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PubMed1991

Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy.

The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.

Albendazole↗

PubMed1989

Changes in anthelmintic resistance status of Haemonchus contortus and Trichostrongylus colubriformis exposed to different anthelmintic selection pressures in grazing sheep.

This experiment was designed to study, over a 5-year-period, the effect of different frequencies of treatment with three different anthelmintic groups, namely, benzimidazoles, levamisole and ivermectin, and different frequencies of alternation between them, on existing levels of anthelmintic resistance in the nematode parasites Haemonchus contortus and Trichostrongylus colubriformis of grazing sheep. No evidence of ivermectin resistance emerged, even in suppressively treated groups. Likewise, H. contortus failed to develop resistance to levamisole under a similar selection regimen. Thiabendazole was shown to select positively against levamisole resistance in T. colubriformis resulting in significantly greater susceptibility to this drug than for the natural reversion which occurred in the untreated control. There was no evidence that an anthelmintic treatment combined with a movement of sheep to pastures of low infectivity selected more rapidly for resistance than where the same number of treatments were given to set-stocked sheep. Rotation between anthelmintic groups at yearly intervals appeared to be more beneficial in delaying resistance than rotation of drugs with each treatment.

Animals↗

PubMed1989

Experimental Haemonchus contortus infections in guinea pigs.

Approximately 40% of exsheathed Haemonchus contortus larvae administered to guinea pigs established in the stomach and developed into fourth stage larvae. Most worms were then lost between 5 and 7 days after infection and the guinea pigs were resistant to a second infection. Haemorrhage, oedema and infiltration with inflammatory cells, especially eosinophils, developed in the stomach wall of infected guinea pigs and reactive hyperplastic changes occurred in the gastric lymph node. H. contortus infection of guinea pigs has some potential as a model for study of the pathology, immunology and chemotherapy of gastric nematodiasis.

Animals↗

PubMed1990

Development and survival of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis on pasture in a tropical environment.

A trial to determine the seasonal pattern of egg hatching and larval survival on pasture was carried out in representative wet and dry zones of Fiji. Fourteen plots were established on parasite-free pasture at each of two sites. One plot at each site was contaminated every month with faeces from naturally infected goats containing a known proportion of Haemonchus contortus and Trichostrongylus colubriformis eggs. Pasture was sampled at regular intervals after contamination and infective larvae identified and counted. Larvae of both species developed throughout the year in the wet zone but development was more sporadic in the dry zone. Larval counts generally declined to below detectable levels within 9 weeks of contamination between September and March but longevity increased during the cooler weather from April to August. The comparatively short larval survival times noted in this experiment may present opportunities for manipulation of parasite population dynamics in the wet tropics.

Animals↗

PubMed1990

Vital staining of glycoprotein secreted by infective third stage larvae of Haemonchus contortus prior to exsheathment.

Staining of infective third stage larvae of Haemonchus contortus prior to exsheathment shows that protein is exuded from the head region and spreads between the cuticles and that a carbohydrate-containing substance is also found between the L2 and L3 cuticles throughout, at exactly the same sites in this nematode. This glycoprotein, which is either secreted from the mouth or the amphids or both is exuded whether or not the larvae have been stimulated to exsheath. Slight staining was also sometimes detected at the secretory-excretory pore and to an even lesser extent at the anus. This glycoprotein is thought to function as a lubricant to prevent abrasion between the cuticles. It accumulates in areas where there is greatest space between the cuticles such as the head, tail and where bending of the nematode body occurs.

Animals↗

PubMed1991

Immunity to Haemonchus contortus and the cellular response to helminth antigens in the mammary gland of non-lactating sheep.

Cellular exudates induced by infusion with helminth antigens were examined in non-lactating mammary glands of ewes immune to infection with the abomasal nematode, Haemonchus contortus. Secondary immunological responsiveness was expressed in two ways. Firstly, antigens from adult H. contortus elicited larger eosinophil-rich cellular exudates in immune compared to non-immune ewes. In this situation, secondary responsiveness in the mammary gland must have been generated through abomasal infection with the parasite. Secondly, repeated infusion with the antigens from adult H. contortus increased the size of cellular exudates in both immune and non-immune ewes. Eosinophils predominated but numbers of macrophages and lymphocytes were also increased. In this second situation, secondary responsiveness must have been either supplemented in immune ewes or derived completely in non-immune ewes by contact with helminth antigens through the mammary gland. The helminth antigens which induce eosinophil exudates in the mammary gland may not be potently protective against H. contortus. Furthermore, eosinophil exudation may not be an in vivo correlate of immunity which is directly useful for discriminating protective antigens and applicable to vaccine development. Infusion with antigens from adult forms of either H. contortus or Trichostrongylus colubriformis elicited cellular exudates equally well in immune ewes primed by infusion with H. contortus adult antigens 7 days beforehand. In addition, antigens from infective larvae of H. contortus elicited cellular exudates more potently than antigens from adult worms. However, vaccination with irradiated larvae has shown that species-specific protective immunity for H. contortus is stronger than cross-protective immunity conferred by T. colubriformis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Effects of cholinergic drugs on longitudinal contraction in levamisole-susceptible and -resistant Haemonchus contortus.

A novel force transducer was used to measure the effects of cholinergic agonists on longitudinal contraction in Haemonchus contortus. Drugs were applied to whole worms or injected via a cannula in the pseudocoelomic cavity. A number of agonists, including nicotine and the anthelmintics m-aminolevamisole, levamisole and morantel, caused contractions in whole worms. Four- to 25-fold increases in concentration of the active compounds were required to cause contractions in each of two levamisole-resistant strains of H. contortus. Of the other compounds tested, bephenium had equivalent activity against susceptible and resistant strains. Anticholinesterase compounds caused contractions after a slight delay in susceptible, but not resistant worms. Numerous cholinergic agonists and other compounds did not cause contraction when applied to whole worms. One of these, acetylcholine, caused contractions in cannulated worms. Compared with the susceptible strain, five- to six-fold higher concentrations of acetylcholine were required to cause equivalent contractions in the resistant strains. Levamisole resistance in adult H. contortus is likely to be due to a change in the characteristics of the cholinergic receptor(s).

Animals↗

PubMed1991

Detection of resistance to ivermectin in Haemonchus contortus.

Infective, third-stage (L3) larvae of Haemonchus contortus isolates resistant to ivermectin (IVM) show a decreased sensitivity to IVM-induced paralysis in vitro. The inhibition of larval motility by IVM can be detected in L3 larvae incubated in the dark on an agar matrix containing IVM, by the failure of affected larvae to move when stimulated by exposure to light. Optimally, avermectin (AVM) potency is quantified after three cycles, each involving storage in the dark for 24 h followed by a brief exposure to light. For IVM-susceptible isolates, a 50% inhibition of motility (LP50) was achieved with IVM concentrations between 0.30 and 0.49 microM, while LP50 values in IVM-resistant isolates ranged from 0.8 to 2.6 microM depending on the in vivo resistance status of the isolate. A limited study of structure-activity relationships within the AVM class indicated that in vitro inhibition of L3 motility was consistent with the known in vivo efficacy of each analogue. Resistance factors for IVM-resistant isolates were dependent on AVM structure with the more polar AVM B2 analogue being a particularly sensitive probe of IVM-resistance status.

Animals↗

PubMed1992

The development and mortality of the free-living stages of Haemonchus contortus in laboratory culture.

Haemonchus contortus eggs were cultured in intact fecal pellets at various temperatures (5-35 degrees C) for 22 days. Temperature and relative humidity were kept constant throughout the incubation period. Nl larval development occurred at 5 degrees C; peak third-stage larval recovery occurred at 20 degrees C. Egg mortality was an age-dependent phenomenon, whereas larval mortality remained constant irrespective of larval age. Development was characterized by a minimum development time followed by a transition to the next stage which occurred at a constant rate. All rates were temperature dependent. The minimum development times reported here are much less than those previously reported. Based on these results a mathematical model was used to describe the demography of the free-living stages of H. contortus at various temperatures.

Animals↗

PubMed1992

The kinetics of mebendazole binding to Haemonchus contortus tubulin.

The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode, Haemonchus contortus, have been characterized. In partially purified preparations, the association of [3H]MBZ to nematode tubulin was rapid, k1 = (2.6 +/- 0.3) x 10(5) M-1 min-1, but dissociation was slow, k-1 = (1.58 +/- 0.02) x 10(-3) min-1. The affinity constant (K(a)) for the interaction, determined by the ratio k1/k-1, was (1.6 +/- 0.2) x 10(8) M-1. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K(a) of (5.3 +/- 1.6) x 10(6) M-1 was obtained. The best estimate for the K(a) of the MBZ-nematode tubulin interaction is considered to be the 'kinetic' value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin, which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs.

Animals↗

PubMed1993

Species-specific PCR for the parasitic nematodes Haemonchus contortus and Trichostrongylus colubriformis.

Reliable species determination of gastrointestinal nematode parasites from sheep is usually carried out on third-stage larvae (L3) by visual differentiation. The culturing of L3 takes about 1 week so a reliable method that could use eggs for the determination would accelerate the procedure. We developed a polymerase chain reaction (PCR), using very small amounts of parasite DNA and an oligonucleotide set according to parts of the DNA sequence of a beta-tubulin gene from Haemonchus contortus, that can discriminate H. contortus DNA and Trichostrongylus colubriformis DNA from each other and from several other sheep nematode parasites. This method can be used for every stage of the parasite, including eggs. For the first time a PCR method is reported to discriminate between sheep nematode parasites.

Animals↗

PubMed1993

The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus contortus.

An extract of adult Haemonchus contortus enriched in the parasite's intestinal microvillar membrane protein H11 and other integral membrane proteins but free of the protein contortin was evaluated as a potential vaccine in two breeds of sheep. The worm burdens of Clun Forest sheep injected with the extract and challenged with 25,000 infective larvae were reduced 89% by weight compared to the average for the controls. The worm burdens of Dorset sheep (challenged with 10,000 infective larvae) were reduced 72%. In both breeds the reduction in the number of female worms, 92 and 71.8%, respectively, was greater than the reduction in the males (86.5 and 46%). Parasite egg output, determined only for the Dorsets, was reduced 92% protection correlated with serum antibody titre. Most of the antibodies were directed against H11.

Animals↗

PubMed1993

Purification and evaluation of the integral membrane protein H11 as a protective antigen against Haemonchus contortus.

A detergent extract of adult Haemonchus contortus enriched in the integral membrane protein H11, previously shown to give protective immunity against the parasite, was fractionated by lectin and ion-exchange chromatography. The fractions were evaluated for their ability to immunize Clun Forest and Dorset Horn sheep against experimental haemonchosis. Most of the protective activity was associated with H11. Used in an approximately 95% pure form it gave a mean reduction in parasite egg output of 94.6% and reduced male and female worm numbers by 86.5 and 93.5%, respectively. Level of protection correlated with serum antibody titre to H11.

Animals↗

PubMed1993

An in vitro assay utilising parasitic larval Haemonchus contortus to detect resistance to closantel and other anthelmintics.

A new assay for detecting resistance to anthelmintics in vitro is described. The assay uses a simple culture system in which the ability of anthelmintics to kill or inhibit the migration of parasitic third and fourth stage Haemonchus contortus larvae through a 50 microns aperture mesh is assessed. The assay detects 2-10-fold resistance to closantel. Resistance to benzimidazoles, levamisole and ivermectin can also be detected.

Animals↗

PubMed1995

Avermectin inhibition of larval development in Haemonchus contortus--effects of ivermectin resistance.

Avermectin (AVM) inhibition of the development of the free-living stages of Haemonchus contortus has been quantified in an assay in which nematode eggs are placed on an agar matrix containing serial dilutions of a drug in the wells of a microtitre plate. Development is allowed to proceed for 6 days by which time larvae in control wells (no drug) have reached the infective, third (L3) stage. At high concentrations (> 30 nM) ivermectin (IVM) paralyses L1 larvae soon after hatching, however, much lower concentrations (approximately 1 nM) are sufficient to inhibit development to the L3 stage which suggests that effects of the drug other than those relating to gross motor activity are responsible for the latter effect. The larval stages of IVM-susceptible H. contortus isolates from both Australia and South Africa, including isolates known to be resistant to levamisole or rafoxanide and/or the benzimidazoles, were equally sensitive to inhibition by AVMs. In contrast, 6 isolates of H. contortus resistant to IVM in vivo showed a reduced sensitivity to AVM inhibition of development. The order of potency of a limited range of AVMs as inhibitors of larval development was consistent with in vivo efficacy. Resistance ratios for IVM-resistant isolates were dependent on AVM structure, with AVM B2 the most sensitive probe for IVM resistance in the isolates tested.

Animals↗

PubMed1995

Relationship of blood loss to worm numbers, biomass and egg production in Haemonchus infected sheep.

Chromium51 labelled erythrocytes were used to measure blood loss in sheep infected with either H. contortus, H. placei or the F1 of their reciprocal cross hybrids (H. contortus males x H. placei females = CXP, H. placei males x H. contortus females = PXC). CXP consisted of sterile males and semi-sterile females and sheep infected with this hybrid had nil egg counts; while PXC consisted of semi-sterile males and females and their hosts had some eggs in their faeces. Immediately after their blood loss was estimated the sheep were killed and their worms counted. One hundred male and 100 female worms from each sheep were dried at 60 degrees C overnight and then weighed to give an estimate of the biomass in each infection. Total daily egg production was calculated from egg counts made on 3 random 2 g samples from the 24 h faecal collection. The relationship of the log blood loss to log worm number, to log biomass of infection in each sheep and to log total egg production in the parental species was linear. Biomass was no better an estimator of blood loss than was worm number. There was no improvement in the estimation of blood loss by regressing the male and female worms separately. In all of the analyses, there was no improvement by regressing H. contortus and H. placei or their hybrids separately. It appeared that egg production was not a major factor in the requirement for blood by Haemonchus. The size of individual female worms was negatively correlated with the number of worms in the infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia↗

PubMed1995

Analysis of proteins related to conditioning for arrested development and differentiation in Haemonchus contortus by two-dimensional gel electrophoresis.

The abundance of the majority of proteins of infectious third-stage larvae (L3) of Haemonchus contortus, conditioned for arrested development, remained unaltered. Only seven proteins showed quantitative differences as observed by two-dimensional gel electrophoresis. These differences were also observed in a laboratory strain which has lost the ability for arrested development. The abundance of two of these proteins increased dramatically during conditioning of larvae for 5-10 weeks. This coincided with the highest percentage of inhibited larvae in experimental infections. Moreover, the abundance of these proteins decreased again after prolonged conditioning (22 weeks). The abundance of the other 5 proteins was not correlated to the percentage of inhibition. We therefore conclude that these proteins are involved in the aging process of larvae. The changes in protein between free-living (L3) and parasitic stages (L4) were large and seem to reflect the large environmental changes experienced by the larvae when entering a mammalian host. Early fourth- (EL4) and late fourth- (LL4) stage larvae differed in 9 proteins. One protein was stage-specific for EL4. These results imply that only minor alterations do occur in these stages notwithstanding the large morphological differences between these larvae.

Animals↗

PubMed1995

Characterisation of an avermectin resistant strain of Australian Haemonchus contortus.

A strain of Haemonchus contortus (CAVR) isolated in Australia was found to be resistant to ivermectin (IVM) with 0.4 mg kg-1 of the anthelmintic failing to significantly reduce worm burdens. Resistance to IVM was sex-influenced in the CAVR strain with adult males showing a greater sensitivity to IVM. Cross resistance to moxidectin was evident with approximately 15% of the population surviving a dose of 0.1 mg kg-1. The free-living stages of the CAVR isolate had a reduced sensitivity to avermectin (AVM) inhibition of development and motility. Similar structure-activity patterns and resistance factors were obtained for a series of related AVMs as inhibitors of larval development and L3 motility in CAVR and White River II, an IVM-resistant H. contortus isolate from South Africa. Further, both isolates were found to be 3 times more sensitive to paraherquamide than a susceptible H. contortus isolate. This suggest that the same resistance mechanism is operating in both isolates. The CAVR strain is susceptible to the benzimidazoles, levamisole and closantel.

Animals↗