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PubMed1996

p-Azidosalicyl-5-amino-6-phenoxybenzimidazole photolabels the N-terminal 63-103 amino acids of Haemonchus contortus beta-tubulin 1.

Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I-salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha-subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta-tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain.

Affinity Labels↗
PubMed1996

Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylated core structures.

Structural studies on the N-linked oligosaccharides of Haemonchus contortus, an economically important nematode that parasitizes domestic ruminants, have revealed core fucosylation of a type not previously observed in any eukaryotic glycoprotein. Mass spectrometric analyses were performed on detergent extracts of homogenized adult H. contortus and on purified H11, a glycoprotein isolated from intestinal brush borders which has been previously shown to be an effective vaccine antigen. The major N-linked glycans identified in the present study have up to three fucose residues attached to their chitobiose cores. The fucoses are found at the 3- and/or 6-positions of the proximal GlcNAc and at the 3-position of the distal GlcNAc. The latter substitution is unique in N-glycans. Most anti-H11 monoclonal antibodies are known to recognize carbohydrate epitopes, and it is possible that the newly discovered multifucosylated core structures are highly immunogenic in this glycoprotein.

Acetylglucosamine↗
PubMed2000

Cathepsin B-like cysteine proteases confer intestinal cysteine protease activity in Haemonchus contortus.

Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN(2), demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion.

Amino Acid Sequence↗
PubMed2003

The predatory capability of three nematophagous fungi in the control of Haemonchus contortus infective larvae in ovine faeces.

The effect of oral administration of three different nematode-trapping fungi, in aqueous suspension containing either Dactylaria sp. or Arthrobotrys oligospora conidia or Duddingtonia flagrans chlamydospores, on the number of Haemonchus contortus infective larvae in sheep faeces, was evaluated. The three selected species of fungi produce three-dimensional adhesive nets in the presence of nematodes. Sixteen Creole sheep were divided into four groups of four animals each. Groups 1 and 2 were orally drenched with a suspension containing 2x10(7) conidia of either A. oligospora or Dactylaria sp. Group 3, received a similar treatment, with D. flagrans chlamydospores, instead of conidia, being administered, at the same dose. Group 4 acted as control, without any fungi. Faecal samples were collected directly from the rectum of each sheep and faecal cultures were prepared and incubated at 15 and 21 days. Larvae were recovered from faecal cultures and counted. The highest reduction of the nematode population occurred in the D. flagrans group, reaching reductions of 96.3% and 91.4% in individual samplings in plates incubated for 15 and 21 days, respectively. Arthrobotrys oligospora showed moderate reductions in the faecal larval population, ranging between 25-64% at 15 days incubation. In general, Dactylaria sp., was less efficient in its trapping ability. Despite the inconsistent results with Dactylaria sp., reduction percentages of 73.4% and 80.7% were recorded in individual samplings during the first and second days, in plates incubated for 15 days. Duddingtonia flagrans, was shown to be a potential biological control agent of H. contortus infective larvae.

Animals↗
PubMed2005

The development and overwintering survival of free-living larvae of Haemonchus contortus in Sweden.

Five complimentary studies were undertaken with the overall aim to examine the ability of free-living stages of Haemonchus contortus to over-winter and tolerate cold stress. Two studies deal with the development and long-term survival of eggs and infective larvae of two geographically different isolates (Kenya and Sweden). Eggs and larvae were monitored in climatic chambers at temperatures that fluctuated daily between -1 degrees C and 15 degrees C, or at constant temperatures of 5 degrees C and 15 degrees C. The development from egg to larvae was dependent on temperatures over 5 degrees C. The long time survival was favoured at lower temperatures. Furthermore, the overwintering capacity of the free-living stages of these isolates was estimated under Swedish field conditions. Two groups of lambs were experimentally infected with different isolates, and kept separated on previously ungrazed plots. In early May the following year, two parasite-naive tracer lambs were turned out on each of the plots to estimate the pick up of overwintered larvae. This experiment was replicated in central and southern Sweden. In addition, two experiments were performed in 2003 on pasture previously grazed by naturally infected sheep. One trial was on a pasture in southern Sweden grazed by a commercial flock, where extreme numbers of H. contortus were found towards the end of the grazing season 2002. The other study was on a pasture plot in central Sweden grazed by a hobby flock in 2002, where three of six lambs died due to haemonchiasis. Overwintered H. contortus was recorded on three of four experimental sites. Worm burdens were in all instances extremely low. No differences in development and survival were found between the isolates. Consequently, overwintering on pasture is of no practical significance in the transmission of H. contortus between grazing-seasons in Sweden.

Animals↗
PubMed1991

Blood gastrin and pepsinogen responses of native village goats in Malaysia to infection with Haemonchus contortus.

Blood gastrin and pepsinogen responses of native village goats in Malaysia to a single dose of 10,500 infective Haemonchus contortus larvae were investigated. Both blood values were significantly elevated within a week of infection and exhibited a highly significant correlation during the study. The magnitude of the blood gastrin response was, however, significantly greater than that of pepsinogen during the period that both blood values were elevated. It is suggested that blood gastrin assay may be of particular value in the diagnosis of chronic haemonchosis in animals harbouring relatively light worm burdens.

Animals↗
PubMed1990

The efficacy of netobimin against a benzimidazole susceptible and a resistant strain of Haemonchus contortus in sheep in The Netherlands.

Netobimin was tested for efficacy against Haemonchus contortus using 7 groups of 5 parasite-free lambs of six months age. The lambs in group 1 and 2 were infected with 10,000 larvae of a benzimidazole susceptible strain and those in groups 3-7 with the same dose of a resistant strain. The following treatment scheme was applied 21 days after infection: lambs in groups 2 and 4-7.5 mg kg-1 netobimin, in group 5-20 mg kg-1 netobimin, in group 6-5 mg kg-1 oxfendazole and in group 7--3.8 mg kg-1 albendazole. The lambs in groups 1 and 3 remained untreated. All lambs were slaughtered 28 days after infection. Egg counts decreased in all lambs after treatment, but increased again in lambs in groups 4, 6 and 7. There was a slight increase in lambs in group 5, while those in group 2 remained negative. Post-mortem worm counts showed a reduction of 99.8 per cent in lambs in group 2 compared to those in group 1. In lambs in group 4-7 the reduction of worm counts was respectively 40.9, 89.5, 24.7 and 40.7 per cent compared to those in group 3. Egg development assays carried out 20 days after infection showed an average LD50 of 0.46 mg ml-1 thiabendazole for the resistant strain. After treatment (day 27) the LD50 was 0.53, 0.48, 0.58, 0.56 and 0.47 in lambs in the groups 3-7. It is concluded that netobimin and other (pro)-benzimidazoles should not be used in cases of benzimidazole resistance and that levamisole, pyrantel tartrate or ivermectin are preferable.

Animals↗
PubMed1990

Efficacy of some commercial formulations of anthelmintics on Haemonchus contortus and Trichostrongylus columbianum.

The anthelmintic action of five commercially formulated anthelmintics: Assiazine, Banminth, Nemafax, piperazine and levamisole has been evaluated by observing the effect on egg hatch, motility and mortality of the larvae of Trichostrongylus columbianum and Haemonchus contortus. Assiazine and levamisole inhibited egg-hatch of the helminth ova dose-dependently. Nemafax, Banminth, and piperazine did not inhibit egg hatch. Levamisole and Banminth affected the motility of the helminth larvae with subsequent death after 24h. The other anthelmintics used did not adversely affect the helminth larvae as compared with the untreated control group.

Animals↗
PubMed1994

Genetic variability of the beta-tubulin genes in benzimidazole-susceptible and -resistant strains of Haemonchus contortus.

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding beta-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two beta-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.

Alleles↗
PubMed2003

Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen.

Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcbeta1,4 (Fucalpha1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response-inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.

Adjuvants, Immunologic↗
PubMed2003

Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus contortus by changing cholesterol content: effects on resistance to anthelmintics.

OBJECTIVES: The efficiency of the anthelmintics used to treat small domestic ruminants infected with nematodes is compromised by the emergence of resistant parasites. Both specific and non-specific mechanisms of resistance exist. The non-specific mechanisms involve multiple resistance phenomena and are dependent on the multidrug resistance (MDR) system, which is also responsible for the development of chemotherapy-resistant tumour cells. We showed previously that the system also exists in nematodes. Membrane 'pumps', known as P-glycoproteins (Pgp), are activated in the MDR system. The nature of the membrane, in particular the lipids, appears to condition the activity of the pumps. Thus, we studied the effects of cholesterol on drug transport activity in the nematode Haemonchus contortus. MATERIALS AND METHODS: We used methyl-beta-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments. The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thiabendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp. We used biochemical assays to estimate the cholesterol concentration in the parasites. RESULTS: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance. These changes also altered the transport of R123. CONCLUSION: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells. The effect appears to be correlated with changes in the function of membrane P-glycoproteins. The lipid environment thus influences the nematode Pgp activity.

Animals↗
PubMed1993

Comparative kinetic disposition of oxfendazole in sheep and goats before and during infection with Haemonchus contortus and Trichostrongylus colubriformis.

The kinetic disposition of [14C]-oxfendazole (OFZ) and its metabolites, fenbendazole (FBZ) and fenbendazole sulphone (FBZ.SO2), in plasma and abomasal fluid were determined in Merino sheep and Angora goats before and during infection with Trichostrongylus colubriformis and Haemonchus contortus. The systemic availability (area under the plasma curve, AUC) of OFZ was significantly lower in goats (13.5 micrograms.h/ml) than in sheep (22.2 micrograms.h/ml) and was reduced with infection in goats (5.6 micrograms.h/ml) and sheep (15.1 micrograms.h/ml). The elimination of plasma [14C] was faster in goats than in sheep. The responses observed for [14C] were a reflection of the behaviour of OFZ. The concentration of OFZ and metabolites in abomasal fluid were similar in both species in the absence or presence of infection. However, as the mean flow rate of abomasal fluid was slower in goats (240 ml/h) than in sheep (488 ml/h), only 7% of the dose passed the pylorus in abomasal fluid of goats compared with 14% in sheep. The presence of gastrointestinal nematodes generally increased abomasal fluid flow rate but neither species nor infection had any effect on the rate or extent of [14C] excretion in urine or faeces. It is suggested that goats possess a faster hepatic metabolism than sheep resulting in more rapid elimination of OFZ.

Abomasum↗
PubMed1989

Parasite-specific T cell responses of sheep following live infection with the gastric nematode Haemonchus contortus.

Helper T cell lines recognizing antigen in the context of MHC Class II molecules were generated from peripheral blood mononuclear cells (PBMC) of sheep undergoing a primary or secondary infection with the nematode parasite Haemonchus contortus. The lines were used to identify 15-18, 25-29, 70-80 and greater than 100 kD immunodominant parasite larval antigens fractionated by SDS-PAGE under reducing conditions. Non-specific mitogenic activity in the antigen fractions was not detected. During primary infection PBMC did not respond to soluble antigens or those fractionated on SDS-PAGE, whereas T cell lines generated from these PBMC responded well. T cell lines and PBMC derived after a secondary infection responded similarly to fractionated larval antigen. This indicated that (a) the T cell lines had retained their antigen specificities (as represented by autologous PBMC reactivity) in spite of in-vitro cycles of restimulation with whole larval soluble antigen and subsequent expansion in recombinant human IL-2, and (b) proliferation of the antigen-specific PBMC was predominantly due to helper T cells. Variations between individual sheep in response to fractionated larval antigens was also observed. The pattern of helper T cell reactivity to SDS-PAGE larval antigens correlated poorly with B cell reactivity in the same animals by serum antibody Western blot analysis. These results demonstrate that helper T cell epitopes can be mapped in complex parasite antigen preparations in sheep and that this approach will be useful in the identification of parasite protective antigens in helminthiasis where the cellular or humoral nature of protection is not clearly defined.

Animals↗
PubMed1991

Protective studies in sheep immunized with cuticular collagen proteins and peptides of Haemonchus contortus.

Twenty-eight nonsibling sheep aged approximately 12 months and raised in a helminth-free environment were used in two protection studies. Immunizations were conducted by two intramuscular injections 30 days apart with a synthetic 18AA cuticle collagen peptide and native cuticle collagens derived from the third- and fourth-stage larvae of Haemonchus contortus. Ten days following the last immunization, the sheep were each given 500 infective H. contortus larvae per day for five consecutive days by intraruminal injection. Both collagen materials induced antibodies reactive with cuticle collagens; however, neither induced reproducible protection to H. contortus infections in vaccinated/infected sheep. In the most extensive test, there were no statistical differences in mean faecal worm egg count for 56 days post worm challenge, in mean numbers of H. contortus and female fecundity ratios at necropsy of immunized and unimmunized sheep. Failure to reproducibly immunize sheep with cuticle collagens may be due to the inability of antibodies or host immune cells to reach the collagen epitopes in the nematode cuticle without prior surface coat removal as postulated in human nematode studies.

Adjuvants, Immunologic↗
PubMed1991

Non-specific resistance of sheep against Haemonchus contortus with Freund's complete adjuvant.

Six groups of four ovines each were injected intraperitoneally with Freund's complete adjuvant (FCA), at various times before, during, or after infection with 10 000 larvae (L3) of Haemonchus contortus per sheep. Animals in groups A and B received FCA on days 14 and 7, respectively, before infection. Ovines in group C were treated with FCA on the same day of infection; while sheep in groups D, E, and F were injected with the adjuvant on days 7, 14, and 21, respectively, after infection. Significant reductions in adult worm numbers compared with non-treated controls, as determined 42 days after infection, were of 30%, 34%, 45%, 52%, 56%, and 55% in groups A, B, C, D, E, and F, respectively. A significant correlation, between the time of FCA administration (referred to the day of infection) and the number of worms recovered at necropsy, was also observed.

Abomasum↗
PubMed1992

Immune response of Clun Forest sheep to vaccination with membrane glycoproteins from Haemonchus contortus.

Clun Forest lambs were injected with a fraction containing integral membrane glycoproteins derived from the intestinal microvilli of Haemonchus contortus in two equal doses of 0.5, 5, 50 or 500 micrograms protein and then challenged with 10,000 infective larvae. The time-course of serum specific antibody responses were determined. Compared to the adjuvant control group, the animals injected with 1000 micrograms protein were better than 84% protected and those injected with 100 micrograms, better than 95% protected by all criteria. For these two groups parasite egg output was reduced 99% on average over the patent period. Two of the animals injected with 10 micrograms protein were partially protected, with 86% reduction in egg output. Two animals in the group injected with 1 microgram protein also showed partial protection. Antibody level correlated with protection.

Animals↗
PubMedDate not supplied

Presence of the LDNF glycan on the host-protective H-gal-GP fraction from Haemonchus contortus.

Immunization of sheep with the gut membrane-associated protein complex H-gal-GP of adult Haemonchus contortus induces high levels of protection against a homologous challenge infection. Protection is correlated with a systemic IgG response against the antigen. Analysis of the antibody response showed that the majority of the antigen-specific IgG was of the IgG2 isotype. A substantial proportion (74%) of this response was directed against the glycan component of H-gal-GP. The high immunogenicity of the H-gal-GP glycans may be due to the presence of the fucosylated LacdiNAc (LDNF) antigen. 2D electrophoresis, Western blotting and mass spectrometry analysis of H-gal-GP showed that this glycan epitope was specifically located on a metalloendopeptidase, MEP3. MEP3 is the most abundant protein in H-gal-GP and has been identified as one of the most likely protective components of the complex. Here, we present evidence that the LDNF glycan does not contribute to the protective capacity of H-gal-GP. Animals vaccinated with reduced and denatured H-gal-GP are not protected against subsequent infection, although the antibody response against the LDNF glycan is very similar to that of animals vaccinated with the native H-gal-GP. In addition, an alternative version of H-gal-GP, H-sialgal-GP, which is equally protective, but isolated by affinity chromatography on jacalin lectin rather than peanut lectin, contains a MEP3 component which has no detectable LDNF glycan.

Animals↗
PubMed2005

Protection induced by vaccinating sheep with a thiol-binding extract of Haemonchus contortus membranes is associated with its protease components.

Previous work has shown that a protein extract enriched for cysteine protease activity (TSBP) prepared from adult Haemonchus contortus using thiol sepharose affinity chromatography confers substantial protection against a single challenge infection. TSBP comprised proteases and other proteins. Here, TSBP were further fractionated using anion-exchange chromatography and fractions pooled on the basis of containing (1) protease activity, (2) a prominent non-protease peptide and (3) material which did not bind to the column. A protection trial showed that only the protease-enriched material conferred protective immunity and this was comparable to that observed in a TSBP-immunized positive control group. Immunization stimulated a marked IgG response with the IgG2 isotype predominating.

Animals↗