Worm Motility Research Articles

In Vitro and in Vivo Anthelmintic Activities of Aqueous Leaf Infusion of Azadirachta indica against Haemonchus contortus

Anthelmintic resistance of Haemonchus contortus worm has become a major problem in ruminant production. Traditional medicinal and potential tropical plants with high tannin contents have a great potency as an alternative source of bio-anthelmintic. The study was carried out to assess the in vitro egg hatches inhibiting (EHI) and adult worm motility (AWM) tests and in vivo anthelmintic activities of aqueous leaf infusion of Azadirachta indica against H. contortus. Leaf infusion at doses of 2%, 4%, and 6% were used for in vitro treatments, and albendazole at a dose of 2 mg/mL was used as a positive control and 0.9% of sodium chloride was used as a negative control. The EHI assay was conducted two times i.e., before treatment and 24 h after treatment. The AWM were monitored 15 and 30 min, and 1, 2, 3, 4, 5, 6, 7, and 8 h post treatment. Sixteen Javanese Thin Tail ewes at the ages of ±12 months that naturally positive of H. contortus with about 1.000 EPG were selected and allocated randomly to 4 groups (n= 4). They were subsequently received the ensuing treatments i.e., A. indica infusion at doses of 0% (A) as a control, 6% (B), 8% (C), and 5 mg/BW albendazole (D) at a single oral dose as a positive control. Experimental ewes in Groups B and C were given oral infusion weekly during 4 weeks of treatment. EPG’s fecal examinations were conducted weekly. The result showed that the leaf infusion of A. indica containing condensed tannin (CT) at a dose of 6% significantly inhibited egg hatching (P<0.05) with a better effect compared to doses of 0%, 2%, and 4%. In AWM test, three doses of infusion significantly difference each other, as well as both negative and positive controls (P<0.05). After treatment, both of the A. indica containing CT levels and albendazole reduced EPG count significantly (P<0.05). Meanwhile, significant difference was not detected in dose of 8% aqueous leaf infusion of A. indica and albendazole treatments on weeks 3, 4, 5, and 6 consecutively. In conclusion, the aqueous leaf infusion of A. indica is fully potential as a bio anthelmintic against H. contortus.

Development of transgenic Caenorhabditis elegans expressing human transthyretin as a model for drug screening

Familial amyloid polyneuropathy is a hereditary systemic amyloidosis caused by a mutation in the transthyretin (TTR) gene. Amyloid deposits in tissues of patients contain not only full-length TTR but also C-terminal TTR fragments. However, in vivo models to evaluate the pathogenicity of TTR fragments have not yet been developed. Here, we generated transgenic Caenorhabditis elegans strains expressing several types of TTR fragments or full-length TTR fused to enhanced green fluorescent protein in the body wall muscle cells and analyzed the phenotypes of the worms. The transgenic strain expressing residues 81–127 of TTR, which included the β-strands F and H, formed aggregates and caused defective worm motility and a significantly shortened lifespan compared with other strains. These findings suggest that the C-terminal fragments of TTR may contribute to cytotoxicity of TTR amyloidosis in vivo. By using this C. elegans model system, we found that (−)-epigallocatechin-3-gallate, a major polyphenol in green tea, significantly inhibited the formation of aggregates, the defective motility, and the shortened lifespan caused by residues 81–127 of TTR. These results suggest that our newly developed C. elegans model system will be useful for in vivo pathological analyses of TTR amyloidosis as well as drug screening.

Effects of Eucalyptus citriodora essential oil and its major component, citronellal, on Haemonchus contortus isolates susceptible and resistant to synthetic anthelmintics

This study aimed to evaluate the effects of Eucalyptus citriodora essential oil and citronellal on a Haemonchus contortus isolate susceptible to anthelmintics, the Inbred-susceptible Edinburgh (ISE) and a resistant Kokstad isolate. The oil was analyzed by gas chromatography (GC). The products were evaluated using an egg hatching test (EHT), a larval development test (LDT) and an adult worm motility test (AWMT). The effective concentrations to inhibit 50% (EC50) of egg hatching and larval development and resistance ratios (RRs) were calculated. The results were analyzed by analysis of variance and Tukey’s or Bonferroni’s test. The major constituent of E. citriodora oil was citronellal (63.9%). According to EHT, EC50 values for the ISE isolate were 0.4 mg/ml and 0.3 mg/ml; for the Kokstad isolate, values were 0.5 mg/ml and 0.4 mg/ml for oil and citronellal, respectively. LDT EC50 values were 2.9 mg/ml and 2.3 mg/ml for the ISE isolate and 3.2 mg/ml and 2.4 mg/ml for the Kokstad isolate for oil and citronellal, respectively. In AWMT, 2 mg/ml of oil and citronellal completely inhibited the motility of both the ISE and Kokstad isolates. RRs between the isolates ranged from 1 to 1.3. No significant differences were observed between the isolates or treatment comparisons (P >0.05). Therefore, E. citriodora oil and citronellal both exhibited ovicidal and larvicidal effects and inhibited the motility of both H. contortus isolates.

Arctigenin: An emerging candidate against infections of Gyrodactylus

Gyrodactylosis is a common parasitic disease that affects both farmed and wild fish. Current strategies of chemotherapy are not always satisfactory, requiring the discovery of new anthelmintic agents. Arctigenin is a natural dibenzylbutyrolactone lignin present in several plants with many biological activities. In this study, in vitro and in vivo anthelminthic efficacy of arctigenin against Gyrodactylus kobayashii in goldfish (Carassius auratus) is reported for the first time. In in vitro trials, exposure to arctigenin caused rapid declines in motility and ultimate death of worms. A positive correlation was seen between arctigenin concentrations and the time to parasite death, which, at the highest concentration of 8 mg/L occurred at 33 min. Bathing G. kobayashii-infected goldfish in arctigenin significantly reduced infection prevalences and intensities as compared to the control, with EC50 values of 1.85 and 1.58 mg/L after 24 and 48-h exposure, respectively, with 100% efficacy against G. kobayashii at 4.0 mg/L after only 4 h. Acute toxicity assay indicated that the LC50 of arctigenin against goldfish after 96-h exposure was 11.63 mg/L, which was 6.29 times higher than the 24 h EC50. The expression of four xenobiotic sensitive genes (cyp1a, hsp70, gst and sod) were significantly altered in the initial 48 h after arctigenin treatment, but had a tendency of returning to the initial level of gene expression after 96 h. In addition, SEM and TEM were used to study the specific action of arctigenin against gyrodactylids. SEM revealed that arctigenin-mediated worm killing was associated with tegumental damage and with damage seen by TEM to the mitochondria may be a cause of parasite death. Collectively, these findings demonstrate the potential of arctigenin as an alternative natural compound for controlling infections of Gyrodactylus. With further medicinal chemistry optimization, more potent arctigenin analogues offer promise for future combinatorial or replacement anthelmintic control

In vitro effects of aqueous extract from Maytenus senegalensis (Lam.) Exell stem bark on egg hatching, larval migration and adult worms of Haemonchus contortus

BackgroundMaytenus senegalensis is a common shrub which is scattered in tropical Africa. Different parts of this plant have been reported to be useful in traditional medicine against gastrointestinal disorders and intestinal worms. This study evaluated the anthelmintic activity of the aqueous stem bark extract of M. senegalensis using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worms’ motility inhibition assay (AMIA).ResultsOn EHA, the extract concentrations tested resulted in a significant (p < 0.01) inhibition of egg hatching in concentration-dependent manner and ranged between 31.86% at 75 μg.mL− 1 to 54.92% at 2400 μg.mL− 1 after a 48 h post-exposure with eggs. For the LMI assays, the aqueous extract of M. senegalensis showed a significant (p < 0.05) inhibition of larval migration in a concentration-dependent manner. The highest concentration used (2400 μg.mL− 1) showed a 37.77% inhibition. The use of polyvinyl polypyrrolidone (PVPP) indicated that tannins and flavonoids were partly involved in the effect since the larval migration was inhibited by 15.5%, but other biochemical compounds were also implicated. On AMIA, M. senegalensis was associated with a reduced worm motility after a 24 h post exposure compared to phosphate buffered saline as control (p < 0.05). By this time 66.66% of the worms’ were found immotile or dead in the wells containing plant extract at 2400 μg.mL− 1. The Phytochemical analysis of aqueous extract of M. senegalensis by HPLC-ESI-MSn detected the presence of proanthocyanidins (20%) and flavonoids (> 50%).ConclusionsThese in vitro results suggest the presence of some anthelmintic properties in M. senegalensis extract, which is traditionally used by small farmers in west and central Africa. These effects may be due to the flavonoids and proanthocyanidins present in the extract and need to be studied under in vivo conditions.

Essential oils of Citrus aurantifolia, Anthemis nobile and Lavandula officinalis: in vitro anthelmintic activities against Haemonchus contortus

BackgroundInfections of sheep with gastrointestinal parasites, especially Haemonchus contortus, have caused serious losses in livestock production, particularly after the emergence of resistance to conventional anthelmintics. The search for new anthelmintic agents, especially those of botanical origin, has grown substantially due to the perspective of less contamination of meat and milk, as well as other advantages related to their cost and accessibility in less developed countries. The aim of this study was to evaluate the in vitro anthelmintic activity of essential oils of the plant species Citrus aurantifolia, Anthemis nobile and Lavandula officinalis against the main developmental stages of the parasite H. contortus.ResultsPlant species were selected based on substantial ethnopharmacological information. Analysis of the composition of each oil by gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of limonene (56.37%), isobutyl angelate (29.26%) and linalool acetate (35.97%) as the major constituents in C. aurantifolia, A. nobile and L. officinalis, respectively. Different concentrations of each oil were tested in vitro for their capacity to inhibit egg hatching (EHT), larval development (LDT) and adult worm motility (AWMT) using a multidrug-resistant strain of H. contortus (Embrapa 2010). The IC50 values obtained for the oils of C. aurantifolia, A. nobile and L. officinalis were 0.694, 0.842 and 0.316 mg/ml in the EHT and 0.044, 0.117 and 0.280 mg/ml in the LDT, respectively. The three oils were able to inhibit adult worm motility completely within the first 8–12 h of observation in the AWMT.ConclusionsThe present results demonstrate significant anthelmintic activity of the three oils against the different developmental stages of H. contortus. Furthermore, this study is of ethnopharmacological importance by validating the anthelmintic activity of the oils studied. Although new experiments are necessary, these data contribute to the development of pharmaceutical-veterinary products for sheep farming by opening up new therapeutic possibilities against gastrointestinal infections caused by H. contortus.

Inhibition efficiency of a newly isolated flavonoid compound from Vitex negundo L. leaves against cattle-endosymbiont Setaria cervi: Phytomedicine for lymphatic filariasis

Experimental studies has been carried out to isolate and identify an active antifilarial compound from Vitex negundo L. plant as it has been used for treatment against filariasis in Indian traditional system of medicine. In vitro antifilarial assay has been carried out against adult filarial parasite Setaria cervi worms by both worm motility and MTT reduction assays. Levels of oxidative stress parameters MDA, carbonyl content and nitric oxide levels have been detected. The isolated compound exhibited significant antifilarial activity in dose dependent manner. The active compound has been chemically characterized and identified as 4,5-diethyl-3′-ethoxy-pyro-flavone.

Anthelmintic efficacy of cranberry vine extracts on ovine Haemonchus contortus

The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress gastrointestinal nematode (GIN) infection has provided promise for alternative methods of GIN control in small ruminants. This investigation is the first to examine the anthelmintic potential of cranberry vine (CV) against the GIN Haemonchus contortus. The purpose of this study was to explore the anti-parasitic activity of CV in the form of a specific organic proanthocyanidin extract (CV-PAC) and an aqueous extract (CV-AqE) containing PAC and other compounds. In vitro egg hatching, first (L1) and third (L3) stage larval and adult worm motility and L3 exsheathment were evaluated after a 24-h incubation with CV products. In addition, CV treated worms were observed via scanning electron microscopy, and a preliminary investigation of the efficacy of CV powder against an experimental infection of H. contortus was conducted. The in vivo effect on an experimental infection was determined by administering 21.1 g CV powder to lambs (n = 9 per group) for three consecutive days, and collecting fecal egg count data for four weeks post-treatment. The effect of CV-PAC on egg hatching, L3 motility and exsheathment was limited. However, a substantial effect was observed on motility of post-hatch L1 (EC50 0.3 mg PAC/mL) and adults (EC50 0.2 mg PAC/mL). The CV-AqE showed more effect on egg hatching (EC50 5.3 mg/mL containing 0.6 mg PAC/mL) as well as impacting motility of L1 (EC50 1.5 mg/mL with 0.2 mg PAC/mL) and adults (EC50 3.4 mg/mL with 0.4 mg PAC/mL), but like CV-PAC, did not substantially effect L3 motility or exsheathment. Scanning electron microscopy revealed an accumulation of aggregate on the cuticle around the buccal area of adult worms incubated in CV-AqE and CV-PAC. In the preliminary in vivo study, there was a significant effect of treatment over time (p = .04), although differences in individual weeks were not significant. In summary, both extracts inhibited motility of L1 and adult worms. The higher efficacy of CV-AqE than CV-PAC at levels that contained the same concentrations of PAC tested alone, suggest that other secondary compounds in the CV-AqE contributed to the observed effects on the parasites. This first study of the in vitro and in vivo effects of CV suggest that this readily available plant product may have utility in integrated control of H. contortus and support the need for additional testing to provide further information.

Subrenal Filaria with Macular Hole: A Rare Case

Introduction: In Indonesia, parasitic infection of the eye is rarely reported, however it can cause blindness. A parasitic disease affecting whole part of eye include retinal layers unilaterally or bilaterally. Onchocercia volvulus, Loa-loa, Brugia malayi and Wuchereria bancrofti have been reported as specific species which can infect retina. The organism can survive up to 4 years in the subretinal space. Objective: To report a rare case of a subretinal worm infection and its management. Case Presentation: A 35-year-old male complaint a sudden loss of vision and felt that something moving within his left eye. He had scotoma and BCVA dropped to 1/60. The anterior segment was normal. Indirect ophthalmoscopy revealed a living worm around the macula and a macular hole. Peripheral retina seen as grayish-white lesion. Pars plana vitrectomy were performed with laser photocoagulation to inactivate the worm. 6 mm worm were evacuated through iatrogenic retinectomy in inferior macula. Continued with ILM peeling and gas tamponade for the macular hole. The parasitologist identified an adult filarial worm and suggested for nocturnal blood smear analysis. Infectious disease specialist suggested diethylcarbamazine citrate (DEC) 6 mg/kg body weight for 28 week. Conclusion: Adult filarial worm in subretinal space can cause severe damage and macular hole by worm motility. However, rapid and right management is the primary choice to inhibit this damage and to detect the species.

DPPH antiradical scavenging, anthelmintic and phytochemical studies of Cissus poulnea rhizomes

Objective: To investigate the phytochemical constituents, antioxidant and anthelmintic activities of the crude methanol extract of Cissus populnea (C. populnea) rhizomes. Methods: Phytochemical screening was performed using standard protocols, and column chromatography of silica gel was used for the compounds isolation. DPPH antiradical scavenging assay was performed in order to evaluate the antioxidant activity. Total phenolic content was evaluated using the Folin–Ciocalteu assay. The anthelmintic activity was screened on the bovine adult male forms of parasitic nematode Onchocerca ochengi, by the in vitro evaluation of the inhibition of adult worm motility and mortality. Worms were incubated in the presence of different concentrations of the plant extract and effects on survival were monitored after 24 and 48 h. Results: The preliminary phytochemical screening revealed the presence of phenolic compounds, saponins, steroids, tannins, and terpenoids. Bergenin and a mixture of phytosterol, β -sitosterol and stigmasterol were isolated from this extract and were identified by nuclear magnetic resonance, mass spectrometry and by comparison with published data. The crude methanol extract of C. populnea rhizomes showed a strong DPPH antiradical activity with a good amount of total phenolic content ((20.69±2.13) g gallic acid equivalent/100 g of extract) and significant anthelmintic activity comparable to the standard drug ivermectin. Bergenin was found to be inactive even after 72 h of incubation. Conclusions: This study constitutes the first report on the anthelmintic activity of this plant and supports the traditional use of C. populnea as a natural antioxidant and anthelmintic.

An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes

Parasitic nematodes infect hundreds of millions of people and farmed livestock. Further, plant parasitic nematodes result in major crop damage. The pipeline of therapeutic compounds is limited and parasite resistance to the existing anthelmintic compounds is a global threat. We have developed an INVertebrate Automated Phenotyping Platform (INVAPP) for high-throughput, plate-based chemical screening, and an algorithm (Paragon) which allows screening for compounds that have an effect on motility and development of parasitic worms. We have validated its utility by determining the efficacy of a panel of known anthelmintics against model and parasitic nematodes: Caenorhabditis elegans, Haemonchus contortus, Teladorsagia circumcincta, and Trichuris muris. We then applied the system to screen the Pathogen Box chemical library in a blinded fashion and identified compounds already known to have anthelmintic or anti-parasitic activity, including tolfenpyrad, auranofin, and mebendazole; and 14 compounds previously undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers an effective, high-throughput system for the discovery of novel anthelmintics.

Low virulence potential and in vivo transformation ability in the honey bee venom treated Clinostomum complanatum

The helminth parasites possess great capabilities to adapt themselves within their hosts and also develop strategies to render the commonly used anthelmintics ineffective leading to the development of resistance against these drugs. Besides using anthelmintics the natural products have also been tested for their anti-parasitic effects. Therapeutic efficacy of honey bee venom (HBV) has been tested in various ailments including some protozoal infections but very little is known about its anthelmintic properties. To investigate the anthelmintic effect of HBV the excysted progenetic metacercariae of Clinostomum complanatum, a heamophagic, digenetic trematode with zoonotic potential, infecting a wide variety of hosts, were obtained from Trichogaster fasciatus, a forage fish, which serves as the intermediate host. The metacercarial worms were in vitro incubated in RPMI-1640 medium containing HBV along with the controls which were devoid of HBV for the analysis of worm motility, enzyme activity, polypeptide profile and surface topographical changes.The motility of the worms was significantly reduced in a time dependent manner with an increase in the concentration of HBV. Following incubation of worms the release of cysteine proteases was inhibited in the presence of HBV as revealed by gelatine substrate gel zymography. As well as the polypeptide profile was also significantly influenced, particularly intensity/expression of Mr 19.4 kDa, 24 kDa and 34 kDa was significantly reduced upon HBV treatment. The HBV treatment also inhibited antioxidant enzyme, superoxide dismutase (SOD) and Glutathione-S-transferase (GST) significantly (p < 0.05) in the worms. The scanning electron microscopy of the HBV treated worms revealed tegumental disruptions and erosion of papillae as well as spines showing vacuolation in the tegument. The HBV treated worms also showed a marked decline in the transformation rate when introduced into an experimental host which further reflect the anthelmintic potential of HBV.

Simvastatin exerts antifibrotic effect and potentiates the antischistosomal effects of praziquantel in a murine model: Role of IL10

Previous studies on simvastatin use in experimental schistosomiasis in mice did not provide a full explanation of its mechanism as antischisome. In this study, we tried to find out the role of IL-10 in the mechanism of action of simvastatin. We used 50 clear mice. Ten were used as normal not treated while 40 were infected with shistosome mansoni then divided into 4 groups; 3 treated groups by praziquantel, simvastatin and combined (praziquantel plus simvastatin) respectively and one group non-treated. The simvastatin treated group showed shortening and loss of the tubercles and disappearance of the spines with swelling and twisted shape of the worms. In addition, it also showed mitigation of ovideposition activity of the worms in the liver and reduction of the fibrous component of the liver granuloma producing a protective effect on the liver. This effect was associated with lowering of IL-10. This may explain the role of IL-10 in the protective effect of simvastatin. Combination of treatment with simvastatin plus praziquantel produced more significant effects in different parameters compared with praziquantel treated group. We recommend using simvastatin as add on therapy to standard antischistosomal therapy, praziquantel. Both drugs affect the worm motility and sucker activity and the ova deposition. Simvastatin has an additional pleiotropic effect halting inflammation and decreasing fibrosis due to increasing IL-10 leading to a hepatoprotective effect. Further clinical studies are needed to further validate these findings.

Antifilarial activity of caffeic acid phenethyl ester on Brugia pahangi in vitro and in vivo

Lymphatic filariasis (LF) is a vector borne disease caused by parasitic worms such as Wuchereria bancrofti, Brugia malayi and B. timori, which are transmitted by mosquitoes. Current therapeutics to treat LF are mainly microfilarcidal, and lack activity against adult worms. This set back, poses a challenge for the control and elimination of filariasis. Thus, in this study the activities of caffeic acid phenethyl ester (CAPE) against the filarial worm B. pahangi and its bacterial endosymbiont, Wolbachia were evaluated. Different concentrations (2, 5, 10, 15, 20 μg/ml) of CAPE were used to assess its effects on motility, viability and microfilarial (mf) production of B. pahangi in vitro. Anti-Wolbachial activity of CAPE was measured in worms by quantification of Wolbachial wsp gene copy number using real-time polymerase chain reaction. Our findings show that CAPE was found to significantly reduce adult worm motility, viability, and mf release both in vitro and in vivo. 20 μg/ml of CAPE halts the release of mf in vitro by day 6 of post treatment. Also, the number of adult worms recovered in vivo were reduced significantly during and after treatment with 50 mg/kg of CAPE relative to control drugs, diethylcarbamazine and doxycycline. Real time PCR based on the Wolbachia ftsZ gene revealed a significant reduction in Wolbachia copy number upon treatment. Anti-Wolbachia and antifilarial properties of CAPE require further investigation as an alternative strategy to treat LF.

Anthelmintic effect of thymol and thymol acetate on sheep gastrointestinal nematodes and their toxicity in mice.

Thymol is a monoterpene and acetylation form of this compound can reduce the toxicity and enhance its biological effects. The objective of this study was to evaluate the effect of thymol and thymol acetate (TA) on egg, larva and adult Haemonchus contortus and the cuticular changes, acute toxicity in mice and the efficacy on sheep gastrointestinal nematodes. In vitro tests results were analyzed by analysis of variance (ANOVA) and followed by comparison with Tukey test or Bonferroni. The efficacy of in vivo test was calculated by the BootStreet program. In the egg hatch test (EHT), thymol (0.5 mg/mL) and TA (4 mg/mL) inhibited larval hatching by 98% and 67.1%, respectively. Thymol and TA (8 mg/mL) inhibited 100% of larval development. Thymol and TA (800 µg/mL) reduced the motility of adult worms, by 100% and 83.4%, respectively. Thymol caused cuticular changes in adult worm teguments. In the acute toxicity test, the LD50 of thymol and TA were 1,350.9 mg/kg and 4,144.4 mg/kg, respectively. Thymol and TA reduced sheep egg count per gram of faeces (epg) by 59.8% and 76.2%, respectively. In in vitro tests thymol presented better anthelmintic activity than TA. However TA was less toxic and in in vivo test efficacy was similar.

In vitro and in vivo anthelmintic activity of extracts from Artemisia parviflora and A. sieversiana

Summary In the northern areas of Pakistan, the use of Artemisia based therapeutics is a common practice. Plants of genus Artemisia are known to possess anthelmintic and therapeutic effect. Infections caused by gastrointestinal nematodes are major threat to livestock industry across the world resulting in loss of production and indirect economic losses due to high cost of anthelmintic drugs. Present study was carried out to evaluate in vitro and in vivo effect of Artemisia sieversiana and Artemisia parviflora on Haemonchus contortus, a parasitic nematode of small ruminants. Methanolic plant extract was tested against three different developmental stages using an egg hatch assay, infective larvae and adult worm motility assay. Different concentrations were used for the bioassays and post exposure mortality was recorded after 8 hr for adult worms and infective larvae, while egg inhibition percentage was observed after 27 hr. A highly significant ability to inhibit the egg hatching (100 %) was recorded for both plant extracts while, the highest activity for adult worm assay and larvicidal assay was 90 % for A. sieversiana. The highest activity for adult motility and larvicidal assay for A. parviflora was 89 % and 86.6 % respectively. For in vivo trials maximum parentage reduction was 77.0 % for A. sieversiana and 73.6 % for A. parviflora. It is concluded that selected plant extracts were effective in reducing worm burden in animals.

Evaluation of anthelmintic antimicrobial and antioxidant activity of Chenopodium album.

The objective of this study was to evaluate the anthelmintic, antimicrobial and antioxidant activities of Chenopodium album against gastrointestinal nematodes of sheep and some pathogenic microbes. A worm motility inhibition assay was used for in vitro study, and a faecal egg count reduction assay was used for an in vivo study. Various concentrations ranging from 100 to 500μg/ml of the extract were subjected to antimicrobial screening by disc diffusion method against four selected bacterial (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas multocida and Escherichia coli) and two fungal (Aspergillus flavus and Candida albicans) strains in order to estimate the medicinal potential of the herb. DPPH (1,1-diphenyl-2-picrylhydrazyl), riboflavin photo-oxidation, deoxyribose, lipid peroxidation assays were used for antioxidant activity. The extracts exhibited dose- and time-dependent anthelmintic effects on the Haemonchus contortus as compared to levamisole. The extract showed maximum inhibitory effect against S. aureus (28±0.14mm), while as mild inhibitory effect was observed against E. coli among the selected microbial strains. The effect produced by the different extract concentrations was comparable with the standard antibacterial agent streptomycin sulphate and antifungal agent nystatin, which were used as effective positive control in the study. The antioxidant activity showed that the extracts exhibited scavenging effect in concentration-dependent manner on superoxide anion radicals and hydroxyl radicals leading to the conclusion that the plant has broad spectrum anthelmintic, antimicrobial and antioxidant activities and could be a potential alternative for treating various diseases.

Phenotypic, chemical and functional characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential anthelmintic drug target.

BackgroundReliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer’s disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals).Methodology/Principal findingsA phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite’s genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target.Conclusions/SignificanceThe orthogonal chemical, biological and genetic strategies employed identify SmPDE4A’s contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.

In vitro effect of metrifonate on the indices of oxidative stress in Gigantocotyle explanatum

Helminth infections in general and digenetic trematodes in particular cause a huge economic loss globally to our livestock. Gigantocotyle explanatum is a digenetic amphistome that infects the bile ducts of water buffalo and are highly prevalent in tropical and sub-tropical countries. In the present study, effects of an organophosphate compound, Metrifonate (MF) in three doses, viz., 9.4 × 10−5 M (Dose I), 14.4 × 10−5 M (Dose II), and 19.4 × 10−5 M (Dose III), have been studied in vitro, on the motility and on some enzymatic and non-enzymatic oxidative stress indices in G. explanatum. The worm's motility and their non-enzymatic oxidative stress biomarkers like lipid peroxides measured as thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) were disrupted significantly in a dose-dependent manner. However, the enzymatic oxidative stress biomarkers like glutathione-S-transferase (GST) and superoxide dismutase (SOD) were affected by MF treatment in a biphasic manner. Exposure to Dose I significantly stimulated the activities of both GST and SOD, whereas exposure to Doses II and III resulted into significant inhibition in a dose-dependent manner. Our findings suggest that MF has potential to be a strong and effective anthelmintic, however, further studies in vitro as well as in vivo are needed to explore further these observations and understand the exact mode of MF action in G. explanatum and other trematodes of veterinary economic importance.

Anthelmintic effects of forage chicory (Cichorium intybus) against free-living and parasitic stages of Cooperia oncophora

Chicory shows great promise as an anthelmintic forage for grazing ruminants that can reduce reliance on anti-parasitic drugs. Recently, we reported potent anthelmintic effects of chicory-based diets in infected cattle with significant reductions in worm burdens of the abomasal nematode Ostertagia ostertagi, whilst no apparent activity was observed against the small intestinal parasite Cooperia oncophora. To explore this discrepancy, we investigated direct anthelmintic effects of forage chicory against C. oncophora in vitro. Chicory leaves (cultivar ‘Spadona’) were extracted with methanol in a Soxhlet apparatus and the resulting extract was purified by solid-phase extraction to concentrate bioactive phytochemicals such as sesquiterpene lactones. C. oncophora eggs and adult worms from mono-infected donor calves were exposed to decreasing concentrations of the chicory extract. In an egg hatch assay, the chicory extract induced a marked and dose-dependent inhibition of egg hatching, with 95% inhibition at 2500μg extract/mL (EC50=619 [95% CI: 530–722] μg extract/mL). In the adult motility inhibition assays, the chicory extract induced a potent and dose-dependent worm paralysis. At 12h of incubation, worms exposed to chicory showed a total paralysis at ≥500μg extract/mL, while after 48h of incubation a complete inhibition of worm motility was observed at ≥250μg extract/mL (EC50=80 [95% CI: 67–95] μg extract/mL). We have demonstrated that forage chicory can induce potent inhibitory effects on the egg hatching and exert direct anthelmintic activity against parasitic stages of C. oncophora. These results suggest that the previously reported absence of in vivo effects of chicory towards C. oncophora in infected animals may be related with host-mediated factors and/or inhibitory digestive conditions, rather than an inherent inactivity of chicory and its bioactive phytochemicals.