Rapamycin exposure to host and to adult worms affects life history traits of Heligmosomoides bakeri.

Parasite life history can be affected by conditions of the host and of the external environment. Rapamycin, a known immunosuppressant of mammals, was fed to laboratory mice that were then infected with the Trichostrongylid nematode Heligmosomoides bakeri to determine if host rapamycin exposure would affect parasite survival, growth, and reproduction. In addition, adult worms from control fed mice were directly exposed to rapamycin to assess if rapamycin would affect worm viability and ex vivo reproduction. We found that host ingestion of rapamycin did not affect H. bakeri survival or growth for male or female worms, but female worms had increased reproduction both in vivo and when removed from the host and cultured ex vivo. After direct rapamycin exposure, motility of female worms was greater at low levels of rapamycin compared to high levels of rapamycin or high levels of DMSO (the vehicle used to solubilize rapamycin) in control media, but was similar to females in low levels of DMSO in control media. Male motility was not affected by the presence of rapamycin or DMSO in the media. Ex vivo egg deposition was higher when exposed to rapamycin than when cultured in control media that contained DMSO, regardless of DMSO dose. Overall, we conclude that host ingestion of rapamycin or direct exposure to rapamycin was generally favorable or neutral for parasite life history traits.

The antiparasitic activity of avenacosides against intestinal nematodes.

Avena sativa L., 1753 (Poaceae) is used as feed for livestock and as a crop rotation agent. The purpose of the study was to examine the molecular mechanisms behind the antihelminth activity of the oat saponins avenacoside B (AveB) and 26-desglucoavenacoside B (26DGAveB) by evaluating their effect on Heligmosomoides bakeri, a parasitic nematode of mice. The avenacosides AveB and 26DGAveB were separated and purified from A. sativa green leaves, and their mycotoxic activity was confirmed against the fungus Trichoderma harzianum. The anti-nematode activity of the avenacosides was measured by egg hatching assay. In the surviving L3 larvae exposed to avenacosides, the expression of CED-9, a protein of the apoptosis pathway, was identified by Western blotting. The protein profile of L3 larvae was monitored by High Performance Liquid Chromatography (HPLC). The action of saponins on glycoprotein pump (Pgp) activity in L3 larvae was compared to that of the pump blocker Verapamil (VPL). A mouse model was used to measure the infectivity of L3 larvae exposed to AveB and 26DGAveB, and the outcome of the immune response. Both compounds induced morphological changes in larvae and blocked Pgp activity; however, only 26DGAveB provoked expression of CED-9. The infected mice displayed changes in the molecular pattern of larval proteins and enhanced IL-4 production, indicating that avenacosides reduced the infectivity of H. bakeri larvae. In avenacosides, the residue without glucose at the C26 position demonstrated greater anti-nematode activity. Our findings indicate that A. sativa compounds are natural products with anti-parasitic activity.

In vitro anthelminthic efficacy of Dichrocephala integrifolia (Asteraceae) extracts on the gastro-intestinal nematode parasite of mice: Heligmosomoides bakeri (Nematoda, Heligmosomatidae).

OBJECTIVE: To evaluate the ovicidal and larvicidal activities of aqueous and ethanolic extracts of leaves of Dichrocephala integrifolia (D. integrifolia) against the eggs (fresh and embryonnated), the first and second larval stages of Heligmosomoides bakeri. In order to verify if this medicinal plant possesses active compounds capable of inhibiting the embryonation and hatching of eggs or to induce the mortality of larvae (L1 and L2). METHODS: dried extracts were diluted in distilled FIV water to obtain five different concentrations: 625, 1,250, 2,500, 3,750 and 5,000 µg/mL. Fresh eggs obtained from artificially infected mice feces were exposed to these different concentrations for 48 h. Time of contact for embryonated eggs was 6 h while L1 and L2 larvae were exposed for 24 h. Distilled water (placebo) and 1.5% DMSO were used as negative controls. RESULTS: Distilled water, and 1.5% DMSO had no effect on embryonation, hatching and larval survival. Aqueous extracts of D. integrifolia showed a weak activity against all stages of the parasite at all concentrations tested. On the contrary, the ethanolic extract of D. integrifolia inhibited the embryonation of 87.5% of fresh eggs, the hatching of 81.1% of embryonated eggs and induced the mortality of 98.1% and 98% of L1 and L2 larvae respectively at 5,000 µg/mL. CONCLUSIONS: The results of the present study indicate that the ethanolic extracts of D. integrifolia contained compounds with ovicidal and larvicidal properties. In spite of these results, in vivo tests, studies on toxicity and mechanism of action of active compounds are also needed to validate the utilisation of this medicinal plant by population of Dschang-Cameroon to treat gastro-intestinal parasites.

Anthelmintic potential of three plants used in Nigerian ethnoveterinary medicine.

CONTEXT: The leaves of Irvingia gabonensis Baill. Ex Lanen (Irvingiaceae), Ficus exasperata Vahl (Moraceae), and Vernonia amygdalina Delile (Asteraceae) are folklorically used in treating worm infestation in Eastern Nigeria. The anthelmintic potential of the ethanol extracts of the leaves of I. gabonensis, F. exasperata, and V. amygdalina was investigated. MATERIALS: Acute toxicity tests were done in mice using 10, 100, and 1000 mg/kg/bw of extracts. In vitro larval assays of Heligmosomoides bakeri larvae at various extract concentrations (125, 250, and 500 mg/kg) were done. Mice experimentally infected with H. bakeri were treated with F. exasperata extract (200, 400, 800 mg/kg). RESULTS: At concentrations of 500, 250, and 125 mg/ml F. exasperata caused 100% larval mortality. V. amygdalina extract caused 71.43, 57.14, and 57.14% larval deaths while I. gabonensis extract caused 71.43, 57.14, and 42.9% larval deaths at the same concentrations. There was no significant difference in the fecal egg output, packed cell volumes and body weights of the F. exasperata treated mice when compared with the infected untreated group. DISCUSSION AND CONCLUSION: Leaf extracts of F. exasperata, V. amygdalina, and I. gabonensis exhibited varying degrees of larvicidal activities on the infective stage larvae of H. bakeri in vitro whereas F. exasperata showed no activity on the parasites in vivo.

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.

A comparative study of the ovicidal and larvicidal activities of aqueous and ethanolic extracts of pawpaw seeds Carica papaya (Caricaceae) on Heligmosomoides bakeri.

OBJECTIVE: To assess the ovicidal and larvicidal activities of aqueous and ethanolic extracts of pawpaw seeds Carica papaya (Caricaceae) on the eggs and first stage larvae (L(1)) of Heligmosomoides bakeri. METHODS: Eggs of this parasite were obtained from experimentally infested mice (Mus musculus) and larvae were from eggs after incubation at 25∘C for about 72 hours. The eggs and larvae were exposed to ten different concentrations (0.125, 0.25, 0.375, 0.5, 0.75, 1.0, 1.25, 1.75, 2.25 and 2.75 mg/mL) of both aqueous and ethanolic extracts respectively for 72 hours. Distilled water and 0.05% ethanol used as placebo and negative control, respectively. RESULTS: Placebo and negative control group all showed average 92% embryonnation, 98% egg hatching and 2% larval mortality, and did not affect development and larval survival. The extracts inhibited embryonic development, egg hatching and larval survival. In general, the ovicidal and larvicidal activities increased with increasing concentration of different extracts. The aqueous extract was found to be more potent on eggs than on larvae. At 2.75 mg/mL, only 8% of eggs embryonnated and 50% hatched to L(1) vs 57% embryonic development and 79% hatching occurred in the ethanolic extract. However, this later extract was more efficient in preventing larval development producing 96% mortality as against 68% with the aqueous extract. CONCLUSIONS: These results shows the ovicidal and larvicidal properties of aqueous and ethanolic pawpaw seeds extracts.

Triterpenoid saponins affect the function of P-glycoprotein and reduce the survival of the free-living stages of Heligmosomoides bakeri.

We studied the effect of triterpenoid saponins on the development of free-living stages of Heligmosomoides bakeri, a parasitic nematode of the mouse intestine. We evaluated the effectiveness of oleane-type glucuronides (GlcUAOA) isolated from Calendula officinalis and Beta vulgaris. The rhodamine 123 retention assay was used to detect dysfunctions of the major membrane transporter for xenobiotics, P-glycoprotein (Pgp). Both C. officinals and B. vulgaris GlcUAOA affect the development of the free living stages and function of Pgp in H. bakeri. The GlcUAOA inhibits egg hatching and moulting of larvae and also changes their morphology. These saponin fractions reversed the toxic effect of thiabendazole on the nematode; the function of Pgp was also inhibited.

Nematocidal activity of nitazoxanide in laboratory models.

The nematocidal activity of a broad-spectrum antiparasitic agent, nitazoxanide [( N-(5-nitrothiazol-2-gammal)salicylamide; NTZ], was evaluated in both in vitro and in vivo models using Caenorhabditis elegans, Heligmosomoides polygyrus and Trichinella spiralis. In vitro, NTZ (100 muM) exhibited a low activity against C. elegans and had no effect on embryonation and hatching of H. polygyrus eggs. At concentrations of 100 and 50 muM, the inhibition of excretion/secretion of acetylcholinesterase and acid phosphatase of adult H. polygyrus by NTZ was variable. The in vitro effects of mebendazole (5 muM), albendazole (1 muM) and levamisole (10 muM) were superior to those of NTZ. In mice, NTZ at 1 g/kg proved to be inactive against preadults of T. spiralis whereas mebendazole at 10 mg/kg reduced the worm burden by up to 83%. NTZ at 1 g/kg per day for 3 consecutive days showed a low activity against adults of H. polygyrus (21% reduction). Levamisole, at a single dose of 10 mg/kg, reduced the worm burden by up to 89.9%. The results of this study suggest that NTZ would not have met criteria of a candidate compound.

Dietary pectin, but not cellulose, influences Heligmosomoides polygyrus (Nematoda) reproduction and intestinal morphology in the mouse.

Dietary texture has been reported to influence parasite establishment and survival, but to what degree this relationship is modified by either the type or quantity of dietary fibre is unknown. Using a 2 x 4 factorial design, we explored the relationship between fibre type (soluble pectin vs insoluble = cellulose) and fibre quantity (0, 5, 10 and 20% by dry weight) on parasitic outcomes in BALB/c mice infected with 100 Heligmosomoides polygyrus (Nematoda) larvae. Pectin, but not cellulose, exerted a significant effect on parasite egg production. Following in vitro culture of female worms, increasing levels of dietary pectin were associated with increasing release of eggs. Yet this pattern was not observed in vivo, where per capita egg production peaked at the 10% pectin concentration, but was very low in mice fed 20% pectin. Parasite establishment was elevated in mice fed 20% pectin, but was unaffected by cellulose concentration. Neither type nor quantity of fibre affected H. polygyrus survival or spatial distribution along the gastrointestinal tract. To what degree differences in parasite establishment and reproduction could be attributed to the marked effects of pectin on gut morphology (increased intestinal length, villus length, mucosa thickness and villus/crypt ratio) requires further exploration. Our data indicate that cellulose is preferable to pectin as the source of fibre for experimental diets as cellulose did not affect H. polygyrus establishment, reproduction or survival during a 4-week primary infection.

Nematocide activity of 6,7-diarylpteridines in three experimental models.

The in vitro nematocide activity of seventeen 6,7-diarylpteridines has been tested using three different experimental models, Caenorhabditis elegans, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. The method of evaluation of inhibition in the secretion of acetylcholinesterase by H. polygyrus seems to be the most indicated to avoid false positives. The in vivo activities, against Trichinella spiralis, of the most in vitro active pteridines have been assayed. All pteridine derivatives bearing 6,7-di-p-bromophenyl substituents have shown in vitro nematocide activities in the three experimental models used. Amongst all the pteridines tested in vivo, only 2,4-pteridinedithione derivatives exhibited moderate activity.

N3-arylspiroimidazolidine-2,4-diones N3-arylspiroimidazolidine-2-thio-4-ones and 4-hydroxy derivatives. Synthesis and anthelmintic activity.

The synthesis of new N3-arylciclohexanespiroimidazolidine-2,4-diones, N3-arylciclohexanespiroimidazolidine-2-tio-4-ones and the 4-hydroxy derivatives is described and their structures discussed on the basis of I.R. and 1H-N.R.M. data. The anthelmintic activity of these compounds was tested.

Heligmosomoides polygyrus: effect of exogenous steroid hormones on egg output in vitro.

The effect of exogenous steroid hormones on the egg output of Heligmosomoides polygyrus (Nematoda: Trichostrongylidae) was examined in vitro. Using worms raised in female mice, it was found that estradiol, testosterone, and cortisone each significantly decreased egg output. Although similar trends were found using H. polygyrus raised in male mice, none of the decreases found was significant. No significant differences were found with ecdysone or progesterone treatments using worms from female or male mice. Treatment of worms with cortisone did not significantly affect retention of eggs within the uterus of H. polygyrus. Titration of the effect of cortisone on egg output indicated that levels of reduction were significant for concentrations of 5.6 x 10(-6) M to 5.6 x 10(-3) M in worms from female mice and for concentrations of 5.6 x 10(-8), x 10(-7), x 10(-5) and x 10(-3) in worms from male mice. Radioisotope labelling experiments showed incorporation of 3H-corticosterone in the nucleus of intestinal cells of H. polygyrus suggesting that its effect on egg production may be via a modulatory effect on the intestinal cells.

Differences in sensitivity of Schistosoma mansoni schistosomula, Dirofilaria immitis microfilariae, and Nematospiroides dubius third-stage larvae to damage by the polyamine oxidase-polyamine system.

The effect of the polyamine oxidase (PAO)-polyamine system on some helminths was examined in vitro. Both Schistosoma mansoni schistosomula and Dirofilaria immitis microfilariae were highly sensitive to this system, the latter more so than the former. In contrast, exsheathed third-stage larvae of Nematospiroides dubius were resistant to the effects of the PAO-polyamine system. After incubation of microfilariae with either spermine or spermidine in the presence of serum containing PAO (bovine serum or human retroplacental serum) or partially purified PAO, damage of worms occurred, compatible with our criteria for worm death. Similar results were obtained with schistosomula by using spermine. The damage seemed to be mediated by PAO products other than hydrogen peroxide because catalase did not protect either parasite. Our data demonstrate that helminths may be damaged by products of the PAO-polyamine system.

The efficacy of levamisole administered orally or parenterally against Heligmosomoides polygyrus in mice.

Albino mice (Balb c/nut) experimentally infected with Heligmosomoides polygyrus were treated with levamisole hydrochloride ('Nemicide'--ICI Pharmaceutical PLC) by oral drenching or subcutaneous injection at 5, 10 and 20 mg kg-1. Faecal egg counts monitored for two or three days after dosing and post-mortem worm counts were used to assess the efficacy of these treatments. The lowest dose rate gave poor clearance of adult worms but at 10 and 20 mg kg-1, 91 and 96% reductions in worm burden were achieved. Reduction in post-dosing faecal egg counts were variable. Neither method of administration offered particular advantage in terms of efficacy. Faecal egg count data gave inconsistent differences and at necropsy, worm counts were lower in mice dosed orally but not significantly so.

Antigen stripping from the nematode epicuticle using the cationic detergent cetyltrimethylammonium bromide (CTAB).

The cuticular antigens of adult Nematospiroides dubius were selectively removed using the cationic detergent cetyltrimethylammonium bromide (CTAB). Nonionic, zwitterionic or anionic detergents were ineffective in comparison. The biochemical profile of the antigens removed by detergent was identical to that of surface antigens removed by homogenization, with the added advantage that detergent-stripped antigens lacked many of the background antigens (excretory/secretory--ES and somatic) seen in homogenates. In addition, the detergent was shown to act in a non-invasive manner as electron micrographs failed to reveal any gross damage to the nematode outer cuticle. The observed selective release of significant quantities of relatively clean nematode surface antigen by CTAB in a non-invasive or destructive manner provides the impetus for definitive studies on the relevance of surface antigens (in the absence of ES or somatic antigens) to the overall immunogenicity of this and other parasites.