Ovicidal and larvicidal activity of the crude extracts from Phytolacca icosandra against Haemonchus contortus

The development of anthelmintic resistance has impacted on the success of conventional anthelmintics (AH) for the control of gastrointestinal nematodes in grazing/browsing sheep and goats. Medicinal plants from the traditional herbolary in Mexico may provide new candidates that can be explored as alternative sources of AHs for ruminants. This study evaluated the leaf extracts derived from Phytolacca icosandra against infective L(3) larvae and eggs from Haemonchus contortus collected from sheep. Three extracts of different polarities were obtained from the leaf plants using ethanol, n-hexane and dichloromethane as the solvents. The effectiveness of the in vitro AH activity of the plant extracts was evaluated using larval migration inhibition (LMI) and egg hatch (EHA) assays. For the LMI assays, the ethanolic extract of P. icosandra showed 55.4% inhibition of larval migration at 2mg/mL (p<0.05). The dichloromethane extract of P. icosandra showed 67.1% inhibition of migration at 3mg/mL (p<0.05) and a dose-dependent response with an LD(50) of 0.90 mg/mL. The n-hexane extract failed to show inhibition of larval migration at any concentration explored. In the EHA for the ethanol extract, the lowest concentration tested (0.15 mg/mL) resulted in inhibition of egg hatching greater than 72.6%. Therefore, the LD(50) could not be calculated for this extract. The LD(50) of the dichloromethane extract of P. icosandra was 0. 28 mg/mL. An egg hatch inhibition greater than 90% was observed with both the ethanolic and dichloromethane extracts when using a concentration of 0.90 mg/mL or higher. The n-hexane extract failed to show egg hatch inhibition at any concentration tested. The AH activity reported for P. icosandra could be attributable to the flavonoids, steroids, terpenoids, coumarins and/or saponins that were present in the ethanolic and dichloromethane extracts. A combination of more than one component may also explain the observed AH activity against the H. contortus life stages that were evaluated. In conclusion, the ethanolic and dichloromethane extracts of P. icosandra showed clear in vitro AH activity against the H. contortus eggs and the L(3) larvae. However, the hexanic extract of the plant leaves failed to show any in vitro AH activity.     

Efficacy of anthelmintic properties of medicinal plant extracts against Haemonchus contortus

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This present work evaluated the efficacy of ethyl acetate, acetone, and methanol dried leaf and seed extracts of five medicinal plants were tested in vitro ovicidal and larvicidal activities on Haemonchus contortus. The in vitro assay was based on egg hatch assay (EHA) and larval development assay (LDA), all plant extracts were evaluated at five concentrations 50, 25, 12.5, 6.25 and 3.13 mg/ml. The leaf and seed ethyl acetate, acetone and methanol of Annona squamosa, Eclipta prostrata, Solanum torvum, Terminalia chebula, and Catharanthus roseus extracts were showed complete inhibition (100%) at the maximum concentration tested (50 mg/ml). The overall findings of the present study have shown that our experimental plant extracts contain possible anthelmintic compounds.     

The anthelmintic efficacy of thiabendazole and levamisole against inhibited Haemonchus contortus larvae in sheep

Fifty-five dogs, 6 to 18 months old, from 11 areas of New Zealand were examined for gastro-intestinal helminths. Thirty-eight (69.1%) were infected. Twenty-one (38.2%) had Toxocara canis, 20 (36.4%) Trichuris vulpis, 20 (36.4%) Uncinaria stenocephala, 3 (5.5%) Toxuscaris leonina, 3 (5.5%) Dipylidium caninum and 1 (1.8%) Taenia sp. Helminth infections were less frequent and populations were smaller in females than in males.     

Fecundity of anthelmintic resistant adult Haemonchus contortus after exposure to ivermectin or benzimidazoles in vivo

A strain of Haemonchus contortus resistant to the benzimidazoles, ivermectin and salicylanilides was used to study the effect of exposure to ivermectin or oxfendazole on the number of eggs produced by adult female parasites. After anthelmintic administration there was a reduction in the faecal egg count of the infected lambs. Ivermectin caused a reduction in the number of eggs within adult parasites for up to 72 hours but this returned to higher than pre-treatment levels at 168 hours after treatment. After oxfendazole treatment there was an increase in the number of eggs stored in the adult parasites at 24 hours and then a significant reduction in the number of eggs for over 168 hours. This suggested that both ivermectin and oxfendazole have a deleterious effect on the reproductive potential of female H contortus although the parasites are resistant to these drugs. These results confirm the importance of allowing up to 10 days to elapse between the time of administration of an anthelmintic and the collecting of faecal samples when using the faecal egg count reduction test to identify anthelmintic resistance, especially when using benzimidazoles. The more prolonged effect on fecundity produced by oxfendazole may reflect the antimitotic action of the benzimidazole anthelmintics.     

In vitro and in vivo characterization of p-amino-phenethyl-m-trifluoromethylphenyl piperazine (PAPP), a novel serotonergic agonist with anthelmintic activity against Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis

The neurotransmitter serotonin (5-hydroxy tryptamine or 5HT) regulates key physiological processes in nematodes such as locomotion and feeding. PAPP (p-amino-phenethyl-m-trifluoromethylphenyl piperazine) is a known agonist of the 5-HT(1Hc) receptor of the barber pole worm, Haemonchus contortus. In this study, PAPP was highly active against L3-stage larvae of H. contortus and Trichostrongylus colubriformis in an in vitro larval migration assay, with EC50 values of 9.36 and 11.8 microM, respectively, that were comparable to levamisole (10.2 microM) and superior to pyrantel (55.39 microM). When administered orally or subcutaneously to nematode infected gerbils, PAPP provided >99% efficacy against H. contortus and >98% efficacy against Teladorsagia circumcincta at 100 mg/kg, comparable to levamisole at 10 mg/kg. Drug titration revealed significant activity down to 50 mg/kg against these two species. Spectrum was limited, however, with somewhat lower efficacy (83%) in T. colubriformis infected gerbils at 100 mg/kg. Oral delivery of hydrochloride, acetate and phosphate salts of PAPP to nematode infected gerbils did not result in an increase in either potency or spectrum. The finding that PAPP exhibits significant anthelmintic activity suggests that the nematode-specific serotonergic system is a viable target for future anthelmintic discovery.     

捻转血矛线虫重组H11抗原山羊免疫保护性试验

以重组H11-1、H11-2及H11-1与H11-2混合物分别免疫山羊后,ELISA检测发现血清抗体均在首次免疫后14 d达到较高水平,在第2次免疫后10 d达到高峰。用10 000只捻转血矛线虫第3期幼虫攻击山羊后,对不同免疫组山羊排出虫卵数、虫卵孵化率和成虫数的统计分析结果表明,免疫组与对照组间差异不显著,但免疫组雌虫数显著少于对照组,这对防治捻转血矛线虫病可能会起一定的作用。     

The establishment and development of Haemonchus contortus in goats

Twelve goats were inoculated with 40,000 third-stage Haemonchus contortus larvae and two were killed on each of Days 4, 7, 11, 14, 18 and 21 after inoculation (DAI). The number of worms that established, and the site of development were recorded. More worms established in the fundic, than in the middle or pyloric thirds of the abomasum. Early development occurred within the mucosa; emergence into the lumen started between 7 and 11 days after infection. By 4 DAI, all worms had completed the third moult to the L4 stage. At 11 DAI the majority of the worms were adults. A mean of 13.2% of the female worms had eggs in their uteri at 18 DAI; by 21 DAI more than half of the female worms had eggs in their uteri. The development of H. contortus was essentially similar to that described in sheep.     

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.     

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.     

Cross-infection between tropical goats and heifers with Haemonchus contortus

Developing effective biological control without the systematic use of anthelmintics is necessary to reduce the impact of gastrointestinal nematodes on small ruminants. Therefore, grazing management systems that use different host species to dilute nematodes in pasture appear to be promising for worm control. A trial was carried out to investigate the specificity of Haemonchus contortus for goats and cattle and to evaluate cross-infection between ruminant species. The effect of an experimental infection of 12 heifers by the free-living stages of H. contortus collected from goats (500 larvae per kg liveweight) was evaluated and compared to uninfected controls. After 28 and 35 days, egg excretion was measured. The experimental infection of heifers by H. contortus was not significant, with no egg excretion. These results, i.e., the lack of cross-infection of GIN between goats and cattle, suggest that integrated grazing using such animals could be employed for pasture dilution and decontamination.     

Duration of the persistent activity of moxidectin against Haemonchus contortus in sheep

SUMMARY Weaned lambs were infected with Haemonchus contortus 35,28,21,14 and 7 days after treatment with moxidectin at 0.2 mg/kg and 35 and 14 days after treatment with ivermectin at the same dose rate. Worm counts 14 days after infection showed that moxidectin prevented the establishment of over 99% of infective larvae for 28 days and reduced the establishment rate at 35 days by 96%, relative to ivermectin. There was no difference in the protective efficacy of ivermectin at 14 or 35 days. The persistence of moxidectin is likely to provide advantages in nematode control, particularly when used as a strategic early summer treatment or as a pre-lambing treatment to ewes. Implications of the persistent activity of moxidectin for the development of resistance during the decay phase are discussed.     

Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus

The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 μg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P<0.001) and A. gaumeri (P<0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L(3) larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.     

In vivo anthelmintic activity of an aqueous extract from sisal waste (Agave sisalana Perr.) against gastrointestinal nematodes in goats

The resistance of gastrointestinal nematodes (GINs) of small ruminants to anthelmintics has required the investigation of new alternatives. The aim of the present study was to evaluate the in vivo anthelmintic activity of an aqueous extract from sisal waste (Agave sisalana) (AESW) against GINs in goats and to observe the animals for toxic effects. Thirty animals that were naturally infected with GINs were distributed into three groups: group I, was treated with daily doses of AESW (1.7 g/kg) for eight days; Group II, the positive control, was treated with a single dose of levamisole phosphate (6.3mg/kg); and group III, the negative control, was left untreated. Faecal eggs counts (FECs), coprocultures and post-mortem worm counts were performed to assess the efficacy of the treatments. Clinical and laboratory analyses were performed to evaluate any toxic effects associated with the treatment. In the goats in groups I and II, a significant reduction (p<0.05) of the number of eggs and infective larvae (L(3)) was observed. The maximum reductions of the FECs were 50.3% and 93.6% for groups I and II, respectively, whereas the percent reductions of the total number of L(3) larvae were 80% (group I) and 85.6% (group II). There was no difference between groups I and III with respect to worm burden, and the percent reductions were 28.8% and 63.4% for Oesophagostomum columbianum and Trichostrongylus colubriformis, respectively. No reduction was detected for the Haemonchus contortus. The positive control group demonstrated a 74% reduction of the parasites that were recovered from the digestive tract. There were no changes in clinical and haematological parameters. The levels of serum urea and creatinine were higher in group I, but remained within the normal range. At necropsy, pale mucous membranes, abomasitis and enteritis were associated with parasitism. In addition, a histological analysis of the liver and kidney did not reveal any changes suggestive of toxicity. A chemical analysis of the AESW demonstrated the presence of saponins, which after acid-hydrolyses reaction, gave the sapogenins hecogenin and tigogenin. The AESW had a low efficacy for the parasitic stages and was moderately effective against eggs and free-living stages. Furthermore, the treatment was not toxic to the goats.     

Variability of resistance in goats infected with Haemonchus contortus in Brazil

The variability between and within breeds with respect to nematode egg counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) was studied in 36 yearling female goats of the Caninde (15), Bhuj (6) and Anglo-Nubian (15) breeds, exposed to Haemonchus contortus. Nematode-free goats were turned to a contaminated paddock in late February. From then on, fecal egg per gram counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) were determined at 2-week intervals up to Week 18. The EPG, transformed as [log(EPG + 75)], varied (P < 0.01) between goats within breeds and between weeks of exposure, but not between goat breeds (P > 0.05). PCV and Hb were affected by goat breeds (P < 0.05), by goats within breeds (P < 0.01) and by weeks of exposure (P < 0.01). Anglo-Nubians had higher (P < 0.01) PCV and Hb than Caninde; Bhuj had intermediate values. There were two EPG rises; one between Weeks 6 and 10 and the other between Weeks 14 and 16. The within breed variability was marked during the EPG rise on Week 6, when individual egg counts ranged from 130 to 2500. The EPG rises coincided with drops in Hb. PCV presented a similar trend, though not as marked. Haemonchus was responsible for more than 95% of nematode eggs counted. Considering the goat as experimental unit, the correlation coefficients (r) were: -0.45, P = 0.0064, between log(EPG + 75) and PCV; and -0.53, P = 0.0009, between log(EPG + 75) and Hb. The negative correlation between egg counts and blood values suggested breed differences in PCV and Hb were related to resistance to H. contortus infection and/or to its effects.     

Failure of young goats to acquire resistance to Haemonchus contortus

Two experiments were carried out to investigate the acquisition by goats of resistance to Haemonchus contortus. In Experiment 1, five Saanen wethers reared worm-free and averaging 5 1/2 months of age at the start of the experiment, were dosed with 200 H. contortus infective larvae three times per week for 10 weeks (approximately 23 infective larvae/kg mean initial liveweight/week) and then given anthelmintic treatment. Each goat and an equal number of worm-free controls were then challenged with 10,000 infective larvae. Post mortem worm counts were carried out 30 days later. In Experiment 2, eight worm-free Saanen wethers, 14 months old at the start of the experiment, were dosed with 825 infective larvae per week for 14 weeks (approximately 23 infective larvae/kg mean initial liveweight/week) except for one week when only 300 larvae were given and one week when no larvae were given. After anthelmintic treatment, each received, together with seven worm-free control animals, a challenge dose of 15,000 infective larvae. Post-mortem worm counts were carried out 28 days later. There were no significant differences in post-mortem worm counts between previously infected and uninfected groups in either experiment. In both experiments, serum pepsinogen values rose significantly as a result of infection but there was no significant (p>0.5) correlation between worm counts and pepsinogen values on the day of slaughter.     

The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis

The indiscriminate use of anthelmintics has resulted in the establishment of parasite resistance. Thus, this study aimed to evaluate the in vitro antiparasitic effect of plant extracts on Haemonchus contortus in sheep and the in vivo effect on Strongyloides venezuelensis in Rattus norvegicus. The plant extracts from Piper tuberculatum, Lippia sidoides, Mentha piperita, Hura crepitans and Carapa guianensis, produced at different research institutions, were chemically analyzed and evaluated through the egg hatch test (EHT) and larval development test (LDT) in H. contortus. P. tuberculatum (150 and 250 mg kg(-1) of body weight) was evaluated for its anthelmintic action on R. norvegicus experimentally infected with S. venezuelensis. In the EHT, the LC(50) and LC(90) of the extracts were respectively as follows: 0.031 and 0.09 mg mL(-1) for P. tuberculatum, 0.04 and 0.13 mg mL(-1) for L. sidoides, 0.037 and 0.10 mg mL(-1) for M. piperita, 2.16 and 17.13 mg mL(-1) for H. crepitans and 2.03 × 10(-6) and 1.22 × 10(-12) mg mL(-1) for C. guianensis. In the LDT, the LC(50) and LC(90) were respectively: 0.02 and 0.031 mg mL(-1) for P. tuberculatum, 0.002 and 0.04 mg mL(-1) for L. sidoides, 0.018 and 0.03 mg mL(-1) for M. piperita, 0.36 and 0.91 mg mL(-1) for H. crepitans and 17.65 and 1890 mg mL(-1) for C. guianensis. The extract of P. tuberculatum showed the following substances: piperamides as (Z)-piplartine, (E)-piplartine, 8,9-dihydropiplartine, piperine, 10,11-dihydropiperine, 5,6 dihydropiperlongumine and pellitorine. The major compounds of the oils were thymol (76.6%) for L. sidoides, menthol (27.5%) for M. piperita and oleic acid (46.8%) for C. guianensis. Regarding the in vivo test, neither dose of P. tuberculatum caused any significant reduction (P>0.05) in worm burden and fecal egg counts compared with the control group. We conclude that the extracts of P. tuberculatum, L. sidoides and M. piperita have effective activity when tested in vitro, but the doses of the extract of P. tuberculatum have no effect when employed in in vivo tests.