In vivo anthelmintic activity of Dorycnium rectum and grape seed extract against Ostertagia (Teladorsagia) circumcincta and Trichostrongylus colubriformis in sheep

AIM: To assess the in vivo anthelmintic activity of condensed tannins (CT) in the forage species Dorycnium rectum and Medicago sativa, and in an extract from grape (Vitus vinifera) seeds (GSE), against two species of parasite, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus colubriformis, at different stages of their life cycle, in sheep that were parasite-na?ve or previously exposed to nematodes. METHODS: In Trial 1, a factorial treatment structure was used to compare faecal nematode egg counts (FEC) and worm burdens in 40 weaned Romney lambs fed either the CT-containing forage D. rectum (12% dry matter; DM) or M. sativa (lucerne; 0.2% DM). Twenty na?ve and 20 previously-exposed lambs were drenched free of parasites then reinfected with known species and numbers of parasites, and housed in pens indoors on a diet of lucerne pellets and chaffed hay. Groups of lambs (n=5 lambs per group) were fed one of the forages over one of two time periods within the parasite's life cycle. Six to nine days after the last feeding of fresh forages, faecal samples were collected for FEC, and all lambs were slaughtered and worm counts conducted. In Trial 2, 12 Suffolk x Romney lambs were surgically implanted with an abomasal cannula and then housed indoors in metabolism crates. After infection with parasites, six lambs were infused continuously over a 14-day period with a commercially available CT GSE (96% DM, made up to 34 g/L in water); the remaining lambs were infused with water. During infusion, samples were collected for egg hatch and larval development assays. After infusion, samples were collected for FEC, and all lambs were slaughtered and worm counts conducted. RESULTS: In Trial 1, there was a significant (p<0.001) difference in burdens of O. circumcincta between na?ve lambs and those previously exposed to parasites, but no other differences were recorded. In Trial 2, lambs infused with GSE had significantly (p<0.05) fewer T. colubriformis at slaughter and significantly (p<0.001) fewer eggs hatched in the egg hatch assay (EHA) than for lambs infused with water. Overall, the differences attributable to GSE were small in magnitude, being an 11% drop in egg hatch, and an 18% drop in numbers of adult T. colubriformis after 14 days of continuous infusion. No other differences were recorded. CONCLUSION: The results indicate that the in vivo anthelmintic activity of these CT sources is, at best, modest and is unlikely to be of any practical value. Further, these data emphasise that in vitro activity is an unreliable indicator of in vivo efficacy for CT-containing forages and extracts.     

In vivo anthelmintic activity of Phytolacca icosandra against Haemonchus contortus in goats

Two separate controlled and blinded studies were conducted to confirm the dose and non-interference of spinosad and milbemycin oxime (MO) administered orally in combination or alone to dogs for the treatment and control of experimentally induced flea infestations (Ctenocephalides felis) and adult hookworm infections (Ancylostoma caninum). For each study, dogs were allocated randomly based on pre-treatment adult flea and hookworm egg counts to one of four treatment groups of 10 animals each. In each study, spinosad and MO in combination, using the lower half (30-45 mg/kg spinosad; 0.5-0.75 mg/kg MO) of the US commercial dose band (30-60 mg/kg spinosad; 0.5-1.0mg/kg MO) of each active ingredient, or individually alone using the full dose range, were given orally to dogs on Day 0 using a tablet formulation. A placebo control was treated similarly. In one study, on Days -1, 5, 12, 19, 28 and 35 each dog was infested with approximately 100 unfed adult C. felis obtained from the investigator's established flea colony. All dogs were infested via the same method. Forty-eight hour post-infestation flea comb counts were conducted on Days 1, 7, 14, 21, 30 and 37 and were used to determine the knockdown and residual flea activity. In the second study, on Day -27 each of 48 dogs were experimentally inoculated with 100 third-stage infective larvae of the hookworm, A. caninum. Dogs were treated on Day 0 and necropsied on Day 7 or Day 8. All nematodes in the intestinal tract were collected on Day 7 or Day 8, identified and counted by species and stage. Post-treatment, the geometric mean live flea counts were significantly different (p-value<0.0001) between the spinosad/MO combination and the spinosad only treatment groups as compared to the vehicle control group. The flea counts in the MO only group and the control group were not statistically different. The spinosad and MO combination group and the spinosad only treatment group demonstrated significantly different knockdown (100%) and post-treatment residual flea efficacy at Day 30 was 100% for both groups as compared to the vehicle control. The presence of MO in combination with spinosad did not interfere with the flea efficacy of spinosad as compared to the spinosad only group. MO alone did not demonstrate any flea efficacy. Post-treatment, the geometric mean A. caninum worm counts were significantly different (p-value<0.0001) between the spinosad and MO combination group as compared to the vehicle control group. The worm counts in the MO only group and the combination group were not statistically different. The spinosad and MO combination group (99.8% reduction) and the MO only treatment group (99.5% reduction) both demonstrated significantly different hookworm efficacy as compared to the vehicle control group. The presence of spinosad in combination with MO did not interfere with the hookworm efficacy of MO as compared to the MO only group. Spinosad alone did not demonstrate any hookworm efficacy. In summary, flavored spinosad and MO combination tablets administered orally to dogs at the lower end (30-45 mg/kg spinosad; 0.5-0.75 mg/kg MO) of the US commercial tablet unit dose range (30-60 mg/kg spinosad; 0.5-1.0mg/kg MO) were both safe and highly efficacious delivering 100% knockdown and 30 days of residual adult flea control on experimentally infested dogs as well as >99% adult hookworm efficacy evaluated under laboratory conditions. Interference between either drugs was not demonstrated for both of these dose limiting parasites.     

Duration of activity of topical eprinomectin against experimental infections with Teladorsagia circumcincta and Trichostrongylus colubriformis in goats

The immediate as well as the persistent anthelmintic efficacies of topically applied eprinomectin were evaluated in goats against induced infections with Teladorsagia circumcincta (2800 L3) and Trichostrongylus colubriformis (6000 L3). Twenty-three culled dairy goats were allocated to the following groups: control animals (group 1), animals treated 21 days prior to nematode infection (group 2), animals treated 7 days prior to nematode infection (group 3) and animals treated 21 days after nematode infection (group 4). Eprinomectin was applied at twice the cattle dose rate (1.0 mg/kg BW). According to the groups, necropsies were undertaken 28 days after nematode infection (groups 1-3) or 14 days after the anthelmintic treatment (group 4). Worm counts were determined for abomasum and small intestine. The curative anthelmintic efficacy of eprinomectin at 1.0 mg/kg BW on existing worm burdens was 100% against T. circumcincta and T. colubriformis. Quite similar worm burdens reductions were observed when eprinomectin was administered 7 days before infection whereas they were only 52.4 and 17.8% for T. circumcincta and T. colubriformis, respectively, for an administration of the drug 21 days prior to the nematode infection.     

Direct anthelmintic and immunostimulatory effects of oral dosing semi-purified phytohaemagglutinin lectin in sheep infected with Teladorsagia circumcincta and Trichostrongylus colubriformis

Lectins are plant secondary compounds that can have anthelmintic properties in vitro. In particular, the phytohaemagglutinin lectin extracted from Phaseolus vulgaris has been shown to inhibit the feeding of Trichostrongylus colubriformis and Teladorsagia circumcincta L1 larvae. However, little is known about the potential anthelmintic properties of this lectin in vivo and its suitability to control gastrointestinal parasite infections in lambs. In a 2 × 2 study, lambs were either orally dosed, or not, with 2.3mg semi-purified PHA lectin per kg live weight (LW) per day, whilst concurrently infected, or not, with 1000 T. circumcincta and 1000 T. colubriformis L3 infective larvae per day for 42 days. There were no adverse clinical effects observed with this dose of PHA lectin. Although worm burdens were similar, animals dosed with PHA lectin had reduced concentration of nematode eggs in the faeces compared with their non-lectin dosed counterparts (P=0.026), suggesting that there may be a direct effect of PHA lectin on parasite fecundity. Irrespective of infection, PHA lectin had immune-stimulatory properties with increased eosinophillia in both abomasal and small intestine tissue sections taken at slaughter on day 42 (P<0.02 for both) and a tendency for decreased ability of Teladorsagia larvae to penetrate abomasal tissue explants (P=0.06). Compared with infection alone, concurrent PHA lectin dosing and infection further increased the number of eosinophils (P<0.01), PAS-positive (mucin-producing cells) (P=0.03) and tended to increase the number of T helper cells (P=0.06). No interactions were observed for cell populations in small intestine tissue sections. These results suggest PHA lectin could have two possible modes of action against T. circumcincta and T. colubriformis, a direct anthelmintic effect on nematode fecundity and an indirect effect through enhancing local immune responses in the host.     

The anthelmintic efficacy of albendazole against Fasciola hepatica and benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

The anthelmintic efficacy of albendazole (methyl [5-(propylthio) - 1H - benzimidazole -2 -yl] carbamate) against immature and adult Fasciola hepatica and against standardised strains of benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis was evaluated in experimentally infected sheep. A single intrarumenal treatment of dose rates of 3.8 and 7.6 mg/kg was ineffective against immature (six weeks old) F hepatica. Dose rates of 5.7 and 7.6 mg/kg reduced the number of mature (12 weeks old) F hepatica by 70 and 91 per cent respectively. Dose rates of 5.7 and 7.6 mg/kg removed 92 and 99 per cent of four-week-old, benzimidazole resistant H contortus and 89 and 99 per cent of four-week-old, benzimidazole resistant T colubriformis.     

Evaluation of ivermectin against experimental infections of Haemonchus contortus and Trichostrongylus colubriformis in goats.

Fourteen nematode parasite-na?ve kids were given a mixed larval inoculum (approximately 17,250 infective larvae) via stomach tube. Twenty-two days after infection, the kids were randomly assigned to control and treatment groups of seven animals each. Mean eggs per gram for control and treatment groups were 4304 and 5315, respectively. Kids in the treatment group were given ivermectin (0.2 mg kg-1) administered by subcutaneous injection. At necropsy, the mean numbers of Haemonchus contortus and Trichostrongylus colubriformis for control and treatment groups were 2259 and 0, and 3033 and 773, respectively. This reduction was significant (P less than 0.05) for both species, resulting in an efficacy of 100.0% and 74.5%, respectively. The lower efficacy against T. colubriformis may be a sign of resistance, a reduced effectiveness due to route of administration, or a higher dose may be required with subcutaneous administration, as has been observed previously.     

The dose response of several benzimidazole anthelmintics against resistant strains of Haemonchus contortus and Trichostrongylus colubriformis selected with thiabendazole

Dose response lines for eight benzimidazole anthelmintics and thiophanate were determined, using standardised strains of thiabendazole selected and resistant Haemonchus contortus and Trichostrongylus colubriformis. Against H contortus, thiophanate, thiabendazole, parbendazole and oxibendazole were inactive. Mebendazole was inactive at dose rates of 6.26 and 12.5 mg/kg, although significant activity occurred at 25 mg/kg. Fenbendazole, cambendazole, oxfendazole and albendazole demonstrated significant activity at dose rates equal to or greater than the recommended therapeutic level. Thiophanate was inactive against resistant T colubriformis. The remaining compounds only showed significant activity when used at dose rates in excess of the recommended therapeutic level. These results show that a side resistance exists among the benzimidazole anthelmintics and suggests that changes in dose response lines could be expected to occur if resistant strains are selected with benzimidazoles other than thiabendazole.     

Green tea flavan-3-ols and oligomeric proanthocyanidins inhibit the motility of infective larvae of Teladorsagia circumcincta and Trichostrongylus colubriformis in vitro

The effects of a hot water infusion and an aqueous acetone extract of green tea (Camellia sinensis) on the motility of infective larvae of the sheep nematodes Teladorsagia circumcincta and Trichostrongylus colubriformis were investigated under in vitro conditions. The infusion and extract dose-dependently inactivated the infective larvae as assessed by the larval migration inhibition (LMI) assay. To determine the components responsible for the inhibitory activity, the hot water infusion and aqueous acetone extract of green tea were fractionated on Sephadex LH-20 and the green tea extract fractions (GTE-I-VIII) characterised by mass spectrometry. The larvae were exposed to increasing concentrations of these GTE fractions. Fractions containing epigallocatechin gallate (EGCG) and proanthocyanidin oligomers were most effective. GTE fractions were more effective against T. circumcincta than T. colubriformis larvae as assessed by the LMI assay.     

In vitro analysis of the anthelmintic activity of forage chicory (Cichorium intybus L.) sesquiterpene lactones against a predominantly Haemonchus contortus egg population

The anthelmintic activity of chicory (Cichorium intybus L.) herbage has been attributed to sesquiterpene lactones. Chicory leaves contain significant amounts of lactucin (LAC), 8-deoxylactucin (DOL), and lactucopicrin (LPIC), but the proportions of these three sesquiterpene lactones vary among forage chicory cultivars. To determine whether the individual compounds differ in anthelmintic activity, we prepared sesquiterpene lactone-enriched extracts from leaves of two forage chicory cultivars, 'Grasslands Puna' (Puna) and 'Forage Feast', and tested their effects on the hatching of a predominantly Haemonchus contortus egg population. The dominant constituents in the Puna and Forage Feast extracts were DOL and LAC, respectively; LPIC concentrations in the two extracts were similar. Extracts from both cultivars inhibited egg hatching at all concentrations tested (P<0.001), but there were significant differences in egg responses to the two extracts (P<0.001). With Puna, egg hatching decreased sharply in a linear fashion when the combined LAC, DOL, and LPIC concentrations ranged from 0 to 5.0mg/ml. A biphasic effect on egg hatching occurred with the Forage Feast extract. The fraction of eggs that hatched decreased gradually to 65% as the sesquiterpene lactone concentrations increased from 0 to 6.7 mg/ml. Treatment with higher concentrations resulted in a sharp decline in egg hatchability. Concentrations of sesquiterpene lactones required for 50% lethality were determined by probit dose-effect analysis to be 2.6 mg/ml (95% confidence interval: 2.4-2.8 mg/ml) for the Puna extract and 6.4 mg/ml (95% confidence interval: 5.9-7.2mg/ml) for the Forage Feast extract (P<0.0001). These concentrations provided 1.3 and 1.5mg/ml of DOL and 0.8 and 3.9 mg/ml of LAC for Puna and Forage Feast extracts, respectively. Results suggest that LAC has minimal effect on egg hatching and that DOL or other constituent(s) in the extracts is inhibitory. Quantitative analyses of free sesquiterpene lactones in chicory leaf extracts suggest that Puna may be a better cultivar than Forage Feast for use in bioactive pastures for gastrointestinal parasite control in small ruminants.     

Anthelmintic efficacy of five tropical native Australian plants against Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats (Capra hircus)

The study of the anthelmintic properties of plants rich in plant secondary metabolites can provide ecologically sound methods for the treatment of parasites on grazing animals. The purpose of the present study was to evaluate the anthelmintic effect of five tropical native Australian plant species rich in plant secondary metabolites on adult Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats. Thirty young, nematode-free goats were infected with 2500 H. contortus and 5000 T. colubriformis infective larvae thrice weekly for a week (day 1-7 of the experiment). On day 27 after first infection, the goats were allocated into six groups of five animals per group. From day 28 to day 35, fresh leaves from Acacia salicina, Acacia nilotica, Eucalyptus corymbia, Casuarina cunninghamiana and Eucalyptus drepanophylla were included in the goats diet. Five groups were offered leaves from one of these plant species and one group, the untreated control, received only the basal diet formulated with 20% Medicago sativa and 80% Avena sativa. Following plant material administration, the goats were monitored daily until day 40 and then slaughtered on day 41. Total faecal worm egg output, total production of larvae recovered from faecal cultures, total post-mortem worm burdens and the per capita fecundity of female worms were estimated. The toxicity of the plant species for the goats was measured by histopathological analyses of liver and kidney samples. Results showed that goats feeding on the plant material rich in plant secondary metabolites had significantly lower egg output compared to the control goats (P<0.05). A similar response was found for larval production in both H. contortus and T. colubriformis supporting that egg output was affected in both species. Although the total worm burdens were not affected by the plant material (P>0.05), the per capita fecundity was significantly reduced by E. corymbia, A. nilotica and A. salicina (P<0.05). No signs of toxicity were detected in the liver or kidney samples. It is concluded that goats can benefit from the short-term ingestion of plant secondary metabolites, which reduce the total faecal egg output and thus decrease the potential for re-infection from the pasture.     

Evolution of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a 4-year experiment

Field trials were undertaken to compare nematode population dynamics in two experimental flocks of rams selected for high and for low faecal egg counts (FECs) following two doses with 20000 infective larvae, 12000 Teladorsagia circumcincta (Tcirc) and 8000 Trichostrongylus colubriformis (Tcol) separated by a treatment. The selection was done by dosing 200 ram lambs (INRA 401 breed) and FECs proceeded with egg identification on days 28 and 35 post-each-infection. The 30 lambs with the lowest FECs constituted the group of permanent resistant rams (R), the 30 with the highest values the group of susceptible ones (S). Each group grazed separated pastures from April to November, this during 4 consecutive years. The contamination (number of eggs deposited) was estimated every 3 weeks on each pasture. The larval populations was measured by successive groups of tracer lambs. The infection of the permanent rams was done by slaughtering eight R and S rams on mid and end of each grazing season. Each autumn, new R and S ram lambs were selected to complete the R and S groups before turning out in spring. On third year, a third species (Haemonchus contortus, Hcon) was added to compare its behaviour with the two others.The regulation of parasite populations were studied by comparing what happens in R rams and pasture to S ones. The selected R ram lambs had a FEC of Tcirc 50% lower than S ones. This induced a similar lowered pasture contamination, a 25% decrease of infective larval population in years 2 and 3, and the worm burdens in R rams were the half of that in S rams after 2 years. The FEC of Tcol was 75% lower at the selection and induced similar difference in pasture contamination, but 65 and 96% reduction in L3 population, respectively, after 1 and 2 years. The worm burden was reduced by 99%. Adding Hcon species during the third year, in year 4, the L3 population was half in R pasture compared with S one, and R rams had 60% less worms of this species than S ones.This selection without any drench prevent outbreaks observed in young tracer lambs on S pasture. The comparison of selected resistant rams to selected susceptible ones and not to unselected controls gave more rapid information about the tendency of the regulation of the communities of nematode parasites.