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PubMed2001

Assessment of benzimidazole binding to individual recombinant tubulin isotypes from Haemonchus contortus.

One a- and 2 beta-tubulin isotypes (isotypes 1 and 2) from the parasitic nematode Haemonchus contortus were artificially expressed in E. coli and purified to obtain tubulin that was capable of polymerizing into microtubules. Binding of [14C] mebendazole (MBZ), a benzimidazole compound, to each individual unpolymerized isotype and to microtubules polymerized from recombinant alpha- and beta-tubulin was assessed and Kd and Bmax values determined. Mebendazole bound to the individual tubulin isotypes with a stoichiometry of 1:1. Binding occurred with highest affinity to alpha-tubulin followed by beta-tubulin isotype 2 and beta-tubulin isotype 1 indicating that alpha-tubulin may play a role in benzimidazole binding to microtubules. Upon polymerization of alpha- and beta-tubulin isotype 2 into microtubules the stoichiometry of binding increased to 2:1 (mebendazole : tubulin) while binding affinity remained the same. Mebendazole binding to alpha/beta-isotype 1 microtubules remained unchanged following polymerization. The increase in the number of benzimidazole receptors on alpha/beta-isotype 2 microtubules suggests the formation of a new benzimidazole receptor upon polymerization.

Animals↗
PubMed2004

Evaluation of anthelmintic properties of some plants used as livestock dewormers against Haemonchus contortus infections in sheep.

Gastrointestinal helminth infections remain a major constraint to livestock production globally. This study evaluated anthelmintic efficacy of 7 plants used as dewormers by farmers and pastoralists in Kenya. Thus 3 commercial anthelmintics and 7 plant preparations were tested in lambs infected with 5000 or 3000 L3 Haemonchus contortus in 4 experiments. In the first experiment, ivermectin, levamisole and albendazole were tested in 46 lambs. Seven plant preparations of Hagenia abyssinica, Olea europaea var. africana, Annona squamosa, Ananas comosus, Dodonea angustifolia, Hildebrandtia sepalosa and Azadirachta indica were tested in 151 lambs in 3 experiments. All 3 anthelminitics were highly effective in reducing faecal egg counts (FEC) and total worm counts (TWC) in lambs. Plant preparations had varying levels of crude proteins from 2.6% for O. europaea to 18.4% for A. indica. Compared with controls, no significant reductions in FEC were observed for any of the treated groups either 2 or 3 weeks post-treatment. Lambs treated with A. squamosa and A. comosus were slaughtered 4 weeks post-treatment. No significant differences were observed in mean TWC or number of eggs per female worm between treated animals and the controls. No significant improvements in weight gain were observed in treated lambs.

Animals↗
PubMed2005

Characterization of proteinases in different isolates of adult Haemonchus contortus.

A high degree of intra- and inter-geographical variation has been demonstrated previously in the excretory/secretory proteinases released by adult Haemonchus contortus. Proteinase activity has also been associated with host-protective 'hidden' antigens isolated from the gut of adult H. contortus. If similar geographical strain variation also exists within the gut-associated proteinases, this will have important implications for the development of a globally effective vaccine. The proteinases active in integral-membrane protein extracts from 3 different strains of adult H. contortus were characterized on the basis of their pH optima and molecular size. Although enzyme activity was detected over a wide pH range, the majority of proteinase activity was detected at acidic pH. Differences in specific activity and size of enzymes were observed between the 3 different parasite strains at different pH values. A high degree of conservation in reactive peptides was observed when protein extracts were probed with antisera raised to the protective hidden gut-antigen complexes isolated from the Moredun strain of H. contortus, or to bacterially expressed subcomponents thereof. Therefore, despite the observed differences in membrane-bound proteinase profiles, the similarity of the immunogenic response against these hidden antigens may be sufficient to prove protective against different geographical isolates of H. contortus.

Animals↗
PubMed2005

Phenotypic characterization of Haemonchus contortus: a study of isolates from Sweden and Kenya in experimentally infected sheep.

The effects of cold storage of infective third-stage larvae (L3) of different isolates of the parasitic nematode Haemonchus contortus were studied with respect to infectivity, pre-patent period and propensity for larval arrestment. Two complementary experiments were conducted with 2 groups of lambs, each animal being inoculated with 2000 L3 of either Swedish or Kenyan origin. In a first experiment, L3s were cold treated at 5 degrees C for 9 months prior to infection, whereas in a second experiment larvae were newly hatched. Individual faecal egg counts (FECs), and worm burdens were determined for each experiment. The results showed that the greatest differences were associated with the pre-treatment of larvae. The pre-patent period and the FECs differed significantly between the experiments but not between the isolates used in each experiment. However, the extent of hypobiosis was significantly different between the two isolates when fresh larvae were used (36% Kenyan isolate and 70% Swedish). The storage of H. contortus at 5 degrees C had no apparent effect on the infectivity of L3s, as high establishment ranging from 43 to 74% were observed, irrespective of isolates used. This study showed that H. contortus exhibited similar phenotypic traits regardless of geographical origin. Thus, there was limited evidence for adaptations to temperate climatic conditions.

Abomasum↗
PubMed2006

Effects of four tanniferous plant extracts on the in vitro exsheathment of third-stage larvae of parasitic nematodes.

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previous in vivo and in vitro results suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 of Haemonchus contortus and Trichostrongylus colubriformis. Artificial exsheathment was induced in vitro by adding hypochloride solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1.5 to 24.7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0.3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 microg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts for T. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts with H. contortus induced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species.

Animals↗
PubMed1997

Protective immunity to the blood-feeding nematode Haemonchus contortus induced by vaccination with parasite low molecular weight antigens.

Partially purified low molecular weight antigens obtained by gel filtration of whole worm homogenates or total adult excretory-secretory (ES) products were tested in a vaccination experiment to determine their ability to induce protective immunity against Haemonchus contortus in sheep. Sheep were challenged with 20,000 infective 3rd-stage larvae. One animal in the low molecular weight vaccinated group showed no protection against H. contortus, whereas the 4 other sheep in this group showed a mean reduction of 99.9% in faecal egg counts and of 97.6% in abomasal worm burden compared to the non-vaccinated controls and the adjuvant controls. The ES-vaccinated sheep showed a 32.2% reduction in parasite egg production and a 63.7% reduction in abomasal worm counts. Analysis of the humoral immune responses revealed no significant differences in antibody recognition of putative protective antigens between the protected and non-protected vaccinated animals. However, a marked lower lymphocyte proliferation response was found in non-protected sheep.

Animals↗
PubMed1997

Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection.

Part of the C epsilon 3-C epsilon 4 region of the ovine immunoglobulin E (IgE) gene (nucleotides 1111-1575) was amplified by PCR. The recombinant protein (recIgE1-2) was expressed in E. coli and both monoclonal and polyclonal antibodies were produced. These antibodies recognized recIgE1-2 and native IgE on Western blots and in ELISA. The polyclonal serum showed cross-reactivity with other sheep immunoglobulin classes. The monoclonal antibody was specific for ovine IgE and goat IgE. Infection of sheep with the abomasal nematode Haemonchus contortus resulted in elevated IgE levels in serum 2-4 weeks after infection, as measured by sandwich ELISA using the rabbit polyclonal as capture antibody and the monoclonal antibody against ovine IgE as second antibody. A negative correlation between worm counts and total serum IgE levels at the end of the experiment was found in repeatedly infected sheep. Significant increased levels of excretory-secretory antigens specific IgE levels were found after H. contortus infection. In contrast, no significant changes in 3rd-stage larvae (L3) antigen-specific IgE titre in sera could be detected after infection.

Animals↗
PubMed1998

The influence of dietary supplementation with urea on resilience and resistance to infection with Haemonchus contortus.

Previous research has indicated that supplementing an apparently adequate diet with additional protein improves both host resistance and resilience in lambs infected with Haemonchus contortus. The present study tested the influence of supplementation with non-protein nitrogen (urea). Helminth-naive Hampshire Down lambs were given an apparently adequate basal diet or a diet supplemented with urea. The lambs were then infected with Haemonchus contortus for 10 weeks. Supplementation with urea had no discernible effect on resistance to infection; faecal egg counts, worm burdens, worm lengths and mean number of eggs per adult female worm did not differ between the 2 groups. However, lambs on the supplemented diet showed better resilience; they had greater packed red cell volumes, higher plasma albumin concentrations and increased liveweight gain compared to lambs on the basal diet. The loss of appetite following infection was less in lambs fed the urea-supplemented diet. The observed effect of urea supplementation was seemingly due to greater food consumption as well as the better diet.

Animal Nutritional Physiological Phenomena↗
PubMed1998

Extracellular and cytoplasmic Cu/Zn superoxide dismutases from Haemonchus contortus.

Full-length cDNAs encoding cytosolic (SODc) and putative extracellular (SODe) Cu/Zn superoxide dismutases (SODs) from the ovine gastrointestinal parasitic nematode Haemonchus contortus have been isolated and characterized. The predicted sequences of the H. contortus SODs showed strong homology to other helminth SODs, the highest level of sequence similarity was with those of the free-living nematode Caenorhabditis elegans++. The predicted amino acid sequence of the putative extracellular form contained an N-terminal extension with the characteristics of a signal sequence including a potential signal peptidase cleavage site. Transcripts of both classes of Cu/Zn SOD were detected in all life-cycle stages examined. The cytosolic SOD mRNA was approximately 6-fold more abundant than that of the extracellular enzyme in adult parasites. Immunoblotting with antisera raised to in vitro-expressed parasite SODs revealed the presence of 2 proteins in extracts of adult H. contortus, with molecular masses of approximately 19.8 and 18 kDa. An additional protein of approximately 16.8 kDa was detected in adult ES material. Immunofluorescent staining showed Cu/Zn SOD was localized in the body wall musculature and the pharynx in adult worms and in the uterine tract of adult females. The immunogenic properties of recombinant H. contortus Cu/Zn SODs was assessed in a challenge infection experiment in lambs.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of glutamate dehydrogenase (GDH) from the gut of Haemonchus contortus.

Vaccination of lambs with the membrane-bound (S3) thiol-Sepharose binding protein (TSBP) fraction derived from the gut of Haemonchus contortus confers significant protection against homologous challenge. The S3 TSBP peptide profile is dominated by a major protein of ca. 60 kDa which is strongly recognized by antisera from sheep demonstrably protected following immunization with S3 TSBP. In an attempt to identify this protein, sera from protected lambs were employed to screen a lambda gt11 cDNA library of the adult parasite and resulted in the isolation of numerous clones encoding a homologue of the mitochondrial enzyme, glutamate dehydrogenase (GDH). GDH enzyme activity was readily demonstrable in S3 TSBP material and immunolocalization studies showed that the enzyme was localized to the cytoplasm of the parasite's gut. Furthermore, the enzyme appeared to be developmentally regulated, with both GDH mRNA and protein expressed almost exclusively during the blood-feeding parasitic stages.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus.

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a 'proteinase-enriched' Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT-PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

Animals↗
PubMed1999

Cloning and characterization of a beta-galactoside-binding protein (galectin) from the gut of the gastrointestinal nematode parasite Haemonchus contortus.

A cDNA encoding a beta-galactoside-binding lectin (galectin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a membrane protein complex (H-gal-GP) derived from the parasites' gut. The cDNA sequence, exhibiting a tandem repeat structure and designated Hco-gal-2, showed significant levels of similarity with galectins from several species of nematode as well as mammalian galectin type 4. Native galectin was preferentially extracted from the H-gal-GP complex and also from an insoluble membrane fraction prepared from adult worms using lactose-agarose affinity chromatography. The affinity-purified material had apparent molecular mass of around 35 kDa with 3 distinct bands visible on SDS-PAGE. All 3 bands were identified as galectins by reaction with antiserum raised to recombinant Hco-GAL-2 on Western blot. To determine whether H. contortus galectins have any protective capacity against infection lambs were vaccinated with affinity-purified galectin and subsequently given a single challenge infection. Vaccination did not confer any protection against infection with H. contortus as judged by faecal egg output or worm counts.

Amino Acid Sequence↗
PubMed2000

Changes in the lipophilicity of the surfaces of Meloidogyne incognita and Haemonchus contortus during exposure to host signals.

The surfaces of plant and animal parasitic nematodes share certain lipids, which seem to be important in the infection process. The surfaces of 2 parasitic nematodes, Meloidogyne incognita and Haemonchus contortus, were activated by different pH buffers to allow the insertion of different fluorescent probes. The lipid analogue PKH26 and the surface charge indicator, cationized ferritin, were used as probes with these nematodes but labelled only the retaining 2nd-stage moulted cuticle of H. contortus 3rd-stage larvae (L3). Shedding of the second moult of H. contortus L3 was also visualized with PKH26 and cationized ferritin. The fluorescent anionic lipid probe 5-N-(octadecanoyl)-aminofluorescein (AF18) was inserted into the epicuticle layer of M. incognita 2nd-stage juveniles (J2) and H. contortus L3, and also of the second moult of H. contortus L3. Incubation with tomato root diffusate caused modifications of the M. incognita surface allowing the insertion of AF18. Fluorescence with AF18 was significantly decreased after treating M. incognita J2 with amiloride, a potent blocker of hydrogen and sodium (H+/Na+) antiporter. No surface fluidity was observed in M. incognita J2 and H. contortus L3 pre-treated with alkaline buffer when the lipid analogue AF18 was used in fluorescence recovery after photobleaching experiments. The significance of these findings to host infection processes is discussed.

Amiloride↗
PubMed2000

Immunological responses of sheep to Haemonchus contortus.

Infections with Haemonchus contortus are a major constraint on ruminant health world-wide. Young lambs are very sensitive to Haemonchus infection. Older lambs and sheep acquire immunity after a continuous or seasonal exposure to the parasite. The mechanisms underlying immunity are still not completely understood. Antibodies, in particular local IgA and IgE, certainly play a role. The role of IgG is less clear. Lymphocyte proliferation responses seem to correlate to immunity. Sheep that have high antigen-induced lymphocyte responses have a low susceptibility to infection. Furthermore, several studies have demonstrated that immunity against H. contortus is associated with mastocytosis and hyper-sensitivity reactions. More recently, increasing attention is being paid to the role of cytokines (interleukins and gamma-interferon) in the activation of specific defence mechanisms. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays to study cytokine mRNA expression have become available. The inability of young lambs to mount a significant Th2 response, which is normally characterized by high IgE levels, mastocytosis and eosinophilia, may account for the phenomenon of unresponsiveness in these animals.

Aging↗
PubMed1985

Antiparasitic agents. 6. Synthesis and anthelmintic activities of novel isothiocyanatophenyl-1,2,4-oxadiazoles.

The syntheses and anthelmintic activities of 31 3- and 5-(isothiocyanatophenyl)-1,2,4-oxadiazoles are reported. In the primary anthelmintic screen, 3-(4-isothiocyanatophenyl)-1,2,4-oxadiazole (39) showed 100% nematocidal activity and 3-(2-furanyl)-5-(4-isothiocyanatophenyl)-1,2,4-oxadiazole (63), 3-(2-furanyl)-5-(2-chloro-4-isothiocyanatophenyl)-1,2,4-oxadiazole (64), and 3-(2-furanyl)-5-(4-chloro-3-isothiocyanatophenyl)-1,2,4-oxadiazole (66) showed 100% taeniacidal activity when administered orally to mice. The two most active members of this series, 39 and 63, were active against the gastrointestinal nematodes of sheep at 100 mg/kg. In addition, 39 was also found to be active against hookworms in dogs at a single, oral dose of 200 mg/kg.

Ancylostomiasis↗
PubMed1982

Avermectin acyl derivatives with anthelmintic activity.

Avermectins A2a, B1a, and B2a (1, 2, and 3) were acetylated to give 4"- and 23-acetates 4 and 5 and 4",23-diacetate 6 from 1, the 4"-and 5-acetates 7 and 8 and 4",5-diacetate 9 from 2, and triacetate 10 from 3. Structure proof by 300-MHz 1H NMR and mass spectral fragmentation is discussed for 10. Forcing acetylation conditions generated from both 1 and 3 the identical aromatic diacetate 11. Good anthelmintic activities in gerbils and sheep for 4"-acetylated derivatives 4 and especially 7 prompted the preparation of additional 4"-acylated derivatives of 2 with pivaloyl, n-octanoyl, succinoyl, carbamoyl, dimethylcarbamoyl and N-acetylglycyl substituents, prepared from the 5-O-tert-butyldimethylsilyl-protected intermediate 12. Other key intermediates were the trichloroethyoxysuccinoyl derivative 18 and 4-nitrophenyl carbonate 21. Anthelmintic activities against Trichostrongylus colubriformis in gerbils comparable in potency to the natural product 2 are shown by the more polar substituted derivatives 20, 23, and 27. Substitution of the 5-hydroxy group or its loss due to aromatization results in drastically reduced anthelmintic potency.

Animals↗
PubMed1998

Synthesis and endectocidal activity of novel 1-(arylsulfonyl)-1-[(trifluoromethyl)sulfonyl]methane derivatives.

We have recently synthesized a series of novel disulfonylmethane compounds that have shown anthelmintic and insecticidal (endectocidal) activity. Several analogues have shown activity against the internal nematode Haemonchus contortus. In sheep studies, these analogues have shown 100% control of this internal parasite at a 10 mg/kg rate. In vitro activity against the biting flies, Stomoxys calcitrans and Haematobia irritans, has been observed at rates as low as 25 and 2.3 ppm, respectively. Only marginal activity against the liver fluke Fasciola hepatica and Trichostrongylus colubriformis was seen. Respiratory control index values on rat liver mitochondria for this series suggested uncoupling of oxidative phosphorylation as a mechanism of action. Compound 1 is considered to be a promising agent for treatment of parasitized sheep.

Animals↗
PubMed2001

Azadepsipeptides: synthesis and evaluation of a novel class of peptidomimetics.

A general route to azadepsipeptides, a new class of pseudopeptides, has been established. The methodology was applied to the synthesis of a bis-aza analogue of the antiparasitic cyclooctadepsipeptide PF1022A. Comparison of the X-ray crystal structures of natural PF1022A (8) and the chimeric aza analogue 9 revealed that the introduction of nitrogen in the backbone of PF1022A results in almost complete conservation of the 3D structure with only minor deviations at the new nitrogen positions.

Animals↗