Effect of sainfoin (Onobrychis viciifolia) on cyathostomin eggs excretion, larval development, larval community structure and efficacy of ivermectin treatment in horses.

Alternative strategies to chemical anthelmintics are needed for the sustainable control of equine strongylids. Bioactive forages like sainfoin (Onobrychis viciifolia) could contribute to reducing drug use, with the first hints of in vitro activity against cyathostomin free-living stages observed in the past. We analysed the effect of a sainfoin-rich diet on cyathostomin population and the efficacy of oral ivermectin treatment. Two groups of 10 naturally infected horses were enrolled in a 78-day experimental trial. Following a 1-week adaptation period, they were either fed with dehydrated sainfoin pellets (70% of their diet dry matter) or with alfalfa pellets (control group) for 21-days. No difference was found between the average fecal egg counts (FECs) of the two groups, but a significantly lower increase in larval development rate was observed for the sainfoin group, at the end of the trial. Quantification of cyathostomin species abundances with an ITS-2-based metabarcoding approach revealed that the sainfoin diet did not affect the nemabiome structure compared to the control diet. Following oral ivermectin treatment of all horses on day 21, the drug concentration was lower in horses fed with sainfoin, and cyathostomin eggs reappeared earlier in that group. Our results demonstrated that short-term consumption of a sainfoin-rich diet does not decrease cyathostomin FEC but seems to slightly reduce larval development. Consumption of dehydrated sainfoin pellets also negatively affected ivermectin pharmacokinetics, underscoring the need to monitor horse feeding regimes when assessing ivermectin efficacy in the field.

One Health surveillance of West Nile and Usutu viruses: a repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020.

BackgroundWest Nile virus (WNV) and Usutu virus (USUV), two closely related flaviviruses, mainly follow an enzootic cycle involving mosquitoes and birds, but also infect humans and other mammals. Since 2010, their epidemiological situation may have shifted from irregular epidemics to endemicity in several European regions; this requires confirmation, as it could have implications for risk assessment and surveillance strategies.AimTo explore the seroprevalence in animals and humans and potential endemicity of WNV and USUV in Southern France, given a long history of WNV outbreaks and the only severe human USUV case in France in this region.MethodsWe evaluated the prevalence of WNV and USUV in a repeated cross-sectional study by serological and molecular analyses of human, dog, horse, bird and mosquito samples in the Camargue area, including the city of Montpellier, between 2016 and 2020.ResultsWe observed the active transmission of both viruses and higher USUV prevalence in humans, dogs, birds and mosquitoes, while WNV prevalence was higher in horses. In 500 human samples, 15 were positive for USUV and 6 for WNV. Genetic data showed that the same lineages, WNV lineage 1a and USUV lineage Africa 3, were found in mosquitoes in 2015, 2018 and 2020.ConclusionThese findings support existing literature suggesting endemisation in the study region and contribute to a better understanding of USUV and WNV circulation in Southern France. Our study underlines the importance of a One Health approach for the surveillance of these viruses.

Observation of the long-term effects of lifestyle intervention during balneotherapy in metabolic syndrome.

OBJECTIVE: Estimate the effect of lifestyle adjustment activities in patients with metabolic syndrome treated by prescribed balneotherapy. METHODS: Observational pilot cohort study with 12-month follow-up after multidimensional lifestyle training (physical, dietary, educational) during 3-week standard stay in the spa town of Eugénie-les-Bains. RESULTS: Of 145 eligible patients, 97 were included; 63 were followed and analysable. At inclusion all had ≥3 National cholesterol education program-Adult treatment panel III (NCEP-ATPIII) criteria defining metabolic syndrome, 76.2% were female, mean age was 61.2 years. At the end of follow-up (median:10.4 months, Inter-Quartile Range: [6.7;11.4]), 48 of these 63 patients (76.2%) no longer had metabolic syndrome (95%CI [65.7;86.7]). These 48 patients without metabolic syndrome at the end of follow-up represented 49.5% of the 97 included (95%CI [39.5;59.4]). CONCLUSIONS: Future studies of lifestyle interventions taking advantage of the spa environment can be expected to find least one third of patients free of metabolic syndrome at the end of 12-month follow-up in the intervention group.

Patterns of variation in equine strongyle community structure across age groups and gut compartments.

BACKGROUND: Equine strongyles encompass more than 64 species of nematode worms that are responsible for growth retardation and the death of animals. The factors underpinning variation in the structure of the equine strongyle community remain unknown. METHODS: Using horse-based strongyle community data collected after horse deworming (48 horses in Poland, 197 horses in Ukraine), we regressed species richness and the Gini-Simpson index upon the horse's age, faecal egg count, sex and operation of origin. Using the Ukrainian observations, we applied a hierarchical diversity partitioning framework to estimate how communities were remodelled across operations, age groups and horses. Lastly, strongyle species counts collected after necropsy (46 horses in France, 150 in Australia) were considered for analysis of their co-occurrences across intestinal compartments using a joint species distribution modelling approach. RESULTS: First, inter-operation variation accounted for > 45% of the variance in species richness or the Gini-Simpson index (which relates to species dominance in communities). Species richness decreased with horse's age (P = 0.01) and showed a mild increase with parasite egg excretion (P < 0.1), but the Gini-Simpson index was neither associated with parasite egg excretion (P = 0.8) nor with horse age (P = 0.37). Second, within-host diversity represented half of the overall diversity across Ukrainian operations. While this is expected to erase species diversity across communities, community dissimilarity between horse age classes was the second most important contributor to overall diversity (25.8%). Third, analysis of species abundance data quantified at necropsy defined a network of positive co-occurrences between the four most prevalent strongyle genera. This pattern was common to necropsies performed in France and Australia. CONCLUSIONS: Taken together, these results show a pattern of ß-diversity maintenance across age classes combined with positive co-occurrences that might be grounded by priority effects between the major species.

Species interactions, stability, and resilience of the gut microbiota - Helminth assemblage in horses.

The nature and strength of interactions entertained among helminths and their host gut microbiota remain largely unexplored. Using 40 naturally infected Welsh ponies, we tracked the gut microbiota-cyathostomin temporal dynamics and stability before and following anthelmintic treatment and the associated host blood transcriptomic response. High shedders harbored 14 species of cyathostomins, dominated by Cylicocyclus nassatus. They exhibited a highly diverse and temporal dynamic gut microbiota, with butyrate-producing Clostridia likely driving the ecosystem steadiness and host tolerance toward cyathostomins infection. However, anthelmintic administration sharply bent the microbial community. It disrupted the ecosystem stability and the time-dependent network of interactions, affecting longer term microbial resilience. These observations highlight how anthelmintic treatments alter the triangular relationship of parasite, host, and gut microbiota and open new perspectives for adding nutritional intervention to current parasite management strategies.

Comparison of two molecular barcodes for the study of equine strongylid communities with amplicon sequencing.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

Genetic diversity and relationships of the liver fluke Fasciola hepatica (Trematoda) with native and introduced definitive and intermediate hosts.

Fasciolosis is a worldwide spread parasitosis mainly caused by the trematode Fasciola hepatica. This disease is particularly important for public health in tropical regions, but it can also affect the economies of many developed countries due to large infections in domestic animals. Although several studies have tried to understand the transmission by studying the prevalence of different host species, only a few have used population genetic approaches to understand the links between domestic and wildlife infections. Here, we present the results of such genetic approach combined with classical parasitological data (prevalence and intensity) by studying domestic and wild definitive hosts from Camargue (southern France) where fasciolosis is considered as a problem. We found 60% of domestic hosts (cattle) infected with F. hepatica but lower values in wild hosts (nutria, 19%; wild boars, 4.5%). We explored nine variable microsatellite loci for 1,148 adult flukes recovered from four different populations (non-treated cattle, treated cattle, nutria and wild boars). Populations from the four groups differed, though we found a number of migrants particularly non-treated cattle and nutria. Overall, we detected 729 different multilocus genotypes (from 783 completely genotyped individuals) and only 46 genotypes repeated across samples. Finally, we experimentally infected native and introduced intermediate snail hosts to explore their compatibility with F. hepatica and assess the risks of fasciolosis expansion in the region. The introduced species Galba truncatula and Pseudosuccinea columella attained the higher values of overall compatibility in relation to the European species. However, concerning the origin, sympatric combinations of G. truncatula were more compatible (higher prevalence, intensity and survival) than the allopatric tested. According to our results, we should note that the assessment of epidemiological risks cannot be limited to a single host-parasite system, but should focus on understanding the diversity of hosts in the heterogeneous environment through space and time.

Evidence for unmyelinated C fibres and inflammatory cells in human varicose saphenous vein.

The physiopathology of venous symptoms, such as pain, leg heaviness or swelling sensations, in chronic venous disease (CVD) remains unclear. Localized release of proinflammatory mediators appears to play a key role but the presence of nociceptors sensitive to inflammatory mediators, such as unmyelinated C fibres, needs to be demonstrated. The present study included 10 patients with documented CVD who underwent surgery for saphenectomy. For each patient, five segments of the great saphenous vein were immunostained with anti-S100 protein and anti-CD45 to identify nerve fibres and inflammatory cells, respectively. Light microscopy was completed by electron microscopy. In all patients, S100 immunopositive nerve fibres and CD45 immunopositive cells were observed. Under an electron microscope, advanced signs of wall remodelling were systematically observed. The density of nerve fibres was low and variable from one sample to another. Unmyelinated C fibres were mainly located in the external part of the media and to a lesser extent in the internal part of the adventitia. Inflammatory cells, mainly histiocytes, were scattered in the media. Mast cells were observed in three patients. In conclusion, unmyelinated C fibres and inflammatory cells are present in the varicose saphenous vein wall. Their linked roles in symptoms of CVD should be further explored.

Leukocyte involvement in the signs and symptoms of chronic venous disease. Perspectives for therapy.

Pain intensity in chronic venous disease varies with the stage in the clinical-etiologic-anatomic-pathophysiologic (CEAP) classification but also with patient perception, pain being by definition subjective. The venous hypertension responsible for the varicose veins and trophic changes in CVD has a variety of algogenic repercussions in which leukocytes play a particular role, notably through their ability to roll along the vessel wall. Shear stress, hypoxia and stasis activate the marginated leukocytes to shed L-selectin from their surface and express integrins, matrix metalloproteinase 9, elastase, lactoferrin and free radicals. Meanwhile the endothelium expresses adhesion molecules that permit slow rolling on E-selectin followed by adhesion and tissue transmigration. Vein wall and valve areas in particular attract mast cells, monocyte-macrophages and T lymphocytes, and undergo remodeling. Sympathetic sensory C and Adelta fibers, which wrap around cutaneous venules and are also present in the venous intima and media, are nociceptors sensitive to the pain mediators concentrated within leukocytes, such as mast cell bradykinin, responsible for visceral pain. Neuronal inflammation combined with wall remodeling intensifies symptoms. Yet no direct link has so far been shown between pain and mast cell mediator levels. Leukocyte adhesion is also associated with the increased capillary permeability that leads to edema. Antileukocyte therapies include postural rest and venotonics which alone or in combination with compression have been shown to unstick and inhibit leukocytes. The micronized purified flavonoid fraction (MPFF) protects vascular endothelium against hypoxia and reduces adhesion molecule expression. Unlike other antileukocyte therapies, venotonics do not cause neutropenia.

Effects of armagnac or vodka on platelet aggregation in healthy volunteers: a randomized controlled clinical trial.

BACKGROUND: Cardiovascular mortality is especially low in southwest France (the French Paradox). In previous experimental studies, we found that alcohol-free extracts of armagnac could inhibit human platelet function in vitro and experimental thrombosis in vivo. To test the possible relevance of these findings, we tested the effects of daily use of small quantities of armagnac against same alcohol strength, polyphenol-free vodka in healthy volunteers. METHOD: Randomized controlled trial comparing 5-year old armagnac (30 ml/day for 2 weeks) to same alcoholic strength vodka, in 20 healthy volunteers, on platelet aggregation induced by ADP, collagen, and thrombin, as well as bleeding time, partial thromboplastin time (pTT), and plasma lipids during and after consumption. Platelet testing was done blind. RESULTS: After 14 days, ADP-induced platelet aggregation was inhibited more in armagnac (-31+/-3.2% compared to pretreatment values, p<.01) than in vodka (-11.0+/-6.8%, NS) users (p<.05, armagnac vs. vodka). A rebound increase of aggregation was found 2 weeks later in vodka but not in armagnac users. The same pattern was found for thrombin-induced aggregation, including post-treatment rebound. No effect was found on collagen-induced aggregation, bleeding time, pTT, or plasma lipids. CONCLUSION: The chronic ingestion of moderate quantities of armagnac modified platelet aggregation to ADP in healthy volunteers. The difference with the effects of same alcohol degree vodka is in favour of an effect of the nonalcoholic fraction in the effects of armagnac, rather than just alcohol. All spirits may not be equal for cardioprotection.

Roles of mechanical blood forces in vascular diseases. A clinical overview.

Under the influence of heart and elasticity of arteries, circulating blood constantly acts upon the layers of the vessels, covered by active endothelial cells. The shear stress appears like the most efficient mechanical factor developing a rubbing physical force, the laminar flow. But pulsating and centrifugal forces allow shear to be often unsteady. Mechanobiology investigates receptors and transduction across wall cells, showing that thousands of genes are activated in the endothelium leading to a lot of adaptable functions. Atherosclerosis is due to disturbances of laminar flow in specific areas where low shear allows white cells to adhere and migrate, permeability to increase. Coronaropathies, cerebral vascular accidents, aneurisms are so geometrically local diseases, of which lesions are subsequently enhanced by risk factors. Chronic venous insufficiency is also related to physical forces: hydrostatic and centrifugal pressures, and disruption of shear stress along the wall and around valvulae. Here also similar cells and biochemical phenomenons are the cause of wall remodelling and varicose. At the level of microcirculation the shear stress fall induces hypoxia, accumulation of white cells and hemorheological disorders in microvenulae. This leads to lesions of tissue, small vessel and ulcers. Numerous treatments in vascular diseases tend to restore flow and blood shearing. However a better understanding in the future should open new therapeutic fields and genetic approaches.

Effect of age of Armagnac extract and duration of treatment on antithrombotic effects in a rat thrombosis model.

In a previous study, we had shown that freeze-dried extracts of 12-year-old Armagnac could inhibit ADP-induced platelet aggregation in a dose- and duration-dependent manner, and reduce thrombus weight in an experimental rat arteriovenous shunt thrombosis model after 2-week oral treatment. Polyphenol content could however vary with age and origin of the brandy, and the onset and offset of the effect were not defined. To this end, we studied the effects of extracts of 5-, 10- and 15-year-old Armagnac from two different producers at 1, 5 and 25 mg/kg orally for 15 days, on the same rat arteriovenous shunt thrombosis model. We then studied the effects of 1, 3, 7 and 15 days of oral treatment with 5 mg/kg extracts of a 5-year-old Armagnac, and the effect 1, 3 and 7 days after a 1-week oral treatment of the same extract at the same dose. There was a dose-dependent decrease in thrombus weight, which was similar for both Armagnac origins for all ages. Extracts of 5- and 10-year-old Armagnac were similar, and more potent than extracts from 15-year-old Armagnac. There was a progressive decrease in thrombus weight over duration of treatment to 7 days, to about 50% of initial thrombus weight. The effect disappeared within 3 days after stopping a 7-day treatment. We confirm the dose-, age- and duration-dependent inhibition of arteriovenous shunt thrombosis in vivo by Armagnac extracts in rats.

Effects of armagnac extracts on human platelet function in vitro and on rat arteriovenous shunt thrombosis in vivo.

INTRODUCTION: The "French paradox", a low cardiovascular mortality compared to the prevalent risk factors, has been attributed to the regular use of red wine, and to the polyphenols it contains. These have among other effects an antioxidant and antithrombotic effect. The French paradox is maximal in southwest France, a region which is the region of production of armagnac, an oak cask aged spirit also rich in polyphenols. METHOD: We tested the effects of a freeze-dried extract of 12-year-old armagnac (EA88) on in vitro human platelet adhesion, and on aggregation induced by collagen or ADP, in the presence or absence of hypoxanthine-xanthine oxidase (HX/XO), at concentrations ranging from 5 x 10(-9) to 5 x 10(-3) g/l, after 15-60 min incubation. We also tested the effects of 2-week oral treatment with 1, 5 and 25 mg/kg EA88 in a rat arteriovenous shunt thrombosis model. RESULTS: EA88 inhibited ADP-induced but not collagen-induced human platelet aggregation in vitro in a concentration- and incubation time-dependent manner, which was greater in the presence of HX/XO. In vivo, giving rats a daily oral dose of EA88 for 2 weeks inhibited thrombus formation in a dose-dependent manner, for doses consistent with the habitual human use of armagnac. CONCLUSION: Armagnac extract EA88 had an antiplatelet and antithrombotic effect that if confirmed in man could contribute to explain the intensity of the French paradox in southwest France.

Interactions between aspirin and COX-2 inhibitors or NSAIDs in a rat thrombosis model.

Recent in vitro studies, clinical trials and epidemiological studies have suggested possible interactions between aspirin and other cyclo-oxygenase (COX) inhibitors, such as ibuprofen of the COX-2 inhibitors celecoxib and rofecoxib. The objective of this study was to test the effects of aspirin (1, 2.5 and 5 mg/kg), and ibuprofen (4 and 15 mg/kg), diclofenac (2.5 mg/kg), flurbiprofen (2 mg/kg), celecoxib (7.5 mg/kg), and rofecoxib (1 mg/kg), alone or combined on a rat model of arterial thrombosis. Drugs were given orally daily for 7 days, before insertion of an arterio-venous shunt thrombosis system, left in place for 15 min. Main parameter was thrombus weight. Five to 12 rats were used per experiment, and 35 controls overall. Aspirin inhibited thrombus formation in a dose-dependent manner. All NSAIDS given alone also inhibited thrombus formation to approximately the same level as aspirin 1 mg/kg/day. Ibuprofen, celecoxib and rofecoxib inhibited the effects of aspirin, but not diclofenac or flurbiprofen. The interactions with aspirin do not seem to affect all NSAIDs to equal levels. The clinical impact of this needs to be confirmed in adequately powered clinical trials or pharmaco-epidemiological studies.

Anti-endothelial cell autoantibodies and soluble markers of endothelial cell dysfunction in systemic lupus erythematosus.

OBJECTIVE: To determine if anti-endothelial cell antibodies (AECA) and plasma markers of endothelial cell function are related to disease severity in systemic lupus erythematosus (SLE). METHODS: We measured AECA by human umbilical vein endothelial cell binding, endothelial markers von Willebrand factor, soluble thrombomodulin, and soluble E-selectin by ELISA, and disease severity by SLEDAI and SLICC/ACR in 35 patients with SLE. RESULTS: Despite high levels of IgG AECA (p = 0.001) and von Willebrand factor (p = 0.0007) compared to 21 healthy controls, we found a positive correlation only between IgG AECA and the SLEDAI index (r = 0.393, p = 0.021). CONCLUSION: IgG AECA seem to be related to disease activity in SLE, possibly in a pathogenic role. Conversely, plasma markers of endothelial cell damage seem to be an epiphenomenon and may simply be related to excess inflammation.