contrast, the macrobiota includes parasitic helminths, which are mostly considered detrimental to host health via a range of pathogenic effects that depend on parasite size, location in the GI tract, burden of infection, metabolic activity, and interactions with the host immune system. Sharing the same environment within the vertebrate host, the GI microbiota and parasitic helminths interact with each other, and the results of such interactions may impact, directly or indirectly, on host health and homeostasis. The complex relationships occurring between parasitic helminths and microbiota have long been neglected; however, recent studies point towards a role for these interactions in the overall pathophysiology of helminth disease, as well as in parasite-mediated suppression of inflammation. Whilst several discrepancies in qualitative and quantitative modifications in gut microbiota composition have been described based on host and helminth species under investigation, we argue that attention should be paid to the systems biology of the gut compartment under consideration, as variations in the abundances of the same population of bacteria inhabiting different niches of the GI tract may result in varying functional consequences for host physiology.

Summary This project investigated the clinical value of a human whole blood point-of-care (POC) triglyceride (TG) measurement device to determine equid blood TG concentrations. Equid plasma TG measurements were determined in a commercial laboratory and also using whole blood and plasma on the POC device from equid patients with TG concentrations from within the normal range to severely hypertriglyceridaemic. Results were compared between methods using the Bland-Altman method for validation. Seventy-four samples were collected: 35 from inappetent or starved equid patients (considered ‘at-risk’ of developing hypertriglyceridaemia) and 39 from equids eating with a normal appetite. The POC device readable range is 0.8–6.86 mmol/l; high readings were diluted and measurements repeated and calculated. Inappetent/starved patients had a higher risk of developing raised plasma triglycerides (OR 16.8, 95% CI 4.8–58.4, P

Background Gastrointestinal nematode infections constitute a threat to the health and welfare of donkeys worldwide. Their primary means of control is via anthelmintic treatments; however, use of these drugs has constraints in developing countries, including cost, limited availability, access to cheaper generic forms of variable quality and potential anthelmintic resistance. As an alternative, bioactive plants have been proposed as an option to treat and control gastrointestinal helminths in donkeys. This study aimed to use participatory methodology to explore donkey owner knowledge, attitudes and beliefs relating to the use of plant-based treatments for gastrointestinal parasites of donkeys in Ethiopia. Results In focus groups, 22/29 groups stated they knew of plants used for the treatment of gastrointestinal parasites in donkeys. All groups volunteered plants that were used in cattle and/or small ruminants. In total, 21 plants were named by participants. ‘Koso’ (Hagenia abyssinica) ‘Grawa’ (Vernonia amygdalina) and a mixed roots and leaves preparation were the most frequently named plant preparations. ‘Enkoko’ (Embelia shimperi) and ‘a mixture of roots and leaves’ were ranked highly for effectiveness in donkeys. However, ‘Grawa’ and ‘Koso’ were the highest ranked when taking into account both the rank position and the number of groups ranking the plant. Thematic analysis of participants’ current attitudes and beliefs surrounding traditional plant-based remedies for gastrointestinal parasites revealed that anthelmintics obtained from clinics were generally favoured due to their ease of administration and perceived higher effectiveness. There was doubt surrounding the effectiveness of some plant-based treatments, but there were also perceived advantages including their low cost, ease of cultivation and availability. However, plant-based treatments were considered a “past trend” and people favoured “modern” medicine, particularly among the younger generation. Conclusions There was extensive knowledge of plant-based treatments for gastrointestinal parasites in livestock in Ethiopia. In donkeys, Koso (Hagenia abyssinica), Grawa (Vernonia amygdalina), Enkoko (Embelia shimperi) and ‘mixed roots and leaves’ were the most frequently named and/or highest ranked plants with reported efficacy against gastrointestinal parasites. Further in vitro and in vivo investigation of these plants is now required to determine viable alternatives for the treatment and control of gastrointestinal parasites in Ethiopia.

Reasons for performing study Anthelmintic resistance is a global problem and constitutes a major threat to the welfare of equids worldwide. The cyathostomins are the most numerous and pathogenic gastrointestinal nematode (GIN) of equids in the developed world. Cyathostomins show widespread resistance to 2 out of 3 of the major classes of anthelmintic and recently there are reports of reduced efficacy to the potent macrocyclic lactones (MLs). None of the 3 novel classes of anthelmintic that have emerged in the last decade are licensed for use in equids. The cysteine proteases (CPs) are plant proteins that have shown potent activity against GINs in vivo in sheep and pigs. Objectives This study aimed to evaluate the anthelmintic effect of the CP papain on cyathostomins in vitro using the egg hatch assay (EHA) and larval migration inhibition test (LMIT). Methods Samples of cyathostomin eggs and third stage larvae were collected and cultured from a population of equids that have recently shown reduced ML efficacy in vivo. The EHA and LMIT were performed on repeated samples with increasing concentrations of papain. Dose–response curves were plotted and PROBIT analysis performed on the data to give EC-50 values (concentration that gives 50% of the maximal response). Results Papain caused a dose dependent inhibition of both egg hatch and larval migration. The EC-50 values were 2 μmol/l and 100 μmol/l in the EHA and LMIT respectively, indicating a more potent effect on egg hatch. Conclusions The CP papain shows potent anthelmintic activity against cyathostomins in vitro. Good evidence of anthelmintic effect against GINs in other host species is supportive of its potential use in equids. Further work is indicated to evaluate safety and in vivo efficacy. Ethical animal research: The study was approved by the University of Liverpool and Donkey Sanctuary Ethics Committee. Explicit owner informed consent for participation in this study was not stated. Sources of funding: The Donkey Sanctuary, University of Liverpool – Institute of Infection and Global Health. Competing interests: None.