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.     

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.     

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.     

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.     

Ovicidal and larvicidal activity of Melia azedarach extracts on Haemonchus contortus

Haemonchus contortus is responsible for severe economic losses in sheep and goat breeding in the Northeast of Brazil. However, the effectiveness of control is compromised due to anthelmintic resistance and misuse. In the search for natural anthelmintics, Melia azedarach L., a plant indigenous to India but now distributed throughout Brazil, was selected due to the reported anthelmintic properties of its seeds. The aim of this study was to evaluate the anthelmintic activity of the seed and leaf extracts of the Brazilian adapted plant and investigate the type of organic chemical compounds present in the most active extracts. The ovicidal and larvicidal activity of M. azedarach extracts on H. contortus was evaluated through egg hatching and larval development tests. Hexane and ethanol extracts of seeds and chloroform and ethanol extracts of leaves of M. azedarach were used in the tests. To perform the larval development test, feces of an animal free from parasites were mixed with third instar H. contortus larvae and extracts in several concentrations. The coprocultures were incubated for 7 days at 30 degrees C, then the larvae were recovered and counted. LC50 was calculated by probits using the SPSS 8.0 program. The seed ethanol extract was the most active on eggs (LC50=0.36mgmL(-1)) and the leaf ethanol extract showed the best inhibition of larval development (LC50=9.18mgmL(-1)). Phytochemical analysis of the most active extracts revealed the presence of condensed tannins, triterpenes and alkaloids.     

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.     

In vitro evidence that the pastoral Artemisia campestris species exerts an anthelmintic effect on Haemonchus contortus from sheep

Occurrence of anthelmintic resistant strains of helminths is increasing. The aim of this study was to evaluate the in vitro anthelmintic activity of Artemisia campestris in comparison to albendazole against Haemonchus contortus of sheep. In this respect, in vitro anthelmintic activities of crude aqueous and crude ethanolic extracts of aerial parts of A. campestris were investigated on eggs and adults of Haemonchus contortus. Chemical analyses revealed that overall profile of both extracts samples were dominated by flavonoids among them quercetin and apigenin derivatives were the most abundant phenolics constituents. Both extract types completely inhibited egg hatching at a concentration close to 2 mg/ml. Lethal concentration 50% of A. campestris ethanolic and aqueous extracts were 0.83 and 1.00 mg/ml respectively (p?A. campestris possesses in vitro anti-Haemonchus contortus properties.     

The anthelmintic efficacy of the plant, Albizia anthelmintica, against the nematode parasites Haemonchus contortus of sheep and Heligmosomoides polygyrus of mice

Albizia anthelmintica Brong., belongs to the plant family Mimosaceae. The plant is widely used in East Africa by poor smallholder farmers and pastoralists to treat their livestock against internal parasites. The anthelmintic effects of water extracts from the bark of A. anthelmintica, obtained from three different geographic areas in Kenya and using different methods of preparation, were tested at different doses in sheep and mice infected with the nematode parasites Haemonchus contortus and Heligmosomoides polygyrus, respectively. Lambs were infected with 3000 infective larvae of H. contortus and treated with the plant preparations 28 days later, while mice were infected with 200 infective larvae of H. polygyrus and treated 18 days later. Proximate analysis established high levels of crude proteins in A. anthelmintica bark. Two sheep out of the 45 treated with the plant preparations suffered from transient bloat, which was relieved by dosing with a surfactant. Significant reductions in faecal egg counts were observed in lambs treated with A. anthelmintica in two of the three experiments undertaken, but the efficacy levels achieved were well below the 70% reduction required. Similar values of packed red cell volume and live weight gain were observed for treated and control lambs. There was no overall significant effect of treatment with A. anthelmintica on faecal egg and total worm counts in mice. A dose rate of 1000 mg/kg bodyweight of A. anthelmintica preparation resulted in death of all mice. The results show that A. anthelmintica at the doses and preparations used is not efficacious against H. contortus in sheep or against H. polygyrus in mice.     

The effect of ageing on the antigenicity of Haemonchus contortus larvae.

Viable Haemonchus contortus larvae used as a source of antigen in complement fixation tests showed a decrease in antigenic potency following storage for approximately two months at 5 degrees C. In contrast, larvae killed by freezing and stored at --15 degree C showed no such decline over a similar period. It is suggested that this loss of potency with ageing may be partly responsible for increased worm populations in sheep in spring.     

In vitro larval migration and kinetics of exsheathment of Haemonchus contortus larvae exposed to four tropical tanniniferous plant extracts

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.     

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.     

Studies on Haemonchus contortus. II. The effect of abomasal nematodes on subsequent challenge with H. contortus

Fifty-thousand infective larvae of Trichostrongylus axei dosed to 7-9-month-old Merinos protected them against a subsequent challenge with 50 000 infective larvae of Haemonchus contortus. Protection varied with the period between an initial dosage of T. axei and challenge with H. contortus as follows: After 90 days it was more than 80% effective in more than 80% of sheep; at 119 and 180 days there was still a significant reduction in the numbers of H. contortus recovered (P < 0,05 and P < 0,001, respectively). Prior infestation with either Ostertagia circumcincta or with Haemonchus placei was ineffective against challenge with H. contortus.     

Effects of artemisinin and Artemisia extracts on Haemonchus contortus in gerbils (Meriones unguiculatus)

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC(50) value of 0.045 mg/ml in EHA, 0.063 mg/ml in LDA, 0.009 mg/ml in LFIA, and 24.66 mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil.     

Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. extracts on Haemonchus contortus

The prevalence of antibodies to Neospora caninum was examined in six wild Artiodactyla species, and in five wild Carnivora species from Kenya. Blood sera (104 wild ungulates from Marula Estates (MEs), and 31 wild carnivores from Masai-Mara reserve and from other wildlife areas in northern and Southern Kenya), were screened using a Neospora agglutination test (NAT), with a twofold dilution (1:40-1:320 titres). Presence of NAT antibodies to N. caninun is reported here for the first time in zebra (Equus burchelli), eland (Taurotragus oryx), African buffalo (Syncerus caffer), Thompson gazelle (Gazella thompsoni), impala (Aepyceros melampus), warthog (Phacochoerus aethiopicus), spotted hyena (Crocuta crocuta) and in free-ranging cheetah (Acinonyx jubatus). At 1:80 dilution, prevalence was 61.5% in eland, 58.5% in zebra, 19.2% in Thompson gazelle, 33.3% in warthog, 50% in African buffalo, 30% in lion (Panthera leo), 20% in cheetah, and 33.3% in spotted hyena. Antibodies up to 1:320 titre were detected in eland (38.4%), zebra (19.5%), Thompson gazelle (3.8%) and lion (5%). Amongst herbivores, sero-prevalence was significantly (P<0.05) higher, at all dilutions, in "grazer/digger" species (e.g. eland and zebra) than in non-"grazer/digger" species (e.g. impala and Thompson gazelle). No antibodies to N. caninum were found in two leopards (Panthera pardus) and one serval (Felis serval). Our results indicates a steady presence of N. caninum in wild mammals from Kenya. The hypothesis of a sylvatic cycle of N. caninum could be suggested, but more data are needed to verify the hypothesis, as to evaluate the role of N. caninum infection on the dynamics of wild animals population in the study area.     

Anthelmintic efficacy of hydro-methanolic extracts of Larrea tridentata against larvae of Haemonchus contortus

An in vitro study was conducted to determine the anthelminthic activity of hydro-methanolic extracts of Larrea tridentata on sheathed and exsheathed larvae of Haemonchus contortus. Larvae of the parasite were incubated at 20–25 °C in hydro-methanolic extracts at concentrations of 12.5, 25, 50, 100, and 200 mg/mL for 24, 48, or 72 h. Ivermectin and water were the positive and negative controls, respectively. Total phenolic compounds of leaves of L. tridentata were 97.88?±?10.45 mg/g of dry matter. Other compounds detected in this shrub by HPLC-mass spectrometry were sesamin, galocatechin, peonidin 3-O rutinoside, methyl galangin, epigallocatechin 7-O-glucuronide, and epigalocatechin. Mortality rate of sheathed and exsheathed H. contortus was low (16–34%) with doses ≤?100 mg/mL of the extracts. At 200 mg/ml, the hydro-methanolic extracts of L. tridentata killed 32.1 and 68.4% of sheathed and exsheathed larvae, respectively, regardless of incubation time. The effective concentration of the L. tridentata extract for 50% larvae mortality (EC₅₀) after 24 h of incubation was 36 mg/mL (CI?=?6–94). Microscopic observations revealed damage to the cuticle of this parasite exposed to extracts of L. tridentata. These in vitro results provided evidence that L. tridentata extracts possess anti-Haemonchus contortus properties, particularly during the exsheathed stage of this nematode. It would be necessary to assess the safety of this shrub in vivo and also to carry out in vivo efficacy studies.

     

Effectiveness of strategic anthelmintic dosing in controlling Haemonchus contortus infections in sheep in the United Kingdom

The strategic use of anthelmintics in the control of infections of Haemonchus contortus in ewes and lambs, was investigated in a series of paddock trials. The levels of infection and clinical signs associated with the presence of either benzimidazole-resistant or benzimidazole-susceptible strains of H contortus in lambs were controlled by regular drenching with levamisole or mebendazole, respectively, or by the strategic use of closantel in combination with mebendazole. In the latter case, control was achieved by dosing either the ewes in the early part of the grazing season, or the lambs from June onwards. It was concluded that worm control strategies based on closantel could provide effective control of both benzimidazole-susceptible and benzimidazole-resistant strains of H contortus on farms in the United Kingdom.