Anthelmintic efficacy of Achillea millifolium against gastrointestinal nematodes of sheep: in vitro and in vivo studies

Achillea millifolium Linn., commonly called 'Pehl-ghasa', is used by farmers in traditional animal health care as a vermifuge. The objective of this study was to evaluate the anthelmintic efficacy of crude aqueous extracts and crude ethanolic extracts of entire A. millifolium against the gastrointestinal nematodes of sheep. The worm motility inhibition assay was used for in vitro studies and faecal egg count reduction assay was used for in vivo studies. In vitro studies revealed significant anthelmintic effects of aqueous extracts and ethanolic extracts on live Haemonchus contortus worms (P < 0.05) as evident from their paralysis and/or death at 8 h post exposure. Aqueous extracts of A. millifolium resulted in a mean worm motility inhibition of 94.44%, while ethanolic extracts resulted in mean worm motility inhibition of 88.88%. The mean mortality index of aqueous extracts was 0.95 while for ethanolic extracts it was 0.9. The lethal concentration 50 was 0.05 mg ml(-1) for aqueous extracts and 0.11 mg ml(-1) for ethanolic extracts. The in vivo anthelmintic activity of aqueous and ethanolic extracts of A. millifolium demonstrated a maximum (88.40%) nematode egg count reduction in sheep treated with aqueous extracts at 2 g kg(-1) body weight on day 15 after treatment. Ethanolic extracts resulted in a maximum of 76.53% reduction in faecal egg counts on day 15 after treatment with 2 g kg(-1) body weight. Thus, the aqueous extracts exhibited greater anthelmintic activity under both in vitro and in vivo conditions, and could be due to the presence of water-soluble active principle/s in A. millifolium. It is concluded that the entire plant of A. millifolium possesses significant anthelmintic activity and could be a potential alternative for treating cases of helminth infections in ruminants.     

Haemonchus contortus: in vitro and in vivo anthelmintic activity of aqueous and hydro-alcoholic extracts of Hedera helix

In vitro anthelmintic activity of crude extracts of the ripe fruits of Hedera helix was investigated on eggs and adult nematode parasites Haemonchus contortus. Aqueous extract of H. helix was also evaluated for in vivo anthelmintic activity at dose of 1.13 and 2.25 g/kg in sheep artificially infected with H. contortus. ED(50) for egg hatch inhibition was 0.12 and 0.17 mg/ml for aqueous and hydro-alcoholic extracts, respectively. There was no statistically significant difference in the activity of the two extract types (p>0.05). Hydro-alcoholic extract showed better in vitro activity against adult parasites compared to the aqueous extract. Significant faecal egg count reduction (FECR) was detected in groups treated with both doses of H. helix (p<0.05) on day 2 post-treatment. On day 7 post-treatment significant reduction was detected only for higher dose of H. helix (p<0.05) while on day 14 post-treatment there was no significant FECR in both groups treated with H. helix. The percentage of larvae recovered from culturing faeces obtained from groups of sheep treated with lower and higher doses of H. helix was 47.52% and 36.07%, respectively, which was significantly lower than (p<0.05) that recovered from the control group (60%). Significant (p<0.05), dose dependent total worm count reduction (WCR) was observed for groups of sheep treated with H. helix. Increasing the dose of H. helix improved the efficacy against the male than the female parasites. Treatment with both doses of H. helix helped the animals maintain their packed cell volume (PCV) unlike the untreated control group. The overall findings of the current study indicated that H. helix has a potential anthelmintic benefit and further in vitro and in vivo evaluation of the different parts and fractions is needed to make use of this plant for therapeutic purposes.     

A molecular diagnostic tool to replace larval culture in conventional faecal egg count reduction testing in sheep

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.     

Benzimidazole resistance of sheep nematodes in Norway confirmed through controlled efficacy test

ABSTRACT: BACKGROUND: Resistance against benzimidazoles (BZ) has recently been detected in Norwegian sheep flocks through a large scale prevalence survey based on the faecal egg count reduction test (FECRT). The use of this test in combination with bulk larval culture only gives an indication of which gastrointestinal nematodes genera that are involved and these results have to be confirmed by a controlled efficacy test (CET) to get accurate information about resistant nematodes populations at species level. A CET was therefore performed with larvae from two flocks where BZ resistance was previously detected through FECRT. RESULTS: The latter test confirmed the previous results in both flocks. In flock A, the BZ resistant nematode population consisted solely of Haemonchus contortus, whereas H. contortus and Teladorsagia circumcincta comprised the resistant worm population in flock B. CONCLUSIONS: Some discrepancies that have been recorded between FECRT and CET results regarding time for post-treatment coproscopical examination and a temporary suppression of faecal egg excretion are discussed.     

Efficacy of the cyclooctadepsipeptide PF1022A against Heligmosomoides bakeri in vitro and in vivo

The cyclooctadepsipeptide PF1022A derived from the fungus, Mycelia sterilia, is characterized by a broad spectrum of activity against different parasitic gastrointestinal nematodes of livestock. In the present work the anthelmintic activity of PF1022A against Heligmosomoides bakeri, a widely used laboratory model was studied. Albendazole, ivermectin and levamisole served as reference. In vitro, PF1022A showed low activity on embryonation but significantly inhibited egg hatch (10 and 100 μg/ml), whereas albendazole (10 and 100 μg/ml) revealed statistically significant inhibitions of both embryonation and egg hatch. PF1022A (1-100 μg/ml) completely inhibited larval movement at most examination points. Comparable significant anthelmintic activity on the larval stages of H. bakeri was observed with levamisole (48-100%), while slightly lower activities were observed with ivermectin (20-92%) and albendazole (0-87%) at 1-100 μg/ml. PF1022A and levamisole significantly inhibited motility and egg release of adult worms, while albendazole and ivermectin failed to demonstrate activity. Significant worm burden reductions were achieved with PF1022A, levamisole and ivermectin in vivo. For example, at 0·125 mg/kg PF1022A a worm burden reduction of 91·8% was observed. The use of drug combinations did not further enhance the in vitro and in vivo activity of PF1022A. In conclusion, further investigations are warranted with PF1022A, as the drug is characterized by significant larvicidal and nematocidal activity in vitro and in vivo.     

Anthelmintic efficacy on UK Thoroughbred stud farms

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14–17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n = 429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8–100%; 95% lower confidence intervals range: 96.8–100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7–100%; 95% lower confidence intervals range: 65–100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0–73%; 95% lower confidence intervals range: 0–59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98–99.4%; 95% lower confidence intervals range: 91.8–99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4–41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5–99.9%; 95% lower confidence intervals range: 96.3–99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5–91.2%; 95% lower confidence intervals range: 6.7–82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.     

Adaptation of gastrointestinal nematode parasites to host genotype: single locus simulation models

Background

Breeding livestock for improved resistance to disease is an increasingly important selection goal. However, the risk of pathogens adapting to livestock bred for improved disease resistance is difficult to quantify. Here, we explore the possibility of gastrointestinal worms adapting to sheep bred for low faecal worm egg count using computer simulation. Our model assumes sheep and worm genotypes interact at a single locus, such that the effect of an A allele in sheep is dependent on worm genotype, and the B allele in worms is favourable for parasitizing the A allele sheep but may increase mortality on pasture. We describe the requirements for adaptation and test if worm adaptation (1) is slowed by non-genetic features of worm infections and (2) can occur with little observable change in faecal worm egg count.

Results

Adaptation in worms was found to be primarily influenced by overall worm fitness, viz. the balance between the advantage of the B allele during the parasitic stage in sheep and its disadvantage on pasture. Genetic variation at the interacting locus in worms could be from de novo or segregating mutations, but de novo mutations are rare and segregating mutations are likely constrained to have (near) neutral effects on worm fitness. Most other aspects of the worm infection we modelled did not affect the outcomes. However, the host-controlled mechanism to reduce faecal worm egg count by lowering worm fecundity reduced the selection pressure on worms to adapt compared to other mechanisms, such as increasing worm mortality. Temporal changes in worm egg count were unreliable for detecting adaptation, despite the steady environment assumed in the simulations.

Conclusions

Adaptation of worms to sheep selected for low faecal worm egg count requires an allele segregating in worms that is favourable in animals with improved resistance but less favourable in other animals. Obtaining alleles with this specific property seems unlikely. With support from experimental data, we conclude that selection for low faecal worm egg count should be stable over a short time frame (e.g. 20 years). We are further exploring model outcomes with multiple loci and comparing outcomes to other control strategies.     

Dose-dependent activity of albendazole against benzimidazole-resistant nematodes in sheep: relationship between pharmacokinetics and efficacy

The relationship between the pharmacokinetic behaviour and the anthelmintic efficacy of albendazole (ABZ) against benzimidazole (BZD)-resistant nematodes was studied in sheep. A micronized ABZ suspension was orally administered at two different dose levels to sheep naturally infected with BZD-resistant gastrointestinal (GI) nematodes. The experimental animals were allocated into the following groups (n = 8): (a) untreated control; (b) orally treated with ABZ at 3.8 mg/kg b.w.; and (c) orally treated with ABZ at 7.5 mg/kg b.w. Plasma samples were obtained serially over 72 h post-treatment from both treated groups and analysed by HPLC to measure the concentrations of ABZ and its sulphoxide (ABZSO) and sulphone (ABZSO(2)) metabolites. Faecal egg counts were performed prior to treatment and at the necropsy day. All experimental animals were sacrificed 10 days after treatment to perform GI worm counts. While ABZ parent drug was not recovered in the bloodstream, ABZSO and ABZSO(2) were the molecules found in plasma. ABZSO was the metabolite measured at the highest concentrations in the bloodstream for up to 36 (treatment at 3.8 mg/kg) or 60 h (treatment at 7.5 mg/kg) post-administration. There was a proportional relationship between the administered ABZ dose and the measured plasma concentrations of both ABZ metabolites. Over a 100% increment on the plasma AUC values for the anthelmintically active ABZSO metabolite was observed at the 7.5 mg/kg compared to the 3.8 mg/kg treatment. The low efficacy patterns (< 24%) observed against the GI nematodes investigated indicate a high level of resistance to ABZ given at 3.8 mg/kg an efficacious therapeutic dose rate recommended in some countries. However, the higher and prolonged plasma drug concentration measured after the 7.5 mg/kg treatment resulted in an improved efficacy pattern (estimated by both faecal egg and adult worm counts) against most of the GI nematodes studied compared to that obtained at the lower dose rate. A direct relationship between drug pharmacokinetic behaviour and anthelmintic efficacy against BZD-resistant nematodes in sheep was shown in the current work, although individual variation precluded the observation of statistically significant differences in worm counts.     

Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis

Allergens were identified from the gastrointestinal nematode of sheep, Trichostrongylus colubriformis, by probing Western blots of infective larvae (third stage) somatic antigen with IgE purified from the serum of sheep grazed on worm contaminated pasture. A 31 kDa allergen was frequently recognised by sera from immune sheep, particularly those deriving from a line that has been genetically selected over 23 years for parasite resistance. Using a proteomic approach, the 31 kDa allergen was identified as an aspartyl protease inhibitor homologue. The entire coding sequence of T. colubriformis aspartyl protease inhibitor (Tco-api-1) was obtained and the mature protein expressed in Escherichia coli. Anti-Tco-API-1 antibodies revealed that a commonly observed 21 kDa T. colubriformis allergen species is a truncated form of Tco-API-1. Specific IgE responses to T. colubriformis aspartyl protease inhibitor were significantly correlated with the degree of resistance to nematode infection as measured by faecal egg count in sheep. Surprisingly, IgE responses to Tco-API-1 were not correlated with breech soiling (dag score), which is thought to be caused, in part, by allergic hypersensitivity to worms. Therefore, a specific IgE response to this allergen may be a suitable marker for identifying lambs at an early age that will develop strong immunity to gastrointestinal nematodes.     

Induction of protective immunity to Trichostrongylus colubriformis in neonatal merino lambs.

The premise that any bias of immune reactivity in neonatal lambs towards T-helper (TH)2 responses could benefit the induction of protection against gastrointestinal nematodes was investigated. In two trials, lambs were either trickle-immunised with 2000 infective larvae of Trichostrongylus colubriformis (TcL3), 3 times weekly from the day of birth for 6 weeks or inoculated with a recombinant T. colubriformis 17 kDa antigen in incomplete Freund's adjuvant (IFA). In trial 1, trickle immunised and control neonates challenged at 7 weeks of age had similar worm counts 10 days after challenge, but from 25 days, significant reductions (P<0.01) in mean faecal egg count and worm count in excess of 75% were displayed by the immunised lambs. The results of a second, similar trial, gave 85-91% reductions in parasitism in trickle immunised neonates (P<0.001) and around 50% protection in neonates vaccinated with recombinant 17 kDa antigen. Parasitism in immunised neonates in Trial 2 was significantly reduced (P<0.001) compared to that in 4-month-old animals. Antibody responses in trickle-immunised (protected) and challenge control (infected) neonates were almost exclusively of the IgG1 isotype compared to vaccinated animals which exhibited increased levels of anti-17kD IgG2. Trichostrongylus colubriformis infection, but not specific vaccination, induced interleukin-5 production by mesenteric lymph node cells. The results offer the tantalising prospect of generating protective immunity to gastrointestinal parasites prior to weaning in sheep; this was most effectively generated by viable parasites in this investigation.     

A new diagnostic approach to fast-track and increase the accessibility of gastrointestinal nematode identification from faeces: FECPAKG2 egg nemabiome metabarcoding

Effective gastrointestinal nematode management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major gastrointestinal nematodes. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed gastrointestinal nematode management decisions. The traditional ‘gold-standard’ approach, larval culture followed by morphological differentiation, is laborious and potentially inaccurate. We developed a new diagnostic approach to identify gastrointestinal nematodes that integrates a remote-location digital faecal egg count platform, FECPAK^G2, with internal transcribed spacer 2 (ITS2) nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAK^G2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing. The gastrointestinal nematode compositions and alpha diversity generated by our FECPAK^G2 egg nemabiome metabarcoding approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAK^G2 harvested eggs in either DNA isolation lysis buffer or 80% ethanol (v/v) had no impact on gastrointestinal nematode identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility for nemabiome metabarcoding, in the absence of a cold chain. We discovered that sustained gastrointestinal nematode egg embryonation in the lysis buffer storage solution lead to higher yields of DNA compared with ethanol-stored gastrointestinal nematode eggs or faeces with gastrointestinal nematode eggs. Taking advantage of an already well-established platform such as FECPAK^G2, and providing the livestock producers that use it with the option to identify gastrointestinal nematodesin their samples and contribute to large-scale gastrointestinal nematode distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAK^G2 egg nemabiome metabarcoding approach.     

Mechanisms underlying resistance to nematode infection

Lambs show considerable genetic variation in faecal egg count following natural, predominantly Ostertagia circumcinta infection. This genetic variation is acquired and not innate. Worm length is positively associated with worm fecundity. The genetic variation in faecal egg count is a consequence of genetic variation in worm length and hence worm fecundity, and not of genetic variation in worm burdens. In contrast to lambs, mature sheep may be able to regulate both fecundity and worm numbers. In lambs, three factors account for the majority of the variation in worm length: the strength of the local IgA response against fourth-stage larvae, the specificity of this response against four molecules in particular, and the density-dependent influence of worm number.     

The effect of moxidectin 0,1% vs ivermectin 0,08% on milk production in sheep naturally infected by gastrointestinal nematodes

Background  

Gastrointestinal nematode (GIN) infection is one of the main constraints to sheep production both in temperate and tropical countries. Economic losses caused by GIN are related to decreased production, treatment costs and even animal death. The present paper was aimed at assessing the anthelmintic efficacy (based on faecal egg count reduction) of moxidectin and ivermectin both admistered per os at dose rate of 0.2 mg/Kg body weight and the benefit of anthelmintic treatments on milk production in a commercial dairy sheep farms in central Italy whose animals were naturally infected by GIN.     

In vitro antifilarial activity of extracts of the medicinal plant Cardiospermum halicacabum against Brugia pahangi

The in vitro effects of ethanol and aqueous extracts of the medicinal plant Cardiospermum halicacabum on adult worms and microfilariae of Brugia pahangi were investigated. With or without the plant extracts in culture medium, the motility of adult worms, microfilariae and microfilarial release from female worms were monitored daily. After 7 days of culture, viability or tissue damage of adult worms was assessed using the MTT assay. At > 500 microg ml-1, the aqueous extract significantly reduced motility of adult females after 24 h of exposure and adult males after 3 days. The aqueous extract, at > 500 microg ml-1, also significantly reduced microfilarial release from female worms, starting on day 2. The reduction in the motility of adult worms and the pattern of microfilarial release from female worms were concentration and time dependent. The MTT assay results revealed that adult worms cultured in the presence of aqueous extracts at > 500 microg ml-1 were damaged. However, the aqueous extract did not affect the motility of microfilariae with the exception of those in higher concentration extracts. Higher concentrations of ethanol extracts (2 mg ml-1) inhibited both the motility of adult worms and the release of microfilariae from females. Little effect of ethanol extracts was detected by the MTT assay, as only slight damage was caused to worms exposed only to the highest concentration (2 mg ml-1). However, ethanol extract at 500 microg ml-1 rapidly reduced the motility of microfilariae on day 2. The present study revealed that an aqueous extract of C. halicacabum has mild but definite direct macrofilaricidal action on B. pahangi.     

Faecal soiling and gastrointestinal helminth infection in lambs

The accumulation of faeces in the wool of the breech area (tail, perineum and anus) of lambs, known as faecal soiling, has been shown to be one of the major factors predisposing sheep to blowfly strike. However, the causes of faecal soiling of lambs in the UK are not clearly understood. Hence, in this investigation, the relationships between faecal soiling, gastrointestinal parasitic nematode infection and resultant diarrhoea were examined in a longitudinal study of 200 lambs at two farms in south-west England. Faecal egg counts, pasture worm burdens, faecal soiling and growth rates were recorded for individually tagged lambs over the summer of 2003. Grass growth and nutritional composition (protein and fibre) and weather data were also recorded over this period. Analysis using linear mixed models showed that faecal soiling was associated with higher strongyle-type egg counts, longer periods since worming, lower live-weights, female gender, lower faecal consistency and pasture quality. The data indicate that dag scoring, especially in mid- to late summer, could be used as a rapid, non-invasive technique for selecting animals, particularly lambs, with high faecal egg counts for selective drenching to reduce the incidence of anthelmintic resistance. Selective drenching of lambs with high dag scores would also be expected to aid in the control of blowfly strike.     

A genome scan for quantitative trait loci affecting resistance to Trichostrongylus colubriformis in sheep

A genome linkage scan was carried out using a resource flock of 1029 sheep in six half-sib families. The families were offspring of sires derived by crossing divergent lines of sheep selected for response to challenge with the intestinal parasitic nematode Trichostrongylus colubriformis. All animals in the resource flock were phenotypically assessed for worm resistance soon after weaning using a vaccination/challenge regime. After correcting for fixed effects using a least squares linear model the faecal egg count data obtained following the first challenge and the faecal egg count data obtained after the second challenge were designated Trait 1 and Trait 2, respectively. A total of 472 lambs drawn from the phenotypic extremes of the Trait 2 faecal egg count distribution were genotyped with a panel of 133 microsatellite markers covering all 26 sheep autosomes. Detection of quantitative trait loci (QTL) for each of the faecal egg count traits was determined using interval analysis with the Animap program with recombination rates between markers derived from an existing marker map. No chromosomal regions attained genome-wide significance for QTL influencing either of the traits. However, one region attained chromosome-wide significance and five other regions attained point-wise significance for the presence of QTL affecting parasite resistance.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

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.     

Animal grazing selectivity and plant chemistry issues impact on the potential of Rhagodia preissii as an anthelmintic shrub

Rhagodia preissii had shown significant in vitro anthelmintic activity in a previous study, we examined the effect of including this shrub in the diet of sheep infected with Trichostrongylus colubriformis. Worm-infected merino wethers were grazed for 7 weeks on either R. preissii or annual pasture, and faecal egg counts (FECs) were conducted weekly. Plant material was collected weekly from eaten and uneaten plants, and analysed for levels of plant secondary metabolites (tannins, oxalates, saponins) and in vitro anthelmintic activity. While mean FECs were consistently lower in sheep grazing R. preissii compared to pasture (reductions of 20-74%), the differences were not significant. There was no relationship between grazing preference (eaten or uneaten) and in vitro anthelmintic activity of plant extracts. The levels of saponins and oxalates did not correlate with grazing preference or in vitro anthelmintic activity, while tannins were not responsible for the anthelmintic activity. While the identity of the grazing deterrent and in vitro anthelmintic compounds remain unknown, the presence of plants which were both highly preferred by the sheep and showed in vitro anthelmintic activity indicates a potential to develop the species as an anthelmintic shrub through selection of shrub populations dominated by such plants.