Effect of Gliricidia sepium leaves intake on larval establishment of Cooperia punctata in calves and bio-guided fractionation of bioactive molecules.

The aims of this study were: 1) to assess the anthelmintic effect of Gliricidia sepium on the establishment of C. punctata third-stage larvae (L3) in calves, and 2) to isolate and to elucidate an anti-exsheathment phytochemical from the plant offered during the trial. Twelve ¾ Holstein × Zebu calves were divided in two experimental groups: control (T1) and treatment (T2) (n = 6). After adaptation, each calf was infected with an oral dose of 400 C. punctata L3/Kg LW. Basal diet consisted of Digitaria decumbens hay (6.27% CP) and commercial concentrate (12% CP). In addition, during the experimental period T2 received fresh G. sepium leaves (26.88% CP) ad libitum. On day 9 post-infection, three calves per treatment were randomly selected for slaughter, and worm counts were performed. Larval establishment rates obtained were 13.44 ±â€¯0.13% and 3.1 ±â€¯1.42% for T1 and T2, respectively (P < .05). The reduction of larval establishment was 76.9%. The total length of worms recovered from the animals was also affected by the intake of G. sepium (P < .05). Phytochemicals present in G. sepium leaves offered to calves were isolated through silica gel columns and elucidated through Magnetic Nuclear Resonance (1H and 13C). Bio-guided isolation procedures lead to the elucidation of Oxytroside (Kaempferol 3-O-rhamnopyranosyl-(1 → 6)-ß-D-glucopyranoside-7-O-rhamnopyranoside), which fully inhibited the C. punctata exsheathment process (2400 µg mL-1). Gliricidia sepium represents an alternative to prevent severe C. punctata infections by reducing larval establishment in cattle.

Effects of selected Palestinian plants on the in vitro exsheathment of the third stage larvae of gastrointestinal nematodes.

BACKGROUND: Gastrointestinal parasites are one of the main restrictions to small ruminant production. Their pathological importance is primarily related to the major production losses, in quantity or quality, induced by the direct action of worms. Control of these parasites is based exclusively on the frequent use of anthelmintic drugs. However, the resistance to anthelmintics in worm populations after commercialisation of chemical drugs is now widespread. Therefore, there is a need to find new natural resources to ensure sustainable and effective treatment and control of these parasites. The aim of this study was to evaluate the anthelmintic activity, as minimum inhibitory concentration (IC 50 mg/mL), of different plant extracts using larval exsheathment inhibition assay using a two-species but steady population of parasitic nematodes (ca. 20% Teladorsagia circumcinta and 80% Trichostrongylus colubriformis). RESULTS: The study showed that the ethanolic extracts of 22 out of the 48 plant extracts, obtained from 46 plant species, have an inhibitory effect >50% (at concentrations of 100 mg/mL) on the third stage larvae (L3) of the nematodes exhibited the strongest inhibition activity (94%) with IC 50 of 0.02 mg/mL, where other members of the Rhamnaceae family have shown to possess strong anthelmintic activity (70-89%). CONCLUSIONS: Plant extracts are potential rich resources of anthelmintics to combat helminthic diseases. Our results suggest that extracts from Rhamnus elaternus, Epilobium hirsutum, Leucaena leucocephala and Rhamnus palaestinus have promising anthelmintic activity, with potential applications in animal therapeutics and feed.

Anthelmintic effect of 2H-chromen-2-one isolated from Gliricidia sepium against Cooperia punctata.

Gliricidia sepium is a tropical legume with known anthelmintic-like properties. The aim of this study was to: (1) perform a bio-guided fractionation of an acetonic extract of G. sepium leaves using the egg hatch assay (EHA); (2) elucidate the anthelmintic (AH)-like phytochemical using nuclear magnetic resonance (NMR); and (3) assess the ultrastructural damage of the Cooperia punctata treated eggs. The anthelmintic activity of G. sepium was traced from an acetonic extract using the EHA. Phytochemicals were isolated through silica gel columns and elucidated through spectroscopic measurements (1H and 13C). Final fraction was evaluated with EHA at decreasing concentrations of: 1.100; 0.500, 0.250, 0.125, 0.060, 0.001 and 0.00001 mg mL-1. Egg hatching inhibition was calculated using the formula: 100*(1-HT/HC). The maximal half of effective concentration (EC50) was calculated with GraphPad. Bio-guided isolation procedures lead to the elucidation of 2H-chromen-2-one, which inhibited both hatching and embryo development of C. punctata (EC50 of 0.024 ± 0.082 mg mL-1) (P < 0.05). Scanning and Transmission Electron Microscopy (SEM and TEM) revealed electrodensity alterations and fractures in the eggshell layers. After toxicity evaluations and in vivo assessment, 2H-chromen-2-one can be suggested as a novel AH-phytochemical for reducing larval density in pastures and worm burdens inside the host.

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes.

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity.

Elucidation of Leucaena leucocephala anthelmintic-like phytochemicals and the ultrastructural damage generated to eggs of Cooperia spp.

Leucaena leucocephala is a tropical forage legume suggested as an alternative method to control gastrointestinal parasitism in ruminants. This study: (1) performed a bio-guided fractionation of an aqueous extract of L. leucocephala using the egg hatch assay (EHA) to identify the anthelmintic (AH)-like phytochemicals present in fresh leaves, and (2) assessed the ultrastructural damage to eggs of Cooperia spp. after incubation with the final fraction. Phytochemicals were isolated using silica gel columns and identified using high performance liquid chromatography and standards for comparison. The final fraction was evaluated using EHA at 0.06, 0.125, 0.250, 0.500 and 1.1 mg ml(-1). The lethal concentration to inhibit 50% of Cooperia spp. egg hatching (LC50) was calculated using a Probit analysis. Scanning and transmission electron microscopy revealed the ultrastructural changes present in Cooperia spp. eggs. Bio-guided isolation procedures led to the recognition of an active fraction (LlC1F3) mainly composed of quercetin (82.21%) and caffeic acid (13.42%) which inhibited 90.49 ± 2.8% of Cooperia spp. egg hatching (P<0.05), and an LC50 of 0.06 ± 0.14 mg ml(-1). Scanning electron microscopy (SEM) showed eggs exposed to the active fraction had an irregular external layer with small projections and ruptures of lateral eggshell walls. Transmission electron microscopy (TEM) showed changes to Cooperia spp. eggs in electro-density, including the thickness of the eggshell layers and fractures after incubation with the final fraction (LlC1F3). Changes in bioactivity after purification suggest synergistic interactions between quercetin and caffeic acid.

The effects of condensed tannins extracted from different plant species on egg hatching and larval development of Teladorsagia circumcincta (Nematoda: Trichostrongylidae).

The effects of condensed tannins (CTs) extracted from five species of plants on egg hatching and larval development of Teladorsagia circumcincta (Stadelmann, 1894) (syn. Ostertagia circumcincta) were evaluated using in vitro bioassays. The extracts of CTs were obtained from Lotus pedunculatus (LP), Lotus corniculatus (LC), Dorycnium pentaphyllum (DP), Dorycnium rectum (DR) and Rumex obtusifolius (RO). The results of egg hatching assay showed that about 53%, 68%, 51%, 60% and 46% of the eggs hatched when in vitro incubations contained 900 microg/ml of CTs from LP, LC, DP, DR and RO, respectively (P < 0.001 relative to control incubation), while in control incubations (no CT added) 87% of the eggs hatched. In the larval development assay, development was allowed to proceed for 7 days, by which time 89% of the hatched larvae in control wells (no CTs) had reached the infective third stage (L3). In incubations containing 200 microg CT from LP, LC, DP, DR and RO/ml, about 8%, 15%, 14%, 8% and 4% of the eggs attained full development to L3 larvae, respectively (P < 0.001 relative to control incubation). Only 1% of the eggs were able to develop to L3 larvae in incubations containing 400 microg CT extracted from LC/ml, whilst in the incubations containing the same concentration of other CTs the eggs were not able to develop to L3 larvae. It seems that CTs are not only slowing down the larval development but also kill the undeveloped larvae. At 400 microg/ml, for example, CT from LP, LC, DP, DR and RO killed 67%, 48%, 68%, 93% and 91% of first-stage (L1) and second-stage (L2) larvae, respectively. This study shows that CTs are able to disrupt the life cycle of nematodes.

In vivo anthelmintic activity of Butea monosperma against Trichostrongylid nematodes in sheep.

Seeds of Butea monosperma administered as crude powder (CP) at doses of 1, 2 and 3 g/kg to sheep naturally infected with mixed species of gastrointestinal nematodes exhibited a dose and a time-dependent anthelmintic effect. The maximum reduction of 78.4% in eggs per gram of feces (EPG) was recorded on day 10 after treatment with 3 g/kg. Levamisole (7.5 mg/kg), a standard anthelmintic agent, exhibited 99.1% reduction in EPG.

[Primary anthelminthic screening on the L4 and L5 developmental stages of the lungworm, Dictyocaulus viviparus, in guinea pigs]. / Primární anthelmintický screening na vývojová stadia L4 a L5 plicnivky Dictyocaulus viviparus u morcat.

The study was aimed at expanding the primary anthelmintic screening to cover a model of the group of pulmonary nematodes; in this particular case to introduce the lungworm, D. viviparus, in laboratory animals. A method of primary screening for the fourth and fifth larval states of D. viviparus in guinea-pigs was worked out after the selection of a suitable laboratory host. In the primary screening, three well-known anthelmintics of the benzimidazol series, including fenbendazole, mebendazole and levamisol, were tested by the method of controlled test. The anthelmintics were administered at the recommended doses of 7.5 and 10.0 mg per kg of live weight for two days in succession. The effectiveness of the control of the 4th and 5th larval states of D. viviparus was 93.4% in fenbendazole, 89.0% in mebendazole, and 89.9% in levamisol. It is confirmed by the results of the trials that guinea-pigs can be used for the testing model of the lungworm, D. viviparus, in anthelmintic screening.

Trichostrongylus colubriformis: in vitro culture of parasitic stages and their use for the evaluation of anthelmintics.

Approximately 50 per cent of fourth stage larvae of Trichostrongylus colubriformis taken from the gerbil, Meriones unguiculatus, on day 8 after infection, moulted to the young adult stage when cultured in a complex medium over a seven day period in vitro. Larvae at the late fourth stage of development were highly susceptible to certain benzimidazole, prebenzimidazole, imidazothiazole, pyrimidine, quaternary ammonium, organophosphorus and macrocyclic lactone anthelmintics when any of these were included at very low concentrations in the culture medium. However, few anthelmintics lacking activity against T colubriformis in vivo affected these larvae. An assay employing these larvae in vitro should offer a means for assessing the intrinsic activity of new compounds against T colubriformis in the absence of any complicating host pharmacokinetic factors, and could also be adapted for use as a high capacity preliminary screen. Thus it should now be possible to employ a target parasite at the earliest stages of a lead discovery programme obviating the need to use less relevant free-living nematodes or ones that are natural parasites of rodents.

Comparative efficacy of standard antihookworm drugs against various test nematodes.

The chemotherapeutic responses of three test nematodes, Nippostrongylus brasiliensis, Nematospiroides dubius and Ancylostoma ceylanicum to standard antihookworm drugs were assessed in order to select a suitable host-parasite system for the primary screening of potential antihookworm compounds. N. dubius behaved inconsistently and, with some infections, required more drug to achieve 100% clearance. Nippostrongylus brasiliensis was found to be sensitive to thiabendazole, tetramisole and levamisole but the broad spectrum anthelmintic mebendazole was ineffective. A. ceylanicum was very sensitive to mebendazole, sensitive to tetramisole and levamisole and refractory to thiabendazole. In vitro, none of the compounds had any lethal effect against any of the nematodes, except mebendazole against A. ceylanicum. A. ceylanicum does occur in man and its chemotherapeutic reactions are similar to those of target hookworm infections of economic importance. As such, although not equally sensitive to standard anthelmintics, it is recommended for routine primary screening.

Use of guniea pigs to assay anthelmintic resistance in ovine isolates of Trichostrongylus colubriformis.

Guinea pigs infected with Trichostrongylus colubriformis were used to develop an assay for anthelmintic resistance by determination of worm burdens following treatment with test anthelmintics. To achieve comparable efficacy with the recommended dose of thiabendazole and levamisole in sheep, dose rates in guinea pigs had to be increased two to four fold. For example, thiabendazole at 100 mg/kg in guinea pigs was 96.6 per cent effective against a thiabendazole susceptible (GS) strain of T colubriformis, but had no effect against a thiabendazole-resistant (VRSG) strain. In sheep 50 mg/kg of thiabendazole would have a similar efficacy against each strain respectively. Morantel tartrate at 10 mg/kg in guinea pigs was 99 to 100 per cent effective against the GS strain but only 54 per cent effective against a morantel resistant (PF4) strain. A slope ratio assay was used to calculate the relative potency of anthelmintics by comparing efficacies against resistant strains with efficacy against the GS strain. Resistance of the VRSG strain to thiabendazole was confirmed with a relative potency for this drug of 0.047 in guinea pigs. The PF4 strain was resistant to both thiabendazole and levamisole which had relative potencies of 0.168 and 0.255 respectively. The advantages of this statistical treatment together with the cost and time savings of the guinea pig model over a conventional critical anthelmintic assay in sheep are discussed.