Adult Worm Motility Research Articles

In vitro anthelmintic activity of Chaetomorpha vieillardii ethanolic extract against adult worm motility and egg-hatching of Haemonchus contortus from sheep

This study aimed to evaluate the potential of macroalgae Chaetomorpha vieillardii as an in vitro candidate for bio-anthelmintic. Adult Worm Motility Test (AWMT) was conducted at concentration levels of 2 mg/mL, 4 mg/mL, and 6 mg/mL of C. vieillardii ethanol extract on 150 female H. contortus worms, randomly divided into 6 treatments with 5 replications, using a two way completely randomized factorial design. 0.9% sodium chloride was used as a negative control, and albendazole at 0.5 and 2 mg/mL served as positive controls. Worm motility observations were performed at 0, 0.25, 0.5, 1, 2, and subsequent time intervals until 100% of the worms were deceased. The identical treatment was applied to the egg hatching inhibiting test (EHIT) using a completely randomized design in a one-way pattern by observing the number of eggs at 0 and 24 hours. The results showed that the concentration level of C. vieillardii ethanol extract and the observation time significantly influenced (P<0.01) the motility of H. contortus, and there was an interaction between them (P<0.01). All three levels were able to decrease the motility of H. contortus, but they were unable to match albendazole. Despite of this result, the three levels were equally effective (P<0.01) as albendazole in terms of inhibiting the egg-hatching of H. contortus. The 2 mg/mL level was sufficient to inhibit the motility and egg-hatching of H. contortus. These findings indicated that the ethanol extract of C. vieillardii has the potential to be developed as a bio-anthelmintic for ruminants.

Methanolic Extract of Rhizophora mangle (Rhizophoraceae) Leaves: Phytochemical Characterization and Anthelmintic Evaluation against Schistosoma mansoni.

Rhizophora mangle is commonly used in traditional medicine to treat infections, reduce inflammation, and promote healing. This study aimed to analyze the phytochemical profile of the methanolic extract of R. mangle leaves (MELRm) and evaluate its in vitro schistosomicidal activity against Schistosoma mansoni as well as its cytotoxicity. Plant material was collected in Itamaracá City, Pernambuco, Brazil. The extract was analyzed using UV/Vis spectrophotometry and high-performance liquid chromatography (HPLC). The motility, mortality, and cell viability of adult worms were assessed in a schistosomicidal assay, while cytotoxicity was evaluated through a colorimetric assay with MTT on RAW 264.7 cells. The primary compounds identified in MELRm were phenolic compounds. In the schistosomicidal assay, all concentrations of MELRs induced changes in the motility of adult worms. At a concentration of 400 μg/mL, MELRs resulted in 56.25% mortality after 72 h of incubation. After 120 h, mortality rates of 75%, 62.5%, and 50% were observed at MELRm concentrations of 400, 200, and 100 μg/mL, respectively. No eggs were detected at any MELRm concentration. MELRs did not show cytotoxicity towards RAW 264.7 cells at the concentrations tested. These results indicate that MELRs demonstrate schistosomicidal activity in vitro, suggesting they are promising candidates for in vivo studies.

Anthelmintic activity and pathophysiological effect of anthelmintic drugs against carcinogenic liver fluke, Opisthorchis viverrini.

Human liver fluke, Opisthorchis viverrini poses a significant risk for development of cholangiocarcinoma (CCA) in Thailand, primarily attributed to consumption of undercooked cyprinoid fishes. The current use of anthelmintic drug treatment such as praziquantel (PZQ), as the main therapeutic agent against O. viverrini. There is a need to explore the efficacy of alternative anthelmintic drugs for O. viverrini treatment. This study aimed to assess the efficacy of anthelmintic drugs, which are commonly use in endemic areas of Southeast Asian countries; PZQ, albendazole (AL), niclosamide (NI), and mebendazole (ME) at concentrations of 600, 400, 500, and 500 mg/ml. The study included a negative and positive control group treated with roswell park memorial institute (RPMI) and PZQ. Reactive oxygen species (ROS) levels, indicative of oxidative stress, were quantified using 2',7'-dichlorofluorescein diacetate staining. Morphological changes were observed using scanning electron microscopy. Furthermore, motility assessments were conducted at various time points (0, 5, 30 minutes, 1, 3, 6, 12, and 24 hours), calculating relative motility (RM) and survival index (SI). The results revealed a significant increase of ROS levels with the intensity and corrected total worm fluorescence (CTWF) mostly observed in order of PZQ, followed by NI, ME, and AL, respectively. Morphological damage was presented the tegumental swelling, papillae changes, and disruption of microvilli (Mv), particularly in the group treated with the most effective anthelmintics PZQ, NI, ME, and AL, while negative control group did not exhibit such alterations. Also, the most efficacy for suppressing the motility of adult worms were displayed in PZQ treatment group, followed by NI, ME, and AL, respectively. Overall, first novel findings suggest that apart from NI, ME, and AL demonstrate potential as alternative therapeutic options for O. viverrini infection. Furthermore, animal model is needed to investigate the efficacy of NI, ME, and AL compare with standard treatment.

Anthelmintic Activity of Red Macroalgae Acrocystis sp. and Acanthophora sp. Etanolic Extract Against Haemonchus contortus in Sheep In Vitro

Abstract This study aimed to evaluate the anthelmintic activity of ethanolic extracts from red macroalgae Acrocystis sp. and Acanthophora sp. against the nematode Haemonchus contortus found in sheep, in vitro. The adult worm motility test was conducted in a factorial completely randomized design with two factors: the type of macroalgae as the first factor and the time of observation as the second factor. A total of 100 adult female H. contortus worms were randomly divided into four treatments with five replicates each. Each replicate consisted of 5 worms placed in a petri dish containing 5 mL of 0.9% sodium chloride solution (C: negative control), 1 mg/mL ethanolic extract of Acrocystis sp. (AR), 1 mg/mL ethanolic extract of Acanthophora sp. (AC), and 0.5 mg/mL albendazole (ALB: positive control). Sodium chloride 0.9% was used as the solvent for AR, AC, and ALB. The results demonstrated that, after 24 hour, AC exhibited anthelmintic activity by inhibiting the motility of H. contortus (P<0.05), to a remaining 4.00%, compared to AR (44.00%), although it could not compete with albendazole (P<0.05). Accordingly, the ethanolic extract of red macroalgae Acanthophora sp. holds potential for further investigation as an anthelmintic agent for ruminant livestock.

Evaluation of anthelmintic efficacy of ethanolic leaf extract of Juglans regia L. on Ascaridia galli: a comprehensive in vitro and in vivo study.

Anthelmintic resistance in livestock animals has been spreading across the world in prevalence and severity. As a result, researchers are exploring alternative strategies to combat this issue, and one promising avenue is the utilization of medicinal plants. This study aims to investigate the anthelmintic efficacy of the crude ethanolic extract (CEE) derived from the leaves of Juglans regia against one of the most detrimental nematode parasites affecting poultry, namely Ascaridia galli (A. galli). For the in vitro studies, adult A. galli worms were collected from the naturally infected chickens and the efficacy of CEE was measured at the concentration of 25, 50, and 100mg/ml using adult worm motility inhibition (WMI) assay. In addition, levamisole (0.55mg/ml) was used as the positive control. Likewise, Phosphate buffered saline (PBS) was used as the negative control. For the in vivo studies, CEE of J.regia at the doses of 500, 1000, and 2000mg/kg were evaluated in chickens experimentally infected with A. galli. The anthelmintic efficacy was monitored using faecal egg count reduction (FECR) and worm count reduction (WCR) assays. In vitro studies revealed significant (P < 0.001) anthelmintic effects of CEE of J.regia on the motility of A. galli worms at different hours post-exposure. At the concentration of 100mg/ml, CEE resulted in 96.5% inhibition of worm motility at 24h post-exposure. While the synthetic anthelmintic drug, levamisole caused the highest inhibition of worm motility (100%) at the same time period. The in vivo anthelmintic activity of CEE of J. regia demonstrated a maximum effect on day 14 post-treatment by inducing 67.28% FECR and 65.03% WCR. We observed no significant difference (P > 0.05) in worm counts between the negative control group and the chickens treated with CEE at the dosage of 500mg/kg. Together, the results of the present study suggest that CEE of J. regia leaves possess anthelmintic properties and could be a potential source of novel anthelmintic compounds for controlling helminth parasites.

In vitro and in vivo anthelmintic effect of essential oil obtained from Thymus capitatus flowers against Haemonchus contortus and Heligmosomoides polygyrus

Sheep haemonchosis is a disease that causes serious losses in livestock production, particularly with the increase of cases of anthelmintic resistance around the world. This justifies the urgent need of alternative solutions. The aim of this study was to determine the chemical profile, in vitro, and, in vivo, anthelmintic properties of Thymus capitatus essential oil. To evaluate the, in vitro, anthelmintic activity of the T. capitatus EO on Haemonchus contortus, two tests were used: egg hatch assay (EHA) and adult worm motility (AWM) assay. The nematicidal effect of this oil was evaluated, in vivo, in mice infected artificially with Heligmosomoides polygyrus using faecal egg count reduction (FECR) and total worm count reduction (TWCR). Chromatographic characterization of T.capitatus composition using gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of carvacrol (81.16%), as the major constituents. The IC50 values obtained was 1.9 mg/mL in the EHT. In the AWM assay; T. capitatus essential oil achieved 70.8% inhibition at 1 mg/mL after 8 h incubation. The in vivo, evaluation on H. polygyrus revealed a significant nematicidal effect 7 days post-treatment by inducing 49.5% FECR and 64.5% TWCR, using the highest dose (1600 mg/kg). The results of present study, demonstrate that T.capitatus EO possess a significant anthelmintic properties. Furthermore, it could be an alternative source of anthelmintic agents against gastrointestinal infections caused by H. contortus.

Emerging alternatives to traditional anthelmintics: the in vitro antiparasitic activity of silver and selenium nanoparticles, and pomegranate (Punica granatum) peel extract against Haemonchus contortus

Haemonchus contortus (H. contortus) is one of the most prevalent gastrointestinal nematodes, causing health problems and economic losses in ruminants. Nanotechnology holds great promise as a field of science, with potential applications in veterinary medicine. This study investigated the in vitro anthelmintic activity of silver nanoparticles (AgNPs), selenium nanoparticles (SeNPs), and pomegranate peel extract (Punica granatum; PPE) on different stages of H. contortus: eggs, larvae, and adults. The in vitro anthelmintic efficacy was evaluated using the egg hatching inhibition assay (EHA), the third larval stage paralysis assay (LPA), and the adult worm motility inhibition assay (WMI). Six dilutions of PPE were utilized for EHA, LPA, and WMI, ranging from 0.25 to 6 mg/ml. AgNPs dilutions ranged from 0.00001 to 1.0 μg/ml for EHA and LPA and 1 to 25 μg/ml for WMI. SeNPs were utilized at dilutions of 1, 5, 10, and 15 μg/ml for EHA, LPA, and WMI. The results showed that the lowest concentration of AgNPs, SeNPs, and PPE significantly inhibited egg hatching. To further assess larvicidal activity, AgNPs at the highest concentration of 1 μg/ml induced a strong larvicidal effect, as did SeNPs at the lowest concentration. On the contrary, PPE displayed a significant larvicidal effect at 1 mg/ml compared to the control. The percentage mortality of adult H. contortus was measured as follows (mortality (%) = the number of dead adult H. contortus/total number of adult H. contortus per test × 100). The death of the adult H. contortus was determined by the absence of motility. Adult H. contortus mortality percentage was also significantly affected by all three agents when compared to the control. The AgNPs, SeNPs, and PPE have effective antiparasitic activity on gastrointestinal parasitic nematodes. These results provide evidence of the excellent antiparasitic properties of AgNPs, SeNPs, and PPE, demonstrating their effectiveness in controlling eggs, larvae, and adult H. contortus in vitro.

A drug repurposing screen for whipworms informed by comparative genomics.

Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of ≤50 μM against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug development against whipworms.

In vitro Anthelmintic Activity of Croton tiglium Seeds Extract on Haemonchus contortus

Background: Haemonchus contortus is a blood sucking parasite whose habitat is the abomasum of ruminants, which poses a significant threat to the health and production of sheep and goats in warm temperate and tropical regions. H. contortus leads to anemia, hypoproteinemia, reduced exercise tolerance and edema subcutaneous tissue that can result in the death of animals. In acute infection, animals suddenly become anemic and many are found dead. Even in the chronic phase, the condition is accompanied by a mild but persistent parasitic burden and is characterized by weight loss, widespread malaise and, in some individuals, anemia. The objective of this study was to identify the anthelmintic activity of Croton tiglium seeds extract against Haemonchus contortus and comparison with some drugs used against internal and external parasites. Methods: A study was performed to evaluate the anthelmintic activity of the methanolic extract of Croton tiglium seeds against Haemonchus contortus using an adult worm motility test. Six graduated concentrations of extract (25, 50, 75, 100, 125 and 150 mg/ml), were tested at different periods and changes over time in the viability of worms were registered for 2, 4 and 6 hr. Normal RPMI-1640 medium and ivermectin 0.1% were used as negative and positive control, respectively. Also, a histological study was conducted on the the integumentary tissue of the H. contortus and the changes that occur in it were observed. Result: At 2 and 4 h post treatment, the concentrations of C. tiglium seeds extract (100, 125, and 150 mg/ml) have caused significantly higher mortality compared to the other concentrations, while, similar to ivermectin that caused death for nematodes within 6 h. The lower concentration (25 mg/ml) was significantly more lethal than the negative control (RPMI-1640 medium) at 2, 4 and 6 h of exposure. Each concentrations damages the cuticle and muscles of H. contortus. The increase in concentration is proportional to the increase in damage to the integumentary. The present study indicated that all concentrations of methanolic extract of C. tiglium seeds produced anthelmintic activity.

Chemical Composition, Anthelmintic Activity, and Mechanism of Action of Lippia dominguensis Mold. Essential Oil on Haemonchus contortus.

Gastrointestinal nematode parasitism is a major burden to small ruminant production globally, compounded by increasing anthelmintic resistance. Previous studies have identified essential oils (EOs) from the Lippia genus with antiprotozoal and anthelmintic effects. Lippia dominguensis Moldenke (Ld), an endemic specie from the Dominican Republic, has similar popular uses, however, is chemically and pharmacologically yet uncharacterized. Here, we investigated the in vitro anthelmintic activity of LdEO and its ultrastructural effects on eggs and adult nematodes of Haemonchus contortus multidrug-resistant isolated. The GC/MS analysis showed linalool (33.85 %), 1,8-cineole (30.88 %), and δ-terpineol (10.61 %) as the main EO constituents. The LdEO showed an IC50 =0.523 mg/mL in the egg hatch test, and the motility in the adult worm motility test was 95.8 % at 1 mg/mL. The confocal scanning laser microscopy of eggs indicated permeabilization or disruption of egg cell membranes as the possible mechanism of action of LdEO. The scanning electron microscopy of adult worms showed wrinkling, undulations, and cuticular disruptions. The LdEO displayed significant in vitro anthelmintic activity on eggs and adult worms of H. contortus. Additionally, the LdEO showed low oral toxicity in mice at 2,000 mg/kg. Thus, additional in vivo studies are justified to determine its anthelmintic efficacy in small ruminants.

Anthelmintic-Like Activity and Ultrastructure Changes Produced by Two Polyphenolic Combinations against Cooperia punctata Adult Worms and Infective Larvae.

Cooperia punctata is one of the most prevalent gastrointestinal nematodes affecting cattle under grazing conditions, and the increasing reports of anthelmintic resistance forces researchers to look for novel control measures. Previous reports have proposed the use of polyphenolic compound (PC) combinations (Coumarin:Quercetin (CuQ) and Caffeic-acid:Rutin (CaR)) against free-living stages (L3) of C. punctata. The objective of this study was to assess the in vitro motility inhibition of C. punctata adult worms and infective larvae using the Larval Motility Inhibition Assay (LMIA) and Adult Motility Inhibition Assay (AMIA), and to assess the structural and ultrastructural changes induced by both treatments using Scanning and Transmission Electron Microscopy. For the LMIA, infective larvae were incubated for 3 h in 0.8 mg mL-1 and 0.84 mg mL-1 of CuQ and CaR, respectively. For AMIA, six concentrations and five incubation periods (2, 4, 6, 12 and 24 h) were assessed using each PC combination. Cooperia punctata motility was calculated as a percentage and corrected using control motility percentages. A multiple comparisons Brown-Forsythe and Welch ANOVA test was used to compare larval motility; and to fit the dose-response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism® V.9.2.0. Although larval motility was barely affected by both treatments (p > 0.05), adult worm motility was inhibited 100% and 86.9% after 24 h incubation with CuQ and CaR, respectively (p < 0.05). The best fit EC50 for adult worm motility inhibition were 0.073 ± 0.071 mg mL-1 and 0.051 ± 0.164 mg mL-1 for CuQ and CaR, respectively. Main structural and ultrastructural lesions observed in both biological stages were: (i) L3 sheath-cuticle complex disruption, (ii) collagen fibers degradation; (iii) hypodermic detachment, (iv) seam cell apoptosis and (v) mitochondrial swelling. The alterations observed suggest that the PC combinations interfere with the anatomy and physiology of the locomotive apparatus of the nematodes.

Isotonic, aqueous-based media as simple and suitable test media for short-term Haemonchus placei adult worm motility assay

The solvating power of test media used in anthelmintic assays is critical to the validity of assay results, especially when evaluating plant extracts. High solutes in media lowers its solvating power, altering the range of concentrations available for investigation and assay performance. To identify simplified, well-tolerated media for adult Haemonchus placei with improved solvating power, we investigated the impact of varying solutions of pH (2.5–8.5), salinity (19–154 mM), and normal saline (NS) incorporating dissolution enhancers (acetone, propylene glycol, DMSO and Tween-80; 10–40% v/v) on the nematode over 3 h at room temperature. The performance of identified media, NS and 20% Tween-80 in NS, were evaluated by preparing sample extracts (acetone extract Sarcocephalus latifolius, AESL20&10; and chloroform extract Vernonia amygdalina, CEVA20&10) stock solutions (20 and 10 mg/mL) in them, assessed their apparent dissolution, and each highest stock solution that dissolves the extracts evaluated for anthelmintic activity against H. placei. We found isotonicity to be the critical-to-worm survival factor as H. placei survived 100% in pH solutions 3.5–8.5, and saline solutions 39–154 mM. The dissolution enhancers, at 40%, gave no survival. At 30% and 20%, only Tween-80 gave 92.5% and 100% survival, respectively. At 10%, Tween-80, acetone, DMSO and propylene glycol gave 100%, 100%, 87.5% and 0% survival, respectively. In 20% Tween-80 in NS, AESL20&10 and CEVA20&10 dissolved, furnishing wider concentration range (20-0 mg/mL); whereas only AESL10 dissolved in NS (narrower concentration range, 10-0 mg/mL). The LC50s (mg/mL) of 7.67 (AESL10, NS) and 7.48 (AESL20, Tween-80 in NS) were not significantly different (p > 0.05), while CEVA20 (Tween-80 in NS) gave 2.67. Our findings show that NS and 20% Tween-80 in NS, as isotonic, aqueous-based media, are suitable, and well-tolerated as test media for adult H. placei in a short-term motility assay. Up to 30% Tween-80 could be used to enhance dissolution where necessary.

In vitro Anthelmintic Activity of Vitis vinifera Leaf Extract on Dicrocoelium dendriticum

Background: Helminth parasites of ruminants are a major factor limiting animal production in many parts of the world. Dicrocoelium dendriticum, causes liver fluke disease in ruminants and is of zoonotic and economic importance. D. dendriticum lives in the adult stage, in the bile ducts and gallbladders of cattle, sheep, goats and pigs. Dicrocoeliasis causes severe pathological changes in the liver and bile system such as abscesses, granulomas and fibrosis. The objective of this study was to identify the anthelmintic activity of Vitis vinifera leaf extract against D. dendriticum and comparison with some drugs used against internal parasites. Methods: A study was performed to evaluate the anthelmintic activity of the methanolic extract of V. vinifera leaf against D. dendriticum using an adult worm motility test. Six graduated concentrations of extract (25, 50, 75, 100, a125 and 150 mg/ml), were tested at different periods and changes over time in the viability of worms were registered for 2, 4 and 6 hr. Normal RPMI-1640 medium and Albendazole were used as negative and positive control, respectively. Result: A significant elevation in mean inhibition of adult worm motility beginning 2 h post-exposure with 150 mg/ml of V. vinifera leaf extract and Albendazole. After 4 h of the exposure time, Albendazole and concentrations of 125 and 150 mg/ml of the extract resulted in significantly higher inhibited motility compared to the remaining concentrations below 125 mg/ml. The lower concentration (25 mg/ml) was significantly more lethal than the negative control (RPMI-1640 medium) at 6 h of exposure. Within 6 h after exposure, concentrations of 100, 125 and 150 mg/ml of V. vinifera leaf extract were more effective against adult worms than the remaining concentrations below 100 mg/ml. As the concentration and duration of exposure increased, the mortality of adult worms and also increased death for all parasites (P less than 0.05). Each concentration damages the tegument and muscles of D. dendriticum. The increase in concentration is proportional to the increase in damage to the integument. The results of this investigation demonstrated the anthelmintic action of V. vinifera leaf extract.

In vitro anthelmintic activity of Lippia alba essential oil chemotypes against Haemonchus contortus

The aim of the present study was to evaluate the effects of the essential oils of Lippia alba chemotypes carvone and citral on H. contortus. Chemical characterization was performed by means of gas chromatography coupled to mass spectrometry (GC/MS). The anthelmintic effects of the essential oils were assessed through the egg hatch test (EHT) and the adult worm motility test (AWMT) using a multidrug-resistant H. contortus Kokstad isolate. Confocal laser scanning microscopy (CLSM) of eggs and adults of H. contortus and scanning electron microscopy (SEM) of adults were performed after treatment with oils for qualitative observations of their effects. The carvone chemotype of L. alba (LaCV) presented 70% carvone, and the citral chemotype of L. alba (LaCT) presented 29.4% geranial and 20.4% neral, respectively. In the EHT, the EC50 values of LaCV and LaCT were 0.2 and 0.3 mg/mL, respectively. In AWMT, after 12 h of exposure to 2 mg/mL LaCV and 2 mg/mL LaCT, 100% of adult nematodes were immobile. CLSM showed changes in larval motility inside the egg caused by LaCV, while LaCT promoted changes in larval formation. In adults exposed to both chemotypes, alterations in the anterior portion of the oesophagus were observed. In SEM, morphological changes were observed in the buccal capsule and in the medial portion of H. contortus adults. It is concluded that the two essential oils of L. alba, the chemotypes carvone and citral, caused morphological changes and inhibited the hatching of eggs and the motility of adult H. contortus nematodes.

Microfluidic-Based Formulation of Essential Oils-Loaded Chitosan Coated PLGA Particles Enhances Their Bioavailability and Nematocidal Activity.

In this study, poly (lactic-co-glycolic) acid (PLGA) particles were synthesized and coated with chitosan. Three essential oil (EO) components (eugenol, linalool, and geraniol) were entrapped inside these PLGA particles by using the continuous flow-focusing microfluidic method and a partially water-miscible solvent mixture (dichloromethane: acetone mixture (1:10)). Encapsulation of EO components in PLGA particles was confirmed by Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction, with encapsulation efficiencies 95.14%, 79.68%, and 71.34% and loading capacities 8.88%, 8.38%, and 5.65% in particles entrapped with eugenol, linalool, and geraniol, respectively. The EO components’ dissociation from the loaded particles exhibited an initial burst release in the first 8 h followed by a sustained release phase at significantly slower rates from the coated particles, extending beyond 5 days. The EO components encapsulated in chitosan coated particles up to 5 μg/mL were not cytotoxic to bovine gut cell line (FFKD-1-R) and had no adverse effect on cell growth and membrane integrity compared with free EO components or uncoated particles. Chitosan coated PLGA particles loaded with combined EO components (10 µg/mL) significantly inhibited the motility of the larval stage of Haemonchus contortus and Trichostrongylus axei by 76.9%, and completely inhibited the motility of adult worms (p < 0.05). This nematocidal effect was accompanied by considerable cuticular damage in the treated worms, reflecting a synergistic effect of the combined EO components and an additive effect of chitosan. These results show that encapsulation of EO components, with a potent anthelmintic activity, in chitosan coated PLGA particles improve the bioavailability and efficacy of EO components against ovine gastrointestinal nematodes.

In vitro anthelmintic activity of an R-carvone nanoemulsions towards multiresistant Haemonchus contortus.

This work aimed to evaluate the in vitro anthelmintic effect of carvone nanoemulsions on Haemonchus contortus. Three R-carvone nanoemulsions were prepared: uncoated R-carvone nanoemulsions homogenized in a sonicator (UNAlg-son) and homogenized in an ultrahomogenizer (UNAlg-ultra) and sodium alginate-coated R-carvone (CNAlg-ultra). The physicochemical characterizations of the nanoemulsions were carried out. The anthelmintic activity was evaluated using egg hatch test (EHT), larval development test (LDT) and adult worm motility test (AWMT). Changes in cuticle induced in adult H. contortus were evaluated by scanning electron microscopy (SEM). The results were subjected to analysis of variance and compared using the Tukey test (P < 0.05). The effective concentration to inhibit 50% (EC50) of egg hatching and larval development was calculated. The particle sizes were 281.1 nm (UNAlg-son), 152.7 nm (UNAlg-ultra) and 557.8 nm (CNAlg-ultra), and the zeta potentials were −15 mV (UNAlg-son), −10.8 mV (UNAlg-ultra) and −24.2 mV (CNAlg-ultra). The encapsulation efficiency was 99.84 ± 0.01%. SEM of the nanoemulsions showed an increase in size. In EHT, the EC50 values of UNAlg-son, UNAlg-ultra and CNAlg-ultra were 0.19, 0.02 and 0.17 mg mL−1, respectively. In LDT, they were 0.29, 0.31 and 0.95 mg mL−1 for UNAlg-son, UNAlg-ultra and CNAlg-ultra, respectively. The adult motility inhibition was 100% after 12 h of exposure to UNAlg-ultra and CNAlg-ultra, while for UNAlg-son, it was 79.16%. SEM showed changes in the buccal capsule and cuticular damage. It was concluded that R-carvone nanoemulsions showed antiparasitic action demonstrating promise for the control of infections caused by gastrointestinal nematodes in small ruminants.

Nodulisporic acid produces direct activation and positive allosteric modulation of AVR-14B, a glutamate-gated chloride channel from adult Brugia malayi

Glutamate-gated chloride channels (GluCls) are unique to invertebrates and are targeted by macrocyclic lactones. In this study, we cloned an AVR-14B GluCl subunit from adult Brugia malayi, a causative agent of lymphatic filariasis in humans. To elucidate this channel's pharmacological properties, we used Xenopus laevis oocytes for expression and performed two-electrode voltage-clamp electrophysiology. The receptor was gated by the natural ligand L-glutamate (effective concentration, 50% [EC50] = 0.4 mM) and ivermectin (IVM; EC50 = 1.8 nM). We also characterized the effects of nodulisporic acid (NA) on Bma-AVR-14B and NA-produced dual effects on the receptor as an agonist and a type II positive allosteric modulator. Here we report characterization of the complex activity of NA on a nematode GluCl. Bma-AVR-14B demonstrated some unique pharmacological characteristics. IVM did not produce potentiation of L-glutamate-mediated responses but instead, reduced the channel's sensitivity for the ligand. Further electrophysiological exploration showed that IVM (at a moderate concentration of 0.1 nM) functioned as an inhibitor of both agonist and positive allosteric modulatory effects of NA. This suggests that IVM and NA share a complex interaction. The pharmacological properties of Bma-AVR-14B indicate that the channel is an important target of IVM and NA. In addition, the unique electrophysiological characteristics of Bma-AVR-14B could explain the observed variation in drug sensitivities of various nematode parasites. We have also shown the inhibitory effects of IVM and NA on adult worm motility using Worminator. RNA interference (RNAi) knockdown suggests that AVR-14 plays a role in influencing locomotion in B. malayi.

In vitro effect of three tropical plants on adult Haemonchus placei, an haematophagous nematode from cattle

BackgroundVernonia amygdalina (leaf), Garcinia kola (seed), and Leucaena leucocephala (seed) are three well-known tropical plants used in African ethnomedicine to reduce parasitic worm burdens and are potential sources of alternative solution for controlling parasitic helminths infection in grazing livestock. This study investigated extracts from these plants for anthelmintic activity against adult Haemonchus placei, an haematophagous nematode from cattle abomasa. Powdered plant materials were macerated in acetone and the crude acetone extracts evaluated for anthelmintic activity using H. placei adult worm motility assay. Afterwards, fresh sample of V. amygdalina was macerated successively in chloroform and acetone and the extracts evaluated for anthelmintic activity. The chloroform extract was subjected to phytochemical and FT-IR analyses and fractionated by vacuum liquid chromatography. Anthelmintic data were fitted to a nonlinear regression equation (Log [extract or fraction] vs. lethality; variable slope) to produce best-fit sigmoidal curves and LC50 values computed with associated uncertainty.ResultsOf the three tropical plants, only V. amygdalina was active against adult H. placei with best-fit LC50 of 6.51 mg/mL (95% CI: 5.32–7.75). Evaluation of the two extracts obtained by successive maceration showed that chloroform extract (LC50, 2.46 mg/mL, 95% CI: 1.87–3.28) was 11 times as potent as acetone extract (LC50, 27.01 mg/mL, 95% CI: 21.32–48.57) (α < 0.0001). Chromatographic fractionation of the chloroform extract yielded four fractions (FA-FD) with FB (LC50, 2.38 mg/mL, 95% CI: 1.76–3.28) 2.19 times as potent as FC (LC50, 5.21 mg/mL, 95% CI: 4.40–5.79) against H. placei, while FA and FD were inactive. Phytochemical evaluation of the chloroform extract revealed the presence of saponins, steroids, terpenoids, cardiac glycosides, and the absence of tannins, flavonoids, alkaloids, and anthraquinones. FT-IR structural analysis of chloroform extract indicated the presence of key functional groups which are chemical fragments/ structural motifs known to be present in the two major classes of bioactive compounds (sesquiterpene lactones and steroid glucosides) reportedly to be found in V. amygdalina.ConclusionsThe findings showed that chloroform extract of V. amygdalina leaf possessed relatively good anthelmintic activity against adult H. placei. This could be indicative of its potential usefulness as an anthelmintic phytomedicine to control gastrointestinal nematodes infection in cattle.Key highlightsExtracts of three different plant materials (one leaf, two seeds) were tested against adult Haemonchus placei in vitro;Chloroform extract of Vernonia amygdalina was 11 times as potent as acetone extract;Fractionation of the chloroform extract yielded a bioactive fraction responsible for about 90% of the total lethal effect of the chloroform extract.Bioprocessing of V. amygdalina leaf could produce phytomedicines for organic livestock farming.Graphical abstract

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Schistosome TRPML channels play a role in neuromuscular activity and tegumental integrity

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma. Mono-therapeutic treatment of this disease with the drug praziquantel, presents challenges such as inactivity against immature worms and inability to prevent reinfection. Importantly, ion channels are important targets for many current anthelmintics. Transient receptor potential (TRP) channels are important mediators of sensory signals with marked effects on cellular functions and signaling pathways. TRPML channels are a class of Ca2+-permeable TRP channels expressed on endolysosomal membranes. They regulate lysosomal function and trafficking, among other functions. Schistosoma mansoni is predicted to have a single TRPML gene (SmTRPML) with two splice variants differing by 12 amino acids. This study focuses on exploring the physiological properties of SmTRPML channels to better understand their role in schistosomes. In mammalian cells expressing SmTRPML, TRPML activators elicit a rise in intracellular Ca2+. In these cells, SmTRPML localizes both to lysosomes and the plasma membrane. These same TRPML activators elicit an increase in adult worm motility that is dependent on SmTRPML expression, indicating a role for these channels in parasite neuromuscular activity. Suppression of SmTRPML in adult worms, or exposure of adult worms to TRPML inhibitors, results in tegumental vacuolations, balloon-like surface exudates, and membrane blebbing, similar to that found following TRPML loss in other organisms. Together, these findings indicate that SmTRPML may regulate the function of the schistosome endolysosomal system. Further, the role of SmTRPML in neuromuscular activity and in parasite tegumental integrity establishes this channel as a candidate anti-schistosome drug target.