Gut microbiota-mediated polyphenol metabolism is restrained by parasitic whipworm infection and associated with altered immune function in mice.

Polyphenols are phytochemicals commonly found in plant-based diets which have demonstrated immunomodulatory and anti-inflammatory properties. However, the interplay between polyphenols and pathogens at mucosal barrier surfaces has not yet been elucidated in detail. Here, we show that proanthocyanidin (PAC) polyphenols interact with gut parasites to influence immune function and gut microbial-derived metabolites in mice. PAC intake inhibited mastocytosis during infection with the small intestinal roundworm Heligmosomoides polygyrus, and altered the host tissue transcriptome at the site of infection with the large intestinal whipworm Trichuris muris, with a notable enhancement of type-1 inflammatory and interferon-driven gene pathways. In the absence of infection, PAC intake promoted the expansion of Turicibacter within the gut microbiota, increased fecal short chain fatty acids, and enriched phenolic metabolites such as phenyl-γ-valerolactones in the cecum. However, these putatively beneficial effects were reduced in PAC-fed mice infected with T. muris, suggesting concomitant parasite infection can attenuate gut microbial-mediated PAC catabolism. Collectively, our results suggest an inter-relationship between a phytonutrient and infection, whereby PAC may augment parasite-induced inflammation (most prominently with the cecum dwelling T. muris), and infection may abrogate the beneficial effects of health-promoting phytochemicals.

Dietary non-starch polysaccharides impair immunity to enteric nematode infection.

BACKGROUND: The influence of diet on immune function and resistance to enteric infection and disease is becoming ever more established. Highly processed, refined diets can lead to inflammation and gut microbiome dysbiosis, whilst health-promoting dietary components such as phytonutrients and fermentable fibres are thought to promote a healthy microbiome and balanced mucosal immunity. Chicory (Cichorium intybus) is a leafy green vegetable rich in fibres and bioactive compounds that may promote gut health. RESULTS: Unexpectedly, we here show that incorporation of chicory into semisynthetic AIN93G diets renders mice susceptible to infection with enteric helminths. Mice fed a high level of chicory leaves (10% dry matter) had a more diverse gut microbiota, but a diminished type-2 immune response to infection with the intestinal roundworm Heligmosomoides polygyrus. Furthermore, the chicory-supplemented diet significantly increased burdens of the caecum-dwelling whipworm Trichuris muris, concomitant with a highly skewed type-1 immune environment in caecal tissue. The chicory-supplemented diet was rich in non-starch polysaccharides, particularly uronic acids (the monomeric constituents of pectin). In accordance, mice fed pectin-supplemented AIN93G diets had higher T. muris burdens and reduced IgE production and expression of genes involved in type-2 immunity. Importantly, treatment of pectin-fed mice with exogenous IL-25 restored type-2 responses and was sufficient to allow T. muris expulsion. CONCLUSIONS: Collectively, our data suggest that increasing levels of fermentable, non-starch polysaccharides in refined diets compromises immunity to helminth infection in mice. This diet-infection interaction may inform new strategies for manipulating the gut environment to promote resistance to enteric parasites.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine.

The faecal egg count reduction test (FECRT) remains the method of choice for establishing the efficacy of anthelmintic compounds in the field, including the diagnosis of anthelmintic resistance. We present a guideline for improving the standardization and performance of the FECRT that has four sections. In the first section, we address the major issues relevant to experimental design, choice of faecal egg count (FEC) method, statistical analysis, and interpretation of the FECRT results. In the second section, we make a series of general recommendations that are applicable across all animals addressed in this guideline. In the third section, we provide separate guidance details for cattle, small ruminants (sheep and goats), horses and pigs to address the issues that are specific to the different animal types. Finally, we provide overviews of the specific details required to conduct an FECRT for each of the different host species. To address the issues of statistical power vs. practicality, we also provide two separate options for each animal species; (i) a version designed to detect small changes in efficacy that is intended for use in scientific studies, and (ii) a less resource-intensive version intended for routine use by veterinarians and livestock owners to detect larger changes in efficacy. Compared to the previous FECRT recommendations, four important differences are noted. First, it is now generally recommended to perform the FECRT based on pre- and post-treatment FEC of the same animals (paired study design), rather than on post-treatment FEC of both treated and untreated (control) animals (unpaired study design). Second, instead of requiring a minimum mean FEC (expressed in eggs per gram (EPG)) of the group to be tested, the new requirement is for a minimum total number of eggs to be counted under the microscope (cumulative number of eggs counted before the application of a conversion factor). Third, we provide flexibility in the required size of the treatment group by presenting three separate options that depend on the (expected) number of eggs counted. Finally, these guidelines address all major livestock species, and the thresholds for defining reduced efficacy are adapted and aligned to host species, anthelmintic drug and parasite species. In conclusion, these new guidelines provide improved methodology and standardization of the FECRT for all major livestock species.

Profiling of hepatic transcriptomes reveals modulatory effects of parasitic infection on the metabolic response to dietary polyphenols in pigs.

Polyphenols are a class of bioactive plant compounds with health-promoting properties, however, the interactions between polyphenols and pathogen infection and their cumulative impact on inflammation and metabolic health are not well understood. Here, we investigated if a subclinical parasitic infection modulates the hepatic response to dietary polyphenol supplementation in a porcine model. Pigs were fed a diet with or without 1% grape proanthocyanidins (PAC) for 28 days. During the final 14 days of the experiment, half the pigs in each dietary group were inoculated with the parasitic nematode Ascaris suum. Serum biochemistry was measured and hepatic transcriptional responses were determined by RNA-sequencing coupled with gene-set enrichment analysis. A. suum infection resulted in reduced serum phosphate, potassium, sodium, and calcium, and increased serum iron concentrations. In uninfected pigs, PAC supplementation markedly changed the liver transcriptome including genes related to carbohydrate and lipid metabolism, insulin signaling, and bile acid synthesis. However, during A. suum infection, a separate set of genes were modulated by dietary PAC, indicating that the polyphenol-mediated effects were dependent on infection status. A. suum infection strongly influenced the expression of genes related to cellular metabolism, and, in contrast to the effects of PAC, these changes were mostly identical in both control-fed and PAC-fed pigs. Thus, the hepatic response to infection was mostly unaffected by concurrent polyphenol intake. We conclude that the presence of a commonly occurring parasite substantially influences the outcome of dietary polyphenol supplementation, which may have important relevance for nutritional interventions in populations where intestinal parasitism is widespread.

A statistical framework for calculating prospective sample sizes and classifying efficacy results for faecal egg count reduction tests in ruminants, horses and swine.

The faecal egg count reduction test (FECRT) is the primary diagnostic tool used for detecting anthelmintic resistance at the farm level. It is therefore extremely important that the experimental design of a FECRT and the susceptibility classification of the result use standardised and statistically rigorous methods. Several different approaches for improving the analysis of FECRT data have been proposed, but little work has been published on how to address the issue of prospective sample size calculations. Here, we provide a complete and detailed overview of the quantitative issues relevant to a FECRT starting from basic statistical principles. We then present a new approach for determining sample size requirements for the FECRT that is built on a solid statistical framework, and provide a rigorous anthelminthic drug efficacy classification system for use with FECRT in livestock. Our approach uses two separate statistical tests, a one-sided inferiority test for resistance and a one-sided non-inferiority test for susceptibility, and determines a classification of resistant, susceptible or inconclusive based on the combined result. Since this approach is based on two independent one-sided tests, we recommend that a 90 % CI be used in place of the historically used 95 % CI. This maintains the desired Type I error rate of 5 %, and simultaneously reduces the required sample size. We demonstrate the use of this framework to provide sample size calculations that are rooted in the well-understood concept of statistical power. Tailoring to specific host/parasite systems is possible using typical values for expected pre-treatment and post-treatment variability in egg counts as well as within-animal correlation in egg counts. We provide estimates for these parameters for ruminants, horses and swine based on a re-examination of datasets that were available to us from a combination of published data and other sources. An illustrative example is provided to demonstrate the use of the framework, and parameter estimates are presented to estimate the required sample size for a hypothetical FECRT using ivermectin in cattle. The sample size calculation method and classification framework presented here underpin the sample size recommendations provided in the upcoming FECRT WAAVP guidelines for detection of anthelmintic resistance in ruminants, horses, and swine, and have also been made freely available as open-source software via our website (https://www.fecrt.com).

Garlic-Derived Metabolites Exert Antioxidant Activity, Modulate Gut Microbiota Composition and Limit Citrobacter rodentium Infection in Mice.

The garlic-derived compounds propyl propane thiosulfinate (PTS) and propyl propane thiosulfonate (PTSO) are metabolites with putative health benefits against intestinal inflammation that may be related to their antioxidant activity. However, the underlying mechanisms remain unclear, and whether PTS-PTSO can promote gut health by altering the microbiota and exert protection against enteric pathogens needs further investigation. Here, we explored the antioxidant activity of PTS-PTSO in murine macrophages in vitro, and in an in vivo model of bacterial infection with the bacterial pathogen Citrobacter rodentium. PTS-PTSO attenuated reactive oxygen species in lipopolysaccharide-stimulated macrophages in a nuclear factor erythroid factor 2-related factor 2 (Nrf2)-dependent manner, decreased nitric oxide levels both in macrophages in vitro and in the sera of mice fed PTS-PTSO, and had putatively beneficial effects on the commensal gut microbiota. Importantly, PTS-PTSO decreased faecal C. rodentium counts, concomitant with upregulation of Nrf2-related genes in colon tissue. Thus, PTS-PTSO mediates Nrf2-mediated antioxidant activity and modulates gut microbiota, which may protect the host against C. rodentium colonization. Our results provide further insight into how PTS-PTSO and related bioactive dietary compounds may reduce enteric infections.

Anti-protozoal activity and metabolomic analyses of Cichorium intybus L. against Trypanosoma cruzi.

Chagas disease, caused by the protozoa Trypanosoma cruzi, is a potentially life-threatening parasitic zoonosis infecting 6-7 million people worldwide, mainly in Latin America. Due to the limited numbers of drugs available against this neglected disease and their frequent adverse effects, novel anti-chagasic agents are urgently needed. Cichorium intybus L. (chicory) is a bioactive plant with potent activity against parasitic nematodes, but its effects on protozoans are poorly known and no studies have explored its trypanocidal potential. Here, we investigated the activity of C. intybus against extracellular and intracellular stages of T. cruzi, including the prediction of trypanocidal compounds by metabolomic analyses and bioactivity-based molecular networking. Purified C. intybus extracts were prepared from leaves and roots of five C. intybus cultivars (cv. 'Benulite', 'Goldine', 'Larigot', 'Maestoso' and 'Spadona'). All C. intybus extracts induced concentration-dependent effects against T. cruzi trypomastigotes. C. intybus leaf extracts had higher trypanocidal selectivity and lower cytotoxicity on mammalian cells than root extracts. The leaf extract of C. intybus cv. Goldine also significantly reduced the number of mammalian cells infected with T. cruzi amastigotes. Metabolomic and bioactivity-based molecular networking analyses revealed 11 compounds in C. intybus leaves strongly linked with activity against trypomastigotes, including the sesquiterpene lactone lactucin, and flavonoid- and fatty acid-derivatives. Furthermore, seven distinct C. intybus molecules (including two sesquiterpene lactone-derivatives) were predicted to be involved in reducing the number of mammalian cells infected with amastigotes. This is the first report of the anti-protozoal activity of C. intybus against trypanosomatid parasites and expands our understanding of the anti-parasitic effects of this plant and its bioactive metabolites. Further studies to elucidate the anti-protozoal compound(s) in C. intybus and their mode(s) of action will improve our knowledge of using this bioactive plant as a promising source of novel broad-spectrum anti-parasitic compounds with associated health benefits and biomedical potential.

Assessing the impact of Ascariasis and Trichuriasis on weight gain using a porcine model.

BACKGROUND: Infections with Ascaris lumbricoides and Trichuris trichiura remain significant contributors to the global burden of neglected tropical diseases. Infection may in particular affect child development as they are more likely to be infected with T. trichiura and/or A. lumbricoides and to carry higher worm burdens than adults. Whilst the impact of heavy infections are clear, the effects of moderate infection intensities on the growth and development of children remain elusive. Field studies are confounded by a lack of knowledge of infection history, nutritional status, presence of co-infections and levels of exposure to infective eggs. Therefore, animal models are required. Given the physiological similarities between humans and pigs but also between the helminths that infect them; A. suum and T. suis, growing pigs provide an excellent model to investigate the direct effects of Ascaris spp. and Trichuris spp. on weight gain. METHODS AND RESULTS: We employed a trickle infection protocol to mimic natural co-infection to assess the effect of infection intensity, determined by worm count (A. suum) or eggs per gram of faeces (A. suum and T. suis), on weight gain in a large pig population (n = 195) with variable genetic susceptibility. Pig body weights were assessed over 14 weeks. Using a post-hoc statistical approach, we found a negative association between weight gain and T. suis infection. For A. suum, this association was not significant after adjusting for other covariates in a multivariable analysis. Estimates from generalized linear mixed effects models indicated that a 1 kg increase in weight gain was associated with 4.4% (p = 0.00217) decrease in T. suis EPG and a 2.8% (p = 0.02297) or 2.2% (p = 0.0488) decrease in A. suum EPG or burden, respectively. CONCLUSIONS: Overall this study has demonstrated a negative association between STH and weight gain in growing pigs but also that T. suis infection may be more detrimental that A. suum on growth.

Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation.

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

Use of agro-industrial by-products containing tannins for the integrated control of gastrointestinal nematodes in ruminants.

Previous studies have illustrated that different bioactive legume fodders containing condensed tannins might represent one of the options for integrated sustainable control of gastrointestinal nematodes (GIN) in ruminants, which may help address the worldwide development of resistance to synthetic anthelmintics. More recently, impetus has been given to assess the potential antiparasitic activity of less conventional resources, represented by different agro-industrial by-products (AIBPs). This review presents in vitro and in vivo results obtained with a range of tannin-containing AIBPs of various geographical and botanical origins, namely AIBP of nuts, temperate and tropical barks, carob, coffee and cocoa. They tend to confirm the "proof of concept" for their antiparasitic effects and also for other aspects of ruminant production in an agro-ecological context. Socio-economic aspects of the exploitation of such non-conventional resources are also discussed as potential models of the circular economy, by using waste. The different modes of use of these resources are presented in this review, as well as strengths, weaknesses, opportunities, and threats (SWOT) analyses to illustrate the advantages and limitations of on-farm use.

TITLE: Utilisation de sous-produits agro-industriels contenant des tanins pour le contrôle intégré des nématodes gastro-intestinaux chez les ruminants. ABSTRACT: Plusieurs études antérieures ont illustré le fait que des légumineuses bioactives contenant des tannins condensés peuvent représenter une des alternatives à intégrer avec d'autres options pour une maitrise durable des nématodes gastro-intestinaux en réponse au développement constant et à l'expansion continue à l'échelle mondiale des résistances aux anthelminthiques de synthèse. Des recherches plus récentes se sont intéressées au potentiel d'application de ressources moins conventionnelles que représentent des coproduits agroindustriels (CPAI). Cette revue vise à présenter des résultats in vitro et in vivo obtenus avec une gamme de CPAI d'origines géographiques et botaniques diversifiées (coproduits de l'industrie des noix, du bois (en régions tempérées et tropicales), du caroubier, du café et du cacao). Ces résultats ont confirmé la preuve de concept pour les effets antiparasitaires, et aussi pour d'autres volets de la production des ruminants dans un contexte agro écologique de l'élevage. Par ailleurs, les aspects socio-économiques d'exploitation de ces ressources, considérées jusqu'à présent comme des déchets, dans un modèle de circuits courts sont aussi évoqués. Les avantages et inconvénients des différentes modalités d'exploitation des CPAI sont aussi discutés dans le cadre d'une analyse SWOT.

Garlic-Derived Organosulfur Compounds Regulate Metabolic and Immune Pathways in Macrophages and Attenuate Intestinal Inflammation in Mice.

SCOPE: Garlic is a source of bioactive phytonutrients that may have anti-inflammatory or immunomodulatory properties. The mechanism(s) underlying the bioactivity of these compounds and their ability to regulate responses to enteric infections remains unclear. METHODS AND RESULTS: This study investigates if a garlic-derived preparation (PTSO-PTS) containing two organosulfur metabolites, propyl-propane thiosulfonate (PTSO), and propyl-propane thiosulfinate (PTS), regulate inflammatory responses in murine macrophages and intestinal epithelial cells (IEC) in vitro, as well as in a model of enteric parasite-induced inflammation. PTSO-PTS decreases lipopolysaccharide-induced secretion of TNFα, IL-6, and IL-27 in macrophages. RNA-sequencing demonstrates that PTSO-PTS strongly suppresses pathways related to immune and inflammatory signaling. PTSO-PTS induces the expression of a number of genes involved in antioxidant responses in IEC during exposure to antigens from the parasite Trichuris muris. In vivo, PTSO-PTS does not affect T. muris establishment or intestinal T-cell responses but significantly alters cecal transcriptomic responses. Notably, a reduction in T. muris-induced expression of Tnf, Saa2, and Nos2 is observed. CONCLUSION: Garlic-derived organosulfur compounds exert anti-inflammatory effects in macrophages and IEC, and regulate gene expression during intestinal infection. These compounds and related organic molecules may thus hold potential as functional food components to improve gut health in humans and animals.

The phytonutrient cinnamaldehyde limits intestinal inflammation and enteric parasite infection.

Phytonutrients such as cinnamaldehyde (CA) have been studied for their effects on metabolic diseases, but their influence on mucosal inflammation and immunity to enteric infection are not well documented. Here, we show that consumption of CA in mice significantly down-regulates transcriptional pathways connected to inflammation in the small intestine, and alters T-cell populations in mesenteric lymph nodes. During infection with the enteric helminth Heligomosomoides polygyrus, CA treatment attenuated infection-induced changes in biological pathways connected to cell cycle and mitotic activity, and tended to reduce worm burdens. Mechanistically, CA did not appear to exert activity through a prebiotic effect, as CA treatment did not significantly change the composition of the gut microbiota. Instead, in vitro experiments showed that CA directly induced xenobiotic metabolizing pathways in intestinal epithelial cells and suppressed endotoxin-induced inflammatory responses in macrophages. Collectively, our results show that CA down-regulates inflammatory pathways in the intestinal mucosa and can limit the pathological response to enteric infection. These properties appear to be largely independent of the gut microbiota, and instead connected to the ability of CA to induce antioxidant pathways in intestinal cells. Our results encourage further investigation into the use of CA and related phytonutrients as functional food components to promote intestinal health in humans and animals.

Fox Serum Proteomics Analysis Suggests Host-Specific Responses to Angiostrongylus vasorum Infection in Canids.

Dogs infected with the cardiopulmonary nematode Angiostrongylus vasorum may suffer from respiratory distress and/or bleeding disorders. Descriptions of clinical signs in foxes are rare, despite high prevalence. To evaluate the impact of infection on coagulation and immune response, serum proteins from eight experimentally infected foxes before and after inoculation (day 0, 35, 84, 154) were subjected to differential proteomic analyses based on quantitative data and compared to available data from dogs. The number of proteins with differential abundance compared to the uninfected baseline increased with chronicity of infection. Bone marrow proteoglycan, chitinase 3-like protein 1 and pulmonary surfactant-associated protein B were among the most prominently increased proteins. The abundance of several proteins involved in coagulation was decreased. Enriched pathways obtained from both increased and decreased proteins included, among others, "platelet degranulation" and "haemostasis", and indicated both activation and suppression of coagulation. Qualitative comparison to dog data suggests some parallel serum proteomic alterations. The comparison, however, also indicates that foxes have a more adequate immunopathological response to A. vasorum infection compared to dogs, facilitating persistent infections in foxes. Our findings imply that foxes may be more tolerant to A. vasorum infection, as compared to dogs, reflecting a longer evolutionary host-parasite adaptation in foxes, which constitute a key wildlife reservoir.

A new phenothiazine derivate is active against Clostridioides difficile and shows low cytotoxicity.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 µg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

Structure-function analysis of purified proanthocyanidins reveals a role for polymer size in suppressing inflammatory responses.

Proanthocyanidins (PAC) are dietary compounds that have been extensively studied for beneficial health effects due to their anti-inflammatory properties. However, the structure-function relationships of PAC and their mode-of-action remain obscure. Here, we isolated a wide range of diverse PAC polymer mixtures of high purity from plant material. Polymer size was a key factor in determining the ability of PAC to regulate inflammatory cytokine responses in murine macrophages. PAC polymers with a medium (9.1) mean degree of polymerization (mDP) induced substantial transcriptomic changes, whereas PAC with either low (2.6) or high (12.3) mDP were significantly less active. Short-term oral treatment of mice with PAC modulated gene pathways connected to nutrient metabolism and inflammation in ileal tissue in a polymerization-dependent manner. Mechanistically, the bioactive PAC polymers modulated autophagic flux and inhibited lipopolysaccharide-induced autophagy in macrophages. Collectively, our results highlight the importance of defined structural features in the health-promoting effects of PAC-rich foods.

Association between milk yield and milk anti-Fasciola hepatica antibody levels, and the utility of bulk tank milk samples for assessing within-herd prevalence on organic dairy farms.

Fasciola hepatica is an important disease of livestock that is responsible for substantial economic losses worldwide. Estimates of the impact of infection on milk yield vary, likely reflecting different geographical locations, farm-level management, and diagnostic methods. Measuring anti-Fasciola antibodies on bulk tank milk (BTM) by ELISA provides a convenient herd-level diagnosis, but the utility of this test remains unclear. Therefore, we evaluated the utility of BTM ELISA test results in Danish organic dairy farms, including estimating the association between 305 day energy corrected milk yield (305d ECM) and F. hepatica infection both at individual and herd level. BTM samples from 218 organic farms were analysed using IDEXX ELISA and subsequently the farmers were interviewed during spring 2016 with the aim of characterising their management practices. The corresponding farm-level production data covering the period 2014-2017 were collected from the Danish national cattle registry. In the following year, 284 individual milk samples (4-7 per herd) along with BTM samples were collected from a subset of the same herds (n = 55). Linear mixed models were used to estimate the association between milk production and ELISA value at both individual and farm levels, and a generalised additive model was used to assess the relationship between within-herd prevalence and BTM ELISA. A dichotomised BTM result with positive outcome was associated with a reduction of 580.5 kg in average 305d ECM, and a positive outcome on individual-level ELISA was associated with a 919.5 kg reduction in milk yield for cows in their third or later lactations. A strong relationship between quantitative BTM ELISA sample to positive percentage (S/P%) and apparent within-herd prevalence based on dichotomised individual-level milk ELISA was also observed, although this relationship was non-linear in nature. We conclude that a useful indication of the within-herd prevalence of infection can be obtained from BTM ELISA following categorisation as negative, low, medium or high according to S/P% cut-offs of approximately 30, 80, and 150. This approach represents a cheap and useful diagnostic tool for monitoring the long-term success of control strategies for F. hepatica infections on a dairy farm.

Identification of compounds responsible for the anthelmintic effects of chicory (Cichorium intybus) by molecular networking and bio-guided fractionation.

Increasing resistance towards anthelmintic drugs has necessitated the search for alternative treatments for the control of gastrointestinal nematode parasites. Animals fed on chicory (Cichorium intybus L.), a temperate (pasture) crop, have reduced parasite burdens, hence making C. intybus a potentially useful source for novel anthelmintic compounds or a diet-based preventive/therapeutic option. Here, we utilized in vitro bioassays with the parasitic nematode Ascaris suum and molecular networking techniques with five chicory cultivars to identify putative active compounds. Network analysis predicted sesquiterpene lactones (SL) as the most likely group of anthelmintic compounds. Further bioassay-guided fractionation supported these predictions, and isolation of pure compounds demonstrated that the SL 8-deoxylactucin (8-DOL) is the compound most strongly associated with anti-parasitic activity. Furthermore, we showed that 8-DOL acts in a synergistic combination with other SL to exert the anti-parasitic effects. Finally, we established that chicory-derived extracts also showed activity against two ruminant nematodes (Teladorsagia circumcincta and Cooperia oncophora) in in vitro assays. Collectively, our results confirm the anti-parasitic activity of chicory against a range of nematodes, and pave the way for targeted extraction of active compounds or selective breeding of specific cultivars to optimize its future use in human and veterinary medicine.

Emerging interactions between diet, gastrointestinal helminth infection, and the gut microbiota in livestock.

Increasing evidence suggests that nutritional manipulation of the commensal gut microbiota (GM) may play a key role in maintaining animal health and production in an era of reduced antimicrobial usage. Gastrointestinal helminth infections impose a considerable burden on animal performance, and recent studies suggest that infection may substantially alter the composition and function of the GM. Here, we discuss the potential interactions between different bioactive dietary components (prebiotics, probiotics and phytonutrients) and helminth infection on the GM in livestock. A number of recent studies suggest that host diet can strongly influence the nature of the helminth-GM interaction. Nutritional manipulation of the GM may thus impact helminth infection, and conversely infection may also influence how the GM responds to dietary interventions. Moreover, a dynamic interaction exists between helminths, the GM, intestinal immune responses, and inflammation. Deciphering the mechanisms underlying the diet-GM-helminth axis will likely inform future helminth control strategies, as well as having implications for how health-promoting feed additives, such as probiotics, can play a role in sustainable animal production.

Parasite-Probiotic Interactions in the Gut: Bacillus sp. and Enterococcus faecium Regulate Type-2 Inflammatory Responses and Modify the Gut Microbiota of Pigs During Helminth Infection.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Giardia and Cryptosporidium infections in Danish cats: risk factors and zoonotic potential.

Giardia and Cryptosporidium infections are common in cats, but knowledge is limited about their clinical importance, risk factors, and the role of cats as a reservoir for human infections. Here, we collected faeces and questionnaire data from 284 cats from shelters and veterinary clinics in the Copenhagen Metropolitan Region (= study population). Additionally, 33 samples were analysed separately from catteries with gastrointestinal clinical signs (= cases). (Oo-)cysts were quantified by immunofluorescence microscopy. All Giardia (n = 34) and Cryptosporidium (n = 29) positive samples were analysed by sequencing of the 18S rRNA, gdh and hsp70 loci, and co-infections were detected by McMaster/inverted microscopy. In the study population, 7.0% and 6.7% were positive for Giardia and Cryptosporidium respectively; 48.5% and 36.4% of the breeder cats (cases) were infected. Increased odds of diarrhoea were demonstrated in Giardia (p = 0.0008) and Cryptosporidium (p = 0.034) positive cats. For Giardia, the odds were positively correlated with infection intensity. Co-infection with Cryptosporidium (OR 12.79; p < 0.001), parasitic co-infections other than Cryptosporidium (OR 5.22; p = 0.009), no deworming (OR 4.67; p = 0.035), and male sex (OR 3.63; p = 0.025) were risk factors for Giardia. For Cryptosporidium, co-infection with Giardia was the only risk factor (OR 11.93; p < 0.0001). Genotyping revealed G. duodenalis assemblages A and F, and C. felis, all of them previously detected in humans. In conclusion, excretion of Giardia and Cryptosporidium was associated with clinical disease. Although a public health risk is likely, studies including larger sample sizes, more discriminatory markers and samples from other animals and humans are needed to reveal the full zoonotic potential.