Comparative analysis of the viability of Fasciola hepatica eggs collected from fecal and gallbladder samples in experimentally infected sheep.

The aim of this study was to evaluate the aqueous and ethanolic leaf extracts of Vitex negundo for their anthelmintic potential against the benzimidazole-resistant Haemonchus contortus of sheep. The organized sheep farms in the various districts of Tamil Nadu viz., Thiruvallur, Kancheepuram, Salem, Karur and Kanniyakumari were randomly selected for sampling. 560 faecal samples were collected from organized sheep farms from the above districts before and after deworming with benzimidazole (BZ). The faecal egg count reduction test (FECRT) revealed the development of anthelmintic resistance to BZ in Thiruvallur and Kancheepuram districts. The allele-specific polymerase chain reaction (AS-PCR), the molecular technique employed to amplify the 250bp BZ-resistant gene fragments, had also revealed the BZ resistance. This study evaluated the anthelmintic potential of Vitex negundo leaves against BZ-resistant H. contortus in sheep. The aqueous leaf extracts (ALE) and ethanolic leaf extracts (ELE) of V. negundo were prepared in various concentrations, viz., 5, 10, 20, and 50 mg/mL. The in vitro methods viz., egg hatch assay (EHA) and larval paralysis assay (LPA) were performed to assess the anthelmintic efficacy of the above extracts. EHA revealed maximum efficacy (%) of 11.67±1.67% and 5.00±0.91% in 50 mg/mL concentration of ALE and ELE, respectively. LPA revealed 13.44±1.41% and 7.23±0.55% efficacy in 50 mg/mL concentrations of ALE and ELE, respectively. The results of this study with V. negundo indicated a dose-dependent effect of ALE and ELE in EHA and LPA. It is concluded that V. negundo could be a futuristic phytomedicine for an alternate control strategy to combat the anthelmintic resistance against BZ-resistant strongyle nematodes of sheep.

BackgroundTo investigate the role of long non-coding RNAs (lncRNAs) in the regulation of albendazole resistance in Haemonchus contortus (H. contortus) through competing endogenous RNA (ceRNA) mechanisms, a ceRNA network analysis was performed based on RNA-seq data from albendazole-sensitive and albendazole-resistant strains. Starting with significantly upregulated lncRNAs and incorporating functional enrichment analysis of predicted target genes, a potential regulatory axis—MSTRG.11540.1/novel-m1192-3p/HCON_00073890 (CYP2C70)—was identified from the ceRNA network. Transcriptomic analysis revealed that MSTRG.11540.1 and CYP2C70 were significantly upregulated (fold changes of 1.20 and 4.01, respectively; P < 0.01), while novel-m1192-3p was significantly downregulated (fold change: 5.91; P < 0.05) in resistant strains. This study employed dual-luciferase reporter assays and RNA interference (RNAi) to validate the interactions and functional relevance of the candidate RNA molecules. Furthermore, an egg hatch assay (EHA) was conducted to assess the impact of the lncRNA-mediated ceRNA axis on albendazole resistance. The aim was to provide insights into the molecular mechanisms by which lncRNAs may regulate drug resistance in H. contortus.ResultsA dual-luciferase reporter plasmid containing the predicted binding sites was constructed to investigate the candidate ceRNA axis MSTRG.11540.1/novel-m1192-3p/CYP2C70. The interaction assay revealed that novel-m1192-3p binds to MSTRG.11540.1, but no direct binding was observed between novel-m1192-3p and its predicted target gene CYP2C70. However, overexpression of novel-m1192-3p downregulated the expression of both MSTRG.11540.1 (45.71% reduction compared to NC group, P > 0.05) and CYP2C70 (40.74% reduction versus NC group, P < 0.05). Subsequent RNAi experiments confirmed the existence of the MSTRG.11540.1/novel-m1192-3p/CYP2C70 regulatory axis. Moreover, EHA results showed that knockdown of MSTRG.11540.1 significantly decreased the LD50 value of albendazole (52.91 µg/mL to 32.02 µg/mL).ConclusionsThis study confirmed that the lncRNA-mediated ceRNA axis MSTRG.11540.1/novel-m1192-3p/CYP2C70 is involved in the regulation of albendazole resistance in H. contortus. These findings provide potential molecular targets for further elucidation of anthelmintic resistance mechanisms and the development of novel therapeutic strategies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-025-04966-8.

Parasitic diseases caused by gastrointestinal nematodes (GIN) are considered important limiting factors for livestock production worldwide, as they significantly compromise animal welfare and increase production costs. For years, parasite control has relied exclusively on the use of synthetic drugs. Resistance of GIN to commercial anthelmintics has been increasingly reported throughout the world. Therefore, alternative approaches have emerged for controlling GIN in farm animals, including the exploration of plants with bioactive potential. Tithonia diversifolia can be harvested year-round, and all parts of the plant have been used in folk medicine for a wide range of diseases. This study evaluated the in vitro antioxidant properties of water and 70% aqueous acetone extracts of T. diversifolia aerial parts, including leaves, branches, and flowers, using radical-based methods, and assessed its anthelmintic potential through a parasite model, employing the egg hatching assay (EHA) with water extracts. Additionally, the total polyphenolic content of the extracts was determined. These findings highlight T. diversifolia as a promising natural source of antioxidant and anthelmintic compounds.

BackgroundDifficulty in combating gastrointestinal nematode (GIN) infection in ruminants is increasing worldwide, owing to progressive anthelmintic resistance (AR). This study evaluates the AR current status against benzimidazoles and macrocyclic lactones on 20 sheep and 10 cattle farms in southern Italy that have performed a greater number of anthelmintic treatments in recent years. On each farm, the animals were randomly divided into two groups of 20 animals; one group was treated with ivermectin (IVM, 0.2 mg/kg of body weight), and the other with albendazole (ALB, 3.8 mg/kg of body weight).In each farm, an individual faecal egg count was performed on samples collected on the day of treatment (D0) and after 14 days (D14) using the Mini-FLOTAC technique. For each group, coprocultures were performed on D0 and D14. An in vitro test (egg hatch assay—EHA) and a follow-up FECR test with fenbendazole were performed to confirm the low in vivo efficacy. To better understand farm management and the anthelmintic treatment methods commonly adopted, a questionnaire was formulated and submitted to farmers during the recruitment phase. The content of the questionnaire was identical for cattle and sheep.ResultsThe in vivo test (FECR) showed a high efficacy (from 96.7% to 100%) for both tested anthelmintics in all cattle farms and in 18 sheep farms. A low efficacy for ALB has been detected in two sheep farms, with FECR value of 86.0% for Farm 1 and 92.4% for Farm 2 with predominant GIN genera of Trichostrongylus and Haemonchus on post-treatment samples. The EHA confirmed AR on both sheep farms with low efficacy in Farm 1 of 87.0% and in Farm 2 of 77.0%. At the end of the administration period, questionnaires were collected and a descriptive analysis of data was carried out. Cattle farmers displayed a low perception of the AR problem, while sheep farmers had a higher perception.ConclusionsThe phenomenon of AR in low-prevalence areas certainly needs to be monitored on each farm in conjunction with the usual anthelmintic treatments, so as to be able to promptly identify and solve the problem.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-025-04894-7.

Haemonchus contortus (H. contortus) is a common gastrointestinal nematode in small ruminants, posing a significant threat to the livestock industry. The control of H. contortus often depends on drugs such as albendazole. However, the prolonged and improper use of these drugs by livestock producers has led to widespread resistance among ruminant populations, posing a major challenge to parasite management. It is reported that the resistance of H. contortus to albendazole is related to the single nucleotide polymorphism (SNP) of β-tubulin homologous type I gene, but whether other genes are involved has not been reported. Based on the comparative analysis of the transcriptome sequencing data of albendazole-sensitive and albendazole-resistant strains of H. contortus, HCON_00043720 (Receptor-type guanylate cyclase GCY-12, GCY-12) gene was selected as the research object from the 23 differential genes in the cyclic guanosine monophosphate (cGMP) signaling pathway where the growth and development of H. contortus dauer stage is located. The GCY-12 gene of H. contortus eggs was silenced by RNA interference (RNAi) test. The reaction temperature was optimized, the expression of silenced eggs was detected by quantitative real-time polymerase chain reaction (qRT-PCR) technology, and the drug resistance of silenced eggs was detected by egg hatch assay (EHA). The results showed that the gene expression level decreased significantly after GCY-12 interference, and the sensitivity of H. contortus to albendazole increased. This study highlights the potential role of GCY-12 in modulating albendazole resistance in H. contortus, offering new insights for developing effective therapeutic approaches.

The majority of medications used to treat nematodiasis in animals have become ineffective as a result of helminth drug resistance. Additionally, going back to medicinal plants and their substances is an environmentally beneficial option to cure nematodiasis since anthelmintics, like many medications, have been designated as hazardous environmental contaminants. The current study was carried out to evaluate phytochemicals, antioxidant, antibacterial and anthelmintic properties from locally available plant Balanites aegyptiaca (B. aegyptiaca). An in vitro free radical scavenging activity of the different extracts was determined by 2,2-azino bis (3-ethylbenzothiazoline-6-sulfonate) method. The antibacterial test was performed by agar well diffusion method using crude aqueous and hydro alcohol extracts of immature and mature fruits. The egg hatch assay and larval mortality assay were used to evaluate the extracts' in vitro effectiveness. 25% crude extracts (aqueous, methanol, ethanol, hydro alcohol and acetone) were used at different concentrations (10 mg/ml to 0.01 mg/ml). Thiabendazole was employed as a reference standard for EHA at a dose of 10 mg/ml. For LMA levamisole was used @ 10 mg/ml as reference standard. In both the assays’ distilled water was used as negative control. In methanol extract of immature fruits Geraniol was the main phytochemicals while in its ethanol extract 4-O-Methylmannose was predominant. Pentanoic acid, 3-methyl- (Valeric acid) and Hexanoic acid (Butyric acid) are predominantly present in mature fruits. In both methanol and ethanol extracts of bark 1,4- Dimethyl-7-(prop-1-en-2-yl) decahydroazulen-4-ol (Pogostole) was predominant. Leaves, bark and mature fruits showed remarkable antioxidant activity. Hydro alcohol extract of immaturte fruit showed marked antimicrobial activity against E. coli. At 200 and 300 mg/g of faeces, adding powder to the faecal culture reduced larval growth by more than 97%. All the extracts of studied plant parts were very effective (&gt;95%) in preventing hatching of eggs and killing the larvae (L1 and L2). The in vivo study with ethanol extract of immature fruit (@ 0.5 g per kg body weight once) showed non-significant variation in faecal egg counts in extract treated and infected untreated control groups, however, compared to the control group, the extract-treated group's numerical mean intensity of infection was lower on the majority of days. Nevertheless, the reduction in infective larvae observed in faecal cultures from extract treated lambs was highly significant from day 3 to end of the study. In conclusion the crude extracts of B. aegyptiaca showed terpenoids, flavonoids, fatty acids and carbohydrates and possessed significant antibacterial, ovicidal and larvicidal properties against Haemonchus contortus (H. contortus).

Anthelmintic resistance (AR) is a major problem in the profitable production of livestock. Therefore, this study evaluated AR status in cattle farms at Mymensingh Sadar using in vivo, fecal egg count reduction test (FECRT) and in vitro, egg hatch assay (EHA) techniques. For this, ten cattle farms and forty animals from individual farms with more than 200 eggs per gram (EPG) of feces were selected by employing the McMaster technique. For FECRT, four groups having ten animals in each group were designed and animals of group I were treated with albendazole (ABZ), group II with levamisole (LEV), group III with ivermectin (IVM) according to the bodyweight and group IV was served as control (untreated). Fecal samples were collected at day 0 (pre-treatment) and day 14 of post-treatment (p. t) from animals of all treated and untreated groups directly from the rectum to calculate EPG and subsequently considered for copro-culture to detect resistant parasitic larvae. For EHA, following the collection of pooled fecal samples from ten farms, eggs were isolated and incubated with different concentrations of albendazole such as 0.05, 0.1, 0.2, 0.3 and 0.5µg/ml. In FECRT, the result revealed that all three drugs such as ABZ, LEV and IVM were resistant in all farms except one where IVM was suspected resistance (SR). The identified resistant larvae from copro-culture were Haemonchus, Trichostrongylus and Oesophagostomum. In EHA, the EC50 value of ABZ ranged from 0.14 to 0.29µg ABZ/ml (> 0.1µg/ml designated as resistance) with the coefficient of correlation (R2) > 0.97 confirming ABZ resistance in all cattle farms. The present study suggests that AR is increasing and creates an alarming condition in controlling GI nematodes in Bangladesh.

Canine hookworm (Ancylostoma caninum), a gastrointestinal nematode of domestic dogs, principally infects the small intestine of dogs and has the potential to cause zoonotic disease. In greyhounds and pet dogs in the USA, A. caninum has been shown to be resistant to multiple anthelmintics. We conducted a molecular survey of benzimidazole resistance in A. caninum from dogs at veterinary diagnostic centers in Australia and New Zealand. First, we implemented an internal transcribed spacer (ITS)-2 rDNA deep amplicon metabarcoding sequencing approach to ascertain the species of hookworms infecting dogs in the region. Then, we evaluated the frequency of the canonical F167Y and Q134H isotype-1 β-tubulin mutations, which confer benzimidazole resistance, using the same sequencing approach. The most detected hookworm species in diagnostic samples was A. caninum (90%; 83/92); the related Northern hookworm (Uncinaria stenocephala) was identified in 11% (10/92) of the diagnostic samples. There was a single sample with coinfection by A. caninum and U. stenocephala. Both isotype-1 β-tubulin mutations were present in A. caninum, 49% and 67% for Q134H and F167Y, respectively. Mutation F167Y in the isotype-1 β-tubulin mutation was recorded in U. stenocephala for the first known time. Canonical benzimidazole resistance codons 198 and 200 mutations were absent. Egg hatch assays performed on a subset of the A. caninum samples showed significant correlation between 50% inhibitory concentration (IC50) to thiabendazole and F167Y, with an increased IC50 for samples with>75% F167Y mutation. We detected 14% of dogs with>75% F167Y mutation in A. caninum. Given that these samples were collected from dogs across various regions of Australia, the present study suggests that benzimidazole resistance in A. caninum is widespread. Therefore, to mitigate the risk of resistance selection and further spread, adoption of a risk assessment-based approach to limit unnecessary anthelmintic use should be a key consideration for future parasite control.

Gastrointestinal nematodes (GINs) inflict significant economic losses on sheep and goat farming globally due to reduced productivity and the development of anthelmintic resistance. Sustainable control strategies are urgently needed including the exploration of medicinal plants as safer alternatives to chemical anthelmintics. This genus of plants is used for anti-inflammatory, antioxidant, and antimicrobial activities. In this study, we aimed to evaluate the anthelmintic activities of Nepeta racemosa Lam. MeOH extract, n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), n-buthanol (n-BuOH) and aqueous (H2O) subextracts, and quantify rosmarinic acid in the active extract by the HPLC method, and perform in silico molecular docking studies of rosmarinic acid to examine its binding interactions with tubulin. The anthelmintic activity of the plant extracts on gastrointestinal nematode eggs and larvae (L3) of the sheep was assessed using in vitro test methods such as the egg hatch assay and larval motility assay, conducted over a 24 h period (1, 2, 3, 4, 6, 8, 24). All extracts exhibited 100% effectiveness in the egg hatch inhibition assay, regardless of concentration (50-1.5625 mg/mL). The EtOAc subextract shows the highest effectiveness at 79.66%, followed by the MeOH extract at 74.00%, water at 64.00%, n-hexane at 67.00%, and DCM at 61.00%, and the lowest effectiveness is observed with n-BuOH at 51.66% in the larval motility assay. The major compound of EtOAc extract, the most active extract of N. racemosa, was determined as rosmarinic acid and its amount in the extract was determined as 14.50 mg/100 mg dry extract. The amount of rosmarinic acid in the MeOH extract was found to be 0.21 mg/100 mg dry extract. n-Hexane, DCM, n-BuOH, and H2O extracts' rosmarinic acid content was lower than the LOQ value. As tubulin plays an important role in the mechanism of anthelmintics, the major compound of the most active extract (NR-EtOAc) rosmarinic acid was docked onto the colchicine-binding site of the tubulin (5OV7) protein. Rosmarinic acid showed a similar activity spectrum to the anthelmintic drug albendazole. The discovery of low-cost and low-toxicity anthelmintic compounds is very important.

Aim: This research intended to assess the in vitro anthelmintic properties of Mahaneem leaves (Melia azedarach) and whole plant of Chirata (Swertia chirata). Study Design: In vitro study was conducted aganist Haemonchus contortus eggs and adult stage by egg hatch assay (EHA) and adult motility inhibition test (AMIT). Place and Duration of Study: This study was conducted in the departement of Veterinary Parasitology, WBUAFS, Kolkata-37 between November,2023 to August, 2024 Methodology: The aqeous extract of Mahaneem and Chirata whole plants were prepared by decoction method, dried and desolved in 2% Dimethyl Sulfoxide to make desirable concentration of extract solutions. Adult Haemonchus contortus was collected from freshly slaughter abomasum of sheep for separation of eggs and active motile adult was used for AMIT. Each EHA and AMIT was performed in triplicate. Results: The results revealed that both Chirata and Mahaneem extracts shows anthelmintic activity against Haemonchus contortus. Chirata extract had better efficacy against both eggs and adult stage of Haemonchus contortus compare to Mahaneem leave extract. In EHA, Chirata extract efficacy at the dose of 50 mg/ml and 25 mg/ml was more statistically significant (p&lt;0.05) than Mahaneem extract. In AMIT, also Chirata shows significantly more efficacy at 50mg/ml and 25 mg/ml than Mahaneem,but at the dose rate of 10 mg Mahaneem shows better efficacy (38.88 2.22) than Chirata (31.11 2.22) extract. At 50 mg/ml concentration after 10 hours of experiment Chirata shows higest efficacy (82.22 2.22) compare to Mahaneem (57.77 2.22). Conclusion: Both Chirata and Mahaneem aqueous extract can potentially be effective against Haemonchus contortus and other GINs in small rumunats when administer properly with selected doses.

Abstract A study was conducted to evaluate the in vitro anthelmintic effect of different crude extracts of Citrus sinensis (orange) and Citrus nobilis (mandarin) fruit peel. In vitro free radical scavenging activity of the different extracts was determined by the 2,2-azino bis (3-ethylbenzothiazoline-6-sulfonate) method. In vitro anthelmintic activity of different extracts was assessed against Haemonchus contortus by egg hatch assay (EHA) and larval mortality assay (LMA). C. sinensis had higher antioxidant activity as compared to C. nobilis. Poor embryonicidal effect was observed on H. contortus eggs by extracts of C. sinensis fruit peel. However, acetone extract of C. nobilis peel (@ 10 mg/ml) exhibited excellent embryonicidal effect (&gt;90%). Though all the extracts of C. sinensis (except ethanol extract) and C. nobilis (except methanol extract) showed excellent inhibition of egg hatching at a minimum concentration of 0.15 mg/ml, among all the extracts acetone and hydroalcohol extracts were found to be the best for inhibition of egg hatching. In larval mortality assay, all the extracts of both fruit peels exhibited marked larvicidal activity at a minimum concentration of 0.63 mg/ml. The results indicated that acetone extract of both C. sinensis and C. nobilis fruit peels has potent ovicidal activity while all the extracts possessed larvicidal properties against H. contortus that need to be investigated in vivo for the management of this parasite.

Abstract A study was aimed to evaluate the different extracts of Ailanthus excelsa (Ardu) bark and pods for their antioxidant, antibacterial and anthelmintic activities. Phytochemical analysis showed higher concentrations of phenols, flavonoids, total tannin and saponin (6.37, 4.12, 5.70 and 18.94 mg/g, respectively) in methanol extract of pod as compared to bark (0.22, 0.79, 0.09 and 5.21 mg/g, respectively). A total of 12 phyto-compounds were identified with predominance of 5-(hydroxymethyl)-2-(dimethoxymethyl) furan in methanol extract of bark and 4-phyto-compounds with predominance of 2,3-butanediol, [R-(R*R*)]- in methanol extract of pod. Antioxidant activity was 93.61 and 9.83% in methanol extract of pods and bark, respectively. Methanol extract of pod showed good antibacterial activity against E. coli. On in vitro egg hatch assay, &gt;90% inhibition of egg embryonation was observed by both methanol (@ 0.31 to 5.00 mg/ml) and hydroalcohol (@ 1.25 to 5.00 mg/ml) extracts of bark. Except for the aqueous extract, all other extracts caused &gt;95% egg hatch inhibition at a concentration of 0.63 to 10.00 mg/ml. Among extracts of pods, only hydroalcohol extract (@ 5.00 to 10.00 mg/ml) caused &gt;90% inhibitory activity on egg embryonation; however, at similar concentrations, all the extracts showed &gt;98% inhibition of egg hatching. On larval mortality assay, all the extracts (except aqueous) showed excellent (100%) larvicidal activity at 0.63 to 10.00 mg/ml concentrations. The results indicated that A. excelsa bark and pods have higher antioxidant, antibacterial activity against E. coli and anthelmintic activities against infective larvae of H. contortus.

Soybean cyst nematode (SCN) is a major pest of soybean crops, causing significant yield losses and economic impact. Current management strategies primarily rely on resistant varieties, cover crops, and seed treatments. In addition, there is a growing interest in developing sustainable, ecologically based approaches to integrate SCN risk reduction into soybean production systems. This study aimed to evaluate the efficacy of various compost and manure amendments in suppressing SCN populations and promoting soybean productivity. An in vitro egg hatching assay was conducted to screen the inhibitory effects of different compost and manure extracts on SCN egg hatching. Results indicated that poultry manure, Layer Ash Blend, and swine manure extracts significantly inhibited SCN hatching compared with other treatments across multiple time points. Greenhouse trials further validated the effectiveness of Layer Manure, poultry manure, High Carbon Dairy Doo, and Seed Starter 101 in suppressing SCN cysts, eggs, and juveniles. A field microplot trial confirmed the potential of Layer Ash Blend and poultry manure in SCN management, with significant reductions in SCN populations and increased soybean yields. The study also investigated the impact of these amendments on promoting the population of bacterivorous and frugivorous nematodes, contributing to a biological diverse soil ecosystem. Overall, the results indicate that amending SCN-infested soil with specific compost or manure formulations can effectively suppress nematode populations while improving soybean productivity. These findings contribute to the development of sustainable strategies for SCN management in soybean production systems.

Ticks pose a significant threat due to their ability to lay thousands of eggs, which can persist in the environment for extended periods. While the impact of various compounds on adult and larval ticks has been studied, research on their efficacy against tick eggs is limited. This study evaluated the ovicidal activity of commercial acaricides, disinfectants, and natural products against Rhipicephalus annulatus eggs using the egg hatch assay (EHA). Deltamethrin and cypermethrin caused a non-significant inhibition of hatching (IH%), even at concentrations higher than the recommended levels. By contrast, the acaricides chlorpyrifos, phoxim, and amitraz significantly inhibited hatching at all tested concentrations. Ivermectin also demonstrated significant IH% at various concentrations but did not fully inhibit the hatching process. Among the disinfectants tested, Virkon-S®, TH4, and Chlorox showed insignificant effects, whereas formalin achieved an IH% of only 34.1% at a high concentration of 200 mg/mL. Natural products, carvacrol and thymol, exhibited significant ovicidal activity, with a significant IH%. In a semi-field application, phoxim (0.5 mg/mL) and deltamethrin (0.05 mg/mL) were sprayed on tick eggs on pasture soil from a farm. The results indicated that phoxim-treated eggs had a 40% IH%, while deltamethrin-treated eggs showed only an 8.79% IH%. In conclusion, the acaricides amitraz, phoxim, and chlorpyrifos, as well as the natural products carvacrol and thymol, caused significant toxicity to R. annulatus eggs.

Fasciolosis caused by Fasciola hepatica is a major public health and economic problem worldwide. Due to the lack of a successful vaccine and emerging resistance to the drug triclabendazole, alternative phytotherapeutic approaches are being investigated. This study investigated the in vitro anthelmintic activity of Lavender (Lavandula angustifolia) and carob (Ceratonia siliqua L.) essential oils (EOs) against F. hepatica. The in vitro study was based on an egg hatch assay (EHA), adult motility inhibition assays, DNA damage, reactive oxygen species (ROS) level along with several oxidative stress biomarkers including glutathione peroxidase (GSH), and glutathione-S-transferase (GST), superoxide dismutase (SOD) and malondialdehyde (MDA). To this end, different concentrations of L. angustifolia and C. siliqua EOs (1, 5, 10, 25 and 50 mg/mL) were used to assess anthelmintic effects on different life stages including egg, and adults of F. hepatica for 24 hrs. The results indicated that these EOs play a significant role as anthelminthics, and the effect was dependent on time and concentration. The in vitro treatment of F. hepatica worms with both L. angustifolia and C. siliqua EOs increased DNA damage, ROS production and induction of oxidative stress (decreased SOD, GST and GSH, and increased MDA), significantly compared to control. Therefore, it can be concluded that L. angustifolia and C. siliqua EOs have the potential to be used as novel agents for the control and treatment of F. hepatica infections. Further studies are required to investigate their pharmacological potential and effectiveness in vivo for the treatment of parasitic infections.

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

Evaluating anthelmintic activity through Caenorhabditis elegans egg hatching assay

The aim of this study was to evaluate the in vitro anthelmintic effect of crude aqueous, methanol, ethanol, hydro alcohol and acetone extracts of Vitex negundo leaves against Haemonchus contortus eggs and larvae. Phytochemical analysis to identify the number of compounds in extracts was done by chemical tests and gas chromatography coupled to a mass spectrophotometer detector (GC-MS). First off all the effectiveness of dried plant materials was evaluated on larval development by mixing powdered material (no nano particles) to faecal cultures from donor sheep. Adding powder to the faecal culture resulted into 100% inhibition in larval development at 200 and 300mg/g of faeces. The anthelmintic activity was assessed using the egg hatch assay (EHA) and the larval mortality assay (LMA). Comparison of mean inhibition percentage of egg embryonation, mean inhibition percentage of egg hatching and mean percentage of larval mortality at different concentrations with control was performed by one-way ANOVA. The means were compared for statistical significance using DMRT at P < 0.05. For both the assays, 50% inhibitory concentration (IC50) and lethal concentration (LC50) were calculated by probit analysis. Chemical test revealed presence of high concentration of saponin and flavoinoids and moderate concentration of total phenols in leaves. The antioxidant activity (radical scavenging activity, RSA %) measured was 35.47%. On GC-MS, the methanolic leaves extract revealed 30 phyto-compounds. On EHA, there was marked effect on inhibition of egg hatching by aqueous, hydro alcohol and acetone extracts. On LMA all the five extracts showed excellent larvicidal activity. V. negundo leaves methanol extract mediated silver nanoparticles were found very effective at much lower concentrations as compared to crude methanol extract. The results indicated that the V. negundo leaves crude extracts possessed excellent in vitro ovicidal and larvicidal properties against H. contortus which needs more investigation, especially in vivo trials for the control of parasite.

Comprehensive analysis of Mentha longifolia subsp. himalaiensis essential oil: GC MS profiling and biological properties