The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae.

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 µg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 - 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 - 48.06 % exsheathment reduction) compared to the RL treatment. The T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 5.34 - 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts.

Phytochemical analysis and anthelmintic activity of Combretum mucronatum leaf extract against infective larvae of soil-transmitted helminths including ruminant gastrointestinal nematodes.

BACKGROUND: Soil-transmitted helminths (STH) infect more than a quarter of the world's human population. In the absence of vaccines for most animal and human gastrointestinal nematodes (GIN), treatment of infections primarily relies on anthelmintic drugs, while resistance is a growing threat. Therefore, there is a need to find alternatives to current anthelmintic drugs, especially those with novel modes of action. The present work aimed to study the composition and anthelmintic activity of Combretum mucronatum leaf extract (CMLE) by phytochemical analysis and larval migration inhibition assays, respectively. METHODS: Combretum mucronatum leaves were defatted with petroleum ether and the residue was extracted by ethanol/water (1/1) followed by freeze-drying. The proanthocyanidins and flavonoids were characterized by thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UPLC). To evaluate the inhibitory activity of this extract, larval migration assays with STH and GIN were performed. For this purpose, infective larvae of the helminths were, if necessary, exsheathed (Ancylostoma caninum, GIN) and incubated with different concentrations of CMLE. RESULTS: CMLE was found to be rich in flavonoids and proanthocyanidins; catechin and epicatechin were therefore quantified for standardization of the extract. Data indicate that CMLE had a significant effect on larval migration. The effect was dose-dependent and higher concentrations (1000 µg/mL) exerted significantly higher larvicidal effect (P < 0.001) compared with the negative control (1% dimethyl sulfoxide, DMSO) and lower concentrations (≤ 100 µg/ml). Infective larvae of Ascaris suum [half-maximal inhibitory concentration (IC50) = 5.5 µg/mL], Trichuris suis (IC50 = 7.4 µg/mL), and A. caninum (IC50 = 18.9 µg/mL) were more sensitive to CMLE than that of Toxocara canis (IC50 = 310.0 µg/mL), while infective larvae of Toxocara cati were largely unaffected (IC50 > 1000 µg/mL). Likewise, CMLE was active against most infective larvae of soil-transmitted ruminant GIN, except for Cooperia punctata. Trichostrongylus colubriformis was most sensitive to CMLE (IC50 = 2.1 µg/mL) followed by Cooperia oncophora (IC50 = 27.6 µg/mL), Ostertagia ostertagi (IC50 = 48.5 µg/mL), Trichostrongylus axei (IC50 = 54.7 µg/mL), Haemonchus contortus (IC50 = 145.6 µg/mL), and Cooperia curticei (IC50 = 156.6 µg/mL). CONCLUSIONS: These results indicate that CMLE exhibits promising anthelmintic properties against infective larvae of a large variety of soil-transmitted nematodes.

Long-term benefit contribution of chemical and biological nematicide in coffee nematode management in soil microbial diversity and crop yield perspectives.

The plant-parasitic root-knot nematode Meloidogyne exigua causes significant damage and is an important threat in Coffea arabica plantations. The utilization of plant-beneficial microbes as biological control agents against sedentary endoparasitic nematodes has been a longstanding strategy. However, their application in field conditions to control root-knot nematodes and their interaction with the rhizospheric microbiota of coffee plants remain largely unexplored. This study aimed to investigate the effects of biological control agent-based bioproducts and a chemical nematicide, used in various combinations, on the control of root-knot nematodes and the profiling of the coffee plant rhizomicrobiome in a field trial. The commercially available biological products, including Trichoderma asperellum URM 5911 (Quality), Bacillus subtilis UFPEDA 764 (Rizos), Bacillus methylotrophicus UFPEDA 20 (Onix), and nematicide Cadusafos (Rugby), were applied to adult coffee plants. The population of second-stage juveniles (J2) and eggs, as well as plant yield, were evaluated over three consecutive years. However, no significant differences were observed between the control group and the groups treated with bioproducts and the nematicide. Furthermore, the diversity and community composition of bacteria, fungi, and eukaryotes in the rhizosphere soil of bioproduct-treated plants were evaluated. The dominant phyla identified in the 16 S, ITS2, and 18 S communities included Proteobacteria, Acidobacteria, Actinobacteria, Ascomycota, Mortierellomycota, and Cercozoa in both consecutive years. There were no significant differences detected in the Shannon diversity of 16 S, ITS2, and 18 S communities between the years of data. The application of a combination of T. asperellum, B. subtilis, and B. methylotrophicus, as well as the use of Cadusafos alone and in combination with T. asperellum, B. subtilis, and B. methylotrophicus, resulted in a significant reduction (26.08%, 39.13%, and 21.73%, respectively) in the relative abundance of Fusarium spp. Moreover, the relative abundance of Trichoderma spp. significantly increased by 500%, 200%, and 100% at the genus level, respectively, compared to the control treatment. By constructing a co-occurrence network, we discovered a complex network structure among the species in all the bioproduct-treated groups. However, our findings indicate that the introduction of exogenous beneficial microbes into field conditions was unable to modulate the existing microbiota significantly. These findings suggest that the applied bioproducts had no significant impact on the reshaping of the overall microbial diversity in the rhizosphere microbiome but rather recruited selected microrganisms and assured net return to the grower. The results underscore the intricate nature of the rhizosphere microbiome and suggest the necessity for alternate biocontrol strategies and a re-evaluation of agricultural practices to improve nematode control by aligning with the complex ecological interactions in the rhizosphere.

Effect of pomegranate (Punica granatum) anthelmintic treatment on milk production in dairy sheep naturally infected with gastrointestinal nematodes.

Anthelmintic drug resistance has proliferated across Europe in sheep gastrointestinal nematodes (GINs). Sheep welfare and health are adversely impacted by these phenomena, which also have an impact on productivity. Finding alternatives for controlling GINs in sheep is thus of utmost importance. In this study, the anthelmintic effectiveness (AE) of a Calabrian ethnoveterinary aqueous macerate based on Punica granatum (whole fruits) was assessed in Comisana pregnant sheep. Furthermore, an examination, both qualitative and quantitative, was conducted on milk. Forty-five sheep were selected for the investigation. The sheep were divided by age, weight, physiological state (pluripara at 20 days before parturition), and eggs per gram of feces (EPG) into three homogeneous groups of 15 animals each: PG received a single oral dosage of P. granatum macerate at a rate of 50 mL per sheep; AG, treated with albendazole, was administered orally at 3.75 mg/kg/bw; and CG received no treatment. Timelines were as follows: D0, treatments, group assignment, fecal sampling, and AE assessment; D7, D14, D21, fecal sampling, and AE evaluation. The FLOTAC technique was used to evaluate the individual GIN fecal egg count (FEC) using a sodium chloride flotation solution (specific gravity = 1.20) and 100 × (1-[T2/C2]) as the formula for evaluating FEC reduction. Following the lambs' weaning, milk was collected on the following days (DL) in order to quantify production: DL35, DL42, DL49, DL56, DL63, DL70, DL77, and DL84. The amount of milk produced by every animal was measured and reported in milliliters (ml) for quantitative evaluations. Using MilkoScan TM fT + foss electric, Denmark, the quality of the milk (casein, lactose, protein concentration, and fat, expressed as a percentage) was assessed. The macerate demonstrated a considerable AE (51.8%). Moreover, its use has resulted in higher milk production rates quantitatively (15.5%) and qualitatively (5.12% protein, 4.12% casein, 4.21% lactose, and 8.18% fat). The study showed that green veterinary pharmacology could be the easiest future approach to counteracting anthelmintic resistance in sheep husbandry.

Adapting the in vitro rumen incubation method to evaluate the effect of a plant extract on the exsheathment inhibition of Haemonchus contortus infective larvae.

This study adapted the in vitro rumen incubation (IVRI) method to evaluate the biological activity of a Gymnopodium floribundum leaves extract against the exsheathment of Haemonchus contortus infective larvae (L3), and to determine the role of plant polyphenols on the biological activity. The incubation protocol followed the IVRI method, adding polyethylene glycol (PEG) as a polyphenol-blocking agent. The L3 were incubated in ruminal liquor (RL), ruminal liquor with PEG (RL+PEG), ruminal liquor with G. floribundum extract (RLE), and ruminal liquor with G. floribundum extract and PEG (RLE+PEG). Incubation condition controls included phosphate buffered saline (PBS), PBS with PEG (PBS+PEG), incubation medium (without ruminal liquor) (IM), and incubation medium with PEG (IM+PEG). The L3 were recovered after incubation times of 0, 1, 3, 6, 9, and 24 h (39 °C). The respective L3 exsheathment kinetics were estimated for the different treatments (RL, RL+PEG, RLE, and RLE+PEG) using Log-Logistic models. The parameters of the different models were compared to determine the impact of the extract, with or without PEG, on the L3 exsheathment kinetics. The exsheathment in PBS and PBS+PEG remained < 2.71% at each incubation time. The exsheathment in IM and IM+PEG reached 13.58% and 17.18% at 24 h, respectively. The exsheathment percentages for RLE were lower than those for RL at 3, 6 and 9 h of incubation. The inflection point, indicating the time required to reach 50% of the maximal exsheathment (T50), was the only parameter that differed between the ruminal liquor models. The T50 in RLE (7.106 h) was higher than the values obtained for RL (5.385 h) and RL+PEG (4.923 h) (99.99% probability of being different). Such delay resulted in a reduction of exsheathment in RLE of 62% at 3 h, 38% at 6 h, and 12% at 9 h, relative to RL values. When PEG was added with the extract (RLE+PEG), the T50 (5.045 h) was similar to that of RL and RL+PEG. The IVRI method was adapted as an in vitro rumen exsheathment test (IVRET). The IVRET showed that H. contortus L3 exposed to G. floribundum extract delayed their exsheathment kinetics at different time points. The exsheathment delay was attributed to the polyphenol content of the extract.

Evaluating the adverse effects and mechanisms of nanomaterial exposure on longevity of C. elegans: A literature meta-analysis and bioinformatics analysis of multi-transcriptome data.

Exposure to large-size particulate air pollution (PM2.5 or PM10) has been reported to increase risks of aging-related diseases and human death, indicating the potential pro-aging effects of airborne nanomaterials with ultra-fine particle size (which have been widely applied in various fields). However, this hypothesis remains inconclusive. Here, a meta-analysis of 99 published literatures collected from electronic databases (PubMed, EMBASE and Cochrane Library; from inception to June 2023) was performed to confirm the effects of nanomaterial exposure on aging-related indicators and molecular mechanisms in model animal C. elegans. The pooled analysis by Stata software showed that compared with the control, nanomaterial exposure significantly shortened the mean lifespan [standardized mean difference (SMD) = -2.30], reduced the survival rate (SMD = -4.57) and increased the death risk (hazard ratio = 1.36) accompanied by upregulation of ced-3, ced-4 and cep-1, while downregulation of ctl-2, ape-1, aak-2 and pmk-1. Furthermore, multi-transcriptome data associated with nanomaterial exposure were retrieved from Gene Expression Omnibus (GSE32521, GSE41486, GSE24847, GSE59470, GSE70509, GSE14932, GSE93187, GSE114881, and GSE122728) and bioinformatics analyses showed that pseudogene prg-2, mRNAs of abu, car-1, gipc-1, gsp-3, kat-1, pod-2, acdh-8, hsp-60 and egrh-2 were downregulated, while R04A9.7 was upregulated after exposure to at least two types of nanomaterials. Resveratrol (abu, hsp-60, pod-2, egrh-2, acdh-8, gsp-3, car-1, kat-1, gipc-1), naringenin (kat-1, egrh-2), coumestrol (egrh-2) or swainsonine/niacin/ferulic acid (R04A9.7) exerted therapeutic effects by reversing the expression levels of target genes. In conclusion, our study demonstrates the necessity to use phytomedicines that target hub genes to delay aging for populations with nanomaterial exposure.

Different Preharvest Diseases in Garlic and Their Eco-Friendly Management Strategies.

BACKGROUND: garlic reproduces mainly through clove planting, as sexual reproduction via seeds is uncommon. Growers encounter challenges with pathogens due to the larger size and vegetative nature of seed cloves, as well as the storage conditions conducive to fungal growth. Some Phyto-pathogenic fungi, previously unrecognized as garlic infections, can remain latent within bulb tissues long after harvest. Although outwardly healthy, these infected bulbs may develop rot under specific conditions. AIM OF REVIEW: planting diseased seed cloves can contaminate field soil, with some fungal and bacterial infections persisting for extended periods. The substantial size of seed cloves makes complete eradication of deeply ingrained infections difficult, despite the use of systemic fungicides during the preplanting and postharvest phases. Additionally, viruses, resistant to fungicides, persist in vegetative material. They are prevalent in much of the garlic used for planting, and their host vectors are difficult to eliminate. To address these challenges, tissue-culture techniques are increasingly employed to produce disease-free planting stock. Key scientific concepts of the review: garlic faces a concealed spectrum of diseases that pose a global challenge, encompassing fungal threats like Fusarium's vascular wilt and Alternaria's moldy rot, bacterial blights, and the elusive garlic yellow stripe virus. The struggle to eliminate deeply ingrained infections is exacerbated by the substantial size of seed cloves. Moreover, viruses persist in garlic seeds, spreading through carrier vectors, and remain unaffected by fungicides. This review emphasizes eco-friendly strategies to address these challenges, focusing on preventive measures, biocontrol agents, and plant extracts. Tissue-culture techniques emerge as a promising solution for generating disease-free garlic planting material. The review advocates for ongoing research to ensure sustainable garlic cultivation, recognizing the imperative of safeguarding this culinary staple from an array of fungal and viral threats.

Identification of Meloidogyne species on traditional Chinese medicine plants in the Qinling mountain area of China and their aggressiveness to different medicinal herbs.

Root-knot nematodes (Meloidogyne spp.) are plant-parasitic nematodes that cause serious damage on a worldwide basis. There are many species of traditional Chinese medicine (TCM) plants, but only a few have been reported to be infected by Meloidogyne species. From 2020 to 2022, a survey was conducted in the Qinling mountain area, which is the main producing region of TCM plants in China. Obvious galling symptoms were observed on the root systems of fifteen species of TCM plants. Females were collected from diverse diseased TCM plants and subsequently identified at morphological and molecular level. Among the twenty diseased root samples collected, Meloidogyne hapla populations were identified in twelve samples (60%) and Meloidogyne incognita populations were identified in eight samples (40%). Among the fifteen species of diseased TCM plants, eight of them, namely Scutellaria baicalensis, Leonurus japonicus, Dioscorea zingiberensis, Cornus officinalis, Viola philippica, Achyranthes bidentata, Senecio scandens, and Plantago depressa were reported to be infected by Meloidogyne species for the first time. The host status of five species of TCM plants for two M. hapla isolates and one M. incognita isolate from TCM plants in this study was then evaluated. Differences in TCM plants' response to nematode infection were apparent when susceptibility was evaluated by the egg counts per gram fresh weight of root and the reproduction factor of the nematodes. Among the five species of TCM plants tested, Salvia miltiorrhiza and Gynostemma pentaphyllum were the most susceptible, while S. baicalensis and V. philippica were not considered suitable hosts for M. hapla or M. incognita.

Botanicals against some important nematodal diseases: Ascariasis and hookworm infections.

Ascariasis and intestinal parasitic nematodes are the leading cause of mass mortality infecting many people across the globe. In light of the various deleterious side effects of modern chemical-based allopathic drugs, our preferences have currently shifted towards the use of traditional plant-based drugs or botanicals for treating diseases. The defensive propensities in the botanicals against parasites have probably evolved during their co-habitation with parasites, humans and plants in nature and hence their combative interference in one another's defensive mechanisms has occurred naturally ultimately being very effective in treating diseases. This article broadly outlines the utility of plant-based compounds or botanicals prepared from various medicinal herbs that have the potential to be developed as effective therapies against the important parasites causing ascariasis and intestinal hookworm infections leading to ascariasis & infections and thereby human mortality, wherein allopathic treatments are less effective and causes enormous side-effects.

Supplementation of minerals and vitamins influences optimal targeted selective treatment thresholds for the control of gastro-intestinal nematodes in lambs.

Targeted Selective Treatment (TST) is a gastrointestinal nematode (GIN) control strategy where anthelmintic treatment decisions are made at an individual animal level. TST has been proven to reduce anthelmintic use and subsequently slow down anthelmintic resistance development, however questions remain regarding optimal TST methods and their applicability across farms. In this study, the influence of Mineral and Vitamin (MV) supplementation on optimal energy utilisation (EU) TST thresholds was assessed on three Welsh farms. In total, 360 lambs were split into two groups, MV supplemented and control, and were treated with an anthelmintic against GIN at the midway point of the experiment. Lambs that improved their EU efficiency post treatment were deemed to have benefited from anthelmintic treatment. Optimal EU TST thresholds was determined for each treatment group per farm using Youden's J statistic where the treatment threshold retrospectively exhibiting the greatest combined sensitivity and specificity in correctly identifying lambs benefiting from treatment was deemed to be optimal. Results demonstrated that the optimal EU TST threshold was higher in MV supplemented groups at 0.72, 0.71 and 0.56 versus 0.58, 0.67, 0.51 for control groups on each respective farm. Identification of lambs for TST was more effective when using an optimised EU TST threshold, compared to when using the standard EU TST threshold of 0.66. The study highlights that applying standard EU TST thresholds may not be appropriate on all commercial farms with factors including MV status as noted in this study likely to influence optimal EU TST thresholds. Additional refinement of TST systems can further strengthen their applicability across sheep flocks.

Airsacculitis Caused by Enterobacteria and Occurrence of Eggs of the Superfamily Diplotriaenoidea in Feces of Tropical Screech Owl (Megascops choliba) in the Amazon Biome.

This study aims to report the clinical signs, therapeutic strategy, necropsy results, and histopathological findings of airsacculitis caused by enterobacteria and the occurrence of eggs from the superfamily Diplotriaenoidea in the feces of Megascops choliba in the Amazon biome. A tropical screech owl nestling was rescued and admitted for hand-rearing. The animal was kept hospitalized for five months. It was fed a diet based on Zophobas morio larvae and thawed chicken breast meat with vitamin and mineral supplements. On the 37th day of hacking training for release, the owl showed weakness, lack of appetite, regurgitation, cachexia, dyspnea, ruffled feathers, dry droppings in the vent and pericloaca, and diarrhea. The parasitological examination showed eggs of the Diplotriaenoidea superfamily in the feces. The therapy employed included oxytetracycline, sulfamethoxazole, mebendazole, Potenay, sodium chloride 0.9%, and Mercepton. However, five days after starting the treatment, the bird died. Upon necropsy, prominence of the keel, pieces of undigested food in the oral cavity and proventriculus, intestinal gas, and thickened and turbid air sacs were found. The microbiological analysis of air sacs identified Escherichia coli, Klebsiella pneumoniae, and Enterobacter aerogenes. Histopathological examination showed heterophilic bacterial airsacculitis.

Five Different Artemisia L. Species Ethanol Extracts' Phytochemical Composition and Their Antimicrobial and Nematocide Activity.

Among the plants that exhibit significant or established pharmacological activity, the genus Artemisia L. deserves special attention. This genus comprises over 500 species belonging to the largest Asteraceae family. Our study aimed at providing a comprehensive evaluation of the phytochemical composition of the ethanol extracts of five different Artemisia L. species (collected from the southwest of the Russian Federation) and their antimicrobial and nematocide activity as follows: A. annua cv. Novichok., A. dracunculus cv. Smaragd, A. santonica cv. Citral, A. abrotanum cv. Euxin, and A. scoparia cv. Tavrida. The study of the ethanol extracts of the five different Artemisia L. species using the methods of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-MS/MS) allowed establishing their phytochemical profile. The obtained data on the of five different Artemisia L. species ethanol extracts' phytochemical composition were used to predict the antibacterial and antifungal activity against phytopathogenic microorganisms and nematocidal activity against the free-living soil nematode Caenorhabditis elegans. The major compounds found in the composition of the Artemisia L. ethanol extracts were monoterpenes, sesquiterpenes, flavonoids, flavonoid glycosides, coumarins, and phenolic acids. The antibacterial and antifungal activity of the extracts began to manifest at a concentration of 150 µg/mL. The A. dracunculus cv. Smaragd extract had a selective effect against Gram-positive R. iranicus and B. subtilis bacteria, whereas the A. scoparia cv. Tavrida extract had a selective effect against Gram-negative A. tumefaciens and X. arboricola bacteria and A. solani, R. solani and F. graminearum fungi. The A. annua cv. Novichok, A. dracunculus cv. Smaragd, and A. santonica cv. Citral extracts in the concentration range of 31.3-1000 µg/mL caused the death of nematodes. It was established that A. annua cv. Novichok affects the UNC-63 protein, the molecular target of which is the nicotine receptor of the N-subtype.

In vitro anthelmintic activity of Euphorbia forskalii J. Gay aqueous extracts evaluation on different life stages of Haemonchus contortus.

The use of medicinal plants in the control of gastrointestinal parasitosis is a promising solution for improving the productivity of sheep flocks. In order to evaluate the anthelmintic activity of Euphorbia forskallii, in vitro bioassays were performed on three life stages of Haemonchus contortus. Five aqueous extracts concentrations namely 10 mg/mL; 5 mg/mL; 2.5 mg/mL; 1.25 mg/mL and 0.62 mg/mL were used for adult worm mortality tests. Egg hatch inhibition and L3 larval migration inhibition tests were studied at 5 mg/mL; 2.5 mg/mL; 1.25 mg/mL; 0.62 mg/mL and 0.31 mg/mL. A negative control PBS and a positive control levamisole 2.5 mg/mL were established for each test. A phytochemical screening was performed to determine the presence of some secondary metabolites. The results obtained showed the presence of total polyphenols, total flavonoids and condensed tannins within the aqueous extracts of E. forskalii. A high and significant (P < 0.05) morality rate compared to the negative control with an LC50 of 2.30 mg/mL was obtained. Inhibition of egg hatch and larval migration were high and significant (p < 0.05) compared to the negative control. There was an IC50 of 1.03 mg/mL and 0.92 mg/mL respectively for inhibition of egg hatching and L3 larval migration. The present study revealed the in vitro anthelmintic activity of E. forskalii aqueous extracts and allows us to consider in perspective complementary studies to confirm this activity.

Anthelmintic activity of European fern extracts against Haemonchus contortus.

Most drugs used in the treatment of helminthiasis in humans and animals have lost their efficacy due to the development of drug-resistance in helminths. Moreover, since anthelmintics, like many pharmaceuticals, are now recognized as hazardous contaminants of the environment, returning to medicinal plants and their products represents an environmentally friendly way to treat helminthiasis. The goal of the present study was to test the anthelminthic activity of methanol extracts of eight selected European ferns from the genera Dryopteris, Athyrium and Blechnum against the nematode Haemonchus contortus, a widespread parasite of small ruminants. Eggs and adults of H. contortus drug-susceptible strain ISE and drug-resistant strain WR were isolated from experimentally infected sheep. The efficacy of fern extracts was assayed using egg hatch test and adults viability test based on ATP-level measurement. Among the ferns tested, only Dryopteris aemula extract (0.2 mg/mL) inhibited eggs hatching by 25% in comparison to control. Athyrium distentifolium, Dryopteris aemula and Dryopteris cambrensis were effective against H. contortus adults. In concentration 0.1 mg/mL, A. distentifolium, D. aemula, D. cambrensis significantly decreased the viability of females from ISE and WR strains to 36.2%, 51.9%, 32.9% and to 35.3%, 27.0%, 23.3%, respectively in comparison to untreated controls. None of the extracts exhibited toxicity in precise cut slices from ovine liver. Polyphenol's analysis identified quercetin, kaempferol, luteolin, 3-hydroxybenzoic acid, caffeic acid, coumaric acid and protocatechuic acid as the major components of these anthelmintically active ferns.

Nematode community structures in the presence of wastewater treatment plant discharge.

Wastewater treatment plants (WWTPs) represent major point sources of pollution in coastal systems, affecting benthic ecosystems. In the present study, we assessed the potential role that WWTPs have in shaping nematode communities and established baseline knowledge of free-living nematode community structures in St. Andrew Bay, Florida. Sediment samples were collected from four sites representing areas of WWTP outflow and areas with no apparent outflow, during the winter and summer. Nematode communities across sites were significantly different, and the differences were strongly associated with the distance to the nearest WWTP. While the communities were not different along transects at each site, nor across seasons, community dissimilarity across sites was high, implying strong contrasts throughout the bay system. Dominance of tolerant, opportunistic genera and Ecological Quality Status assessments suggest that the system is stressed by organic enrichment, possibly linked to the WWTPs. Our results suggest that knowledge on the life-history of dominant genera is imperative to assess the ecological quality of a benthic system, in addition to taxonomic and functional metrics. Considering the value of marine nematodes as bioindicators, more work should be done to monitor temporal variability in nematode communities in this system as future infrastructure changes alter its dynamics.

Nematicidal activity of o-hydroxybenzaldehyde from common buckwheat methanol extract on Meloidogyne incognita.

The nematicidal activity of buckwheat (Fagopyrum esculentum Moench) on the root-knot nematode Meloidogyne incognita was tested. Dried plant methanol extract presented higher nematicidal activity than fresh plant extracts with an EC50 = 62.6 ± 26.0 and 40.8 ± 26.1 µg/ml after 48 and 72 hours of immersion, respectively. GC-MS analysis showed the presence of 17 aldehydes, with salicylaldehyde (o-hydroxybenzaldehyde) being the most abundant at 16%. Nematicidal activity of the latter refers to salicylaldehyde and other aldehydes with chemical similarities was then assessed. The most active aldehyde was o-hydroxybenzaldehyde followed by m-hydroxybenzaldehyde, p-hydroxybenzaldehyde and benzeneacetaldehyde with an EC50 of about 11.0 ± 1.0, 31.0 ± 22.0, 75.0 ± 23.0 and 168.1 ± 52.3 µg/ml after 1 day of immersion, respectively. Position 2 of the hydroxyl group in the benzene ring seems to be very important for the nematicidal activity, followed by positions 3 and 4. As a complementary experiment, synergistic activity was observed when we added o-hydroxybenzaldehyde to m-hydroxybenzaldehyde and to p-hydroxybenzaldehyde with an EC50 after 24 hours of immersion of 8.0 ± 2.5 and 6.1 ± 2.3 µg/ml, respectively. Antioxidant activity assessment showed that this latter is inversely proportional to nematicidal activity. Our results showed that F. esculentum and its major compound salicylaldehyde could be integrated into the pest management system.

Nematostatic activity of isoprenylated guanidine alkaloids from Pterogyne nitens and their interaction with acetylcholinesterase.

Although new nematicides have appeared, the demand for new products less toxic and more efficient for the control of plant-parasitic nematodes are still high. Consequently, studies on natural secondary metabolites from plants, to develop new nematicides, have increased. In this work, nineteen extracts from eleven Brazilian plant species were screened for activity against Meloidogyne incognita. Among them, the extracts of Piterogyne nitens showed a potent nematostatic activity. The alkaloid fraction obtained from the ethanol extract of leaves of P. nitens was more active than the coming extract. Due to the promising activity from the alkaloid fraction, three isoprenylated guanidine alkaloids isolated from this fraction, galegine (1), pterogynidine (2), and pterogynine (3) were tested, showing similar activity to the alkaloid fraction, which was comparable to that of the positive control Temik at 250 µg/mL. At lower concentrations (125-50 µg/mL), compound 2 showed to be the most active one. As several nematicides act through inhibition of acetylcholinesterase (AChE), the guanidine alkaloids were also employed in two in vitro AChE assays. In both cases, compound 2 was more active than compounds 1 and 3. Its activity was considered moderated compared to the control (physostigmine). Compound 2 was selected for an in silico study with the electric eel (Electrophorus electricus) AChE, showing to bind mostly to the same site of physostigmine in the AChEs, pointing out that this could be the mechanism of action for this compound. These results suggested that the guanidine alkaloids 1,2 and 3 from P. nitens are promising for the development of new products to control M. incognita, especially guanidine 2, and encourage new investigations to confirm the mechanism of action, as well as to determine the structure-activity relationship of the guanidine alkaloids.

Two in vitro anthelmintic assays of four Paraguayan medicinal plants for proof of concept of the role of polyphenols in their biological activities and LC-HRMS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Haemonchus spp., and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. THE AIMS OF OUR STUDY: Four medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occidentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. MATERIALS AND METHODS: To evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without polyvinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). RESULTS: C. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42-41.80 µg/mL) and A. absinthium on egg hatching processes (EC50 = 121.70-137.34 µg/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus, and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20-67.00% of larvae exsheathment, (p < 0.05) and 40.51-24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. CONCLUSIONS: The present study demonstrated that C. occidentalis, A. absinthium, and K. odorata, which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.

Computed tomography and radioactive 32P detected phosphorus impairment in metabolism, reduced bones density and animal performance caused by mixed infection of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

Phosphorus (P) is one of the main minerals present in the animal body and exerts crucial functions in the organism. P is present at all cell membranes and integrates the structure of bones, being necessary its supplementation in ruminants due to the deficiency of this mineral in the pastures. One of the principal factors that compromise its metabolization are gastrointestinal nematodes (GIN). Thus, the objective of this study was evaluate the performance and metabolism of P through its distribution in the animal body, density of bones and muscles, dynamic fluxes, biological availability and half live of P, concentration of P in tissues and bones of lambs simultaneously infected with the most prevalent GIN to sheep, in tropical or subtropical areas, (Haemonchus contortus and Trichostrongylus colubriformis) using the isotopic dilution technique with 32P radioisotope. Twenty Santa Ines sheep with seven months of age and averaging initial weight of 30.8 ± 6.41 kg were used and allocated to one of two treatments. Ten animals were orally infected (a single dose of 30,000 L3 larvae of T. colubriformis + 10,000 L3 larvae of H. contortus), and ten animals were not infected (control group). During the experimental, samples of blood, feces, urine, and diet refusals were collected and weighting were performed. A computed tomography was performed twice, before infection and at the end of the experiment, to evaluate changes in body composition. On 64-d after experimental infection, animals received an intravenous injection of 32P solution, and 7-d after they received radioisotope injection. The experimental animals were slaughtered, and tissue and bones were collected for P concentrations. The results showed that the parasitic infection compromised the absorption of P, impairing the metabolism, decreasing the mineral bioavailability increasing P bones reabsorption, and reducing bones density, also negatively compromising the infected animal performance.

Antiparasitic Tannin-Rich Plants from the South of Europe for Grazing Livestock: A Review.

Internal parasites are one of the main causes of health threats in livestock production, especially in extensive livestock farming. Despite the environmental toxic effects (loss of dung beetles, biodiversity, and other issues) and resistance phenomenon derived from their prolonged use, anti-parasitic chemical pharmaceuticals are frequently used, even in organic farming. Such a situation within the context of climate change requires urgent exploration of alternative compounds to solve these problems and apparent conflicts between organic farming objectives regarding the environment, public health, and animal health. This review is focused on some plants (Artemisia spp., Cichorium intybus L., Ericaceae family, Hedysarum coronarium L., Lotus spp., Onobrychis viciifolia Scop.) that are well known for their antiparasitic effect, are voluntarily grazed and ingested, and can be spontaneously found or cultivated in southern Europe and other regions with a Mediterranean climate. The differences found between effectiveness, parasite species affected, in vitro/in vivo experiments, and active compounds are explored. A total of 87 papers where antiparasitic activity of those plants have been studied are included in this review; 75% studied the effect on ruminant parasites, where gastrointestinal nematodes were the parasite group most studied (70%), and these included natural (31%) and experimental (37%) infections.