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PubMed1993

Laboratory selection for ivermectin resistance in Haemonchus contortus.

A strain of Haemonchus contortus, susceptible to ivermectin (IVM), was submitted to a selection procedure using IVM at 0.02 mg kg-1 during nine successive passages in lambs. Treatment reduced worm egg counts (epg) by more than 99% up to the seventh passage, but thereafter there was no significant reduction in epg. The efficacy of ivermectin against the ninth selected generation compared with the original susceptible strain was assessed. The results showed that animals infected with the selected strain and treated with IVM did not show any significant reduction (P > 0.05) in epg compared with a 99.9% reduction in the susceptible group. At necropsy, post-treatment worm burdens confirmed that the selection pressure applied to the original strain had been successful in stimulating resistance to IVM.

Animals↗

PubMed1993

Effects of time, from collection to processing, on the recovery of Haemonchus contortus third-stage larvae from herbage.

Being able to obtain accurate estimates for the number of Haemonchus contortus third-stage larvae (L3) on pasture is essential to any type of strategy intended for control. The objective of this experiment was to study the effect of the time, from collection to processing, on the recovery rate of H. contortus L3 from herbage. Separate herbage samples were inoculated with two treatments of a known amount of larvae on Day 1 and then samples were processed from Day 1 to Day 30. This simulated infective herbage was collected, refrigerated, and processed over a period of 1 month. A drop in the recovery rate over time was found. Treatment 2 (5000 L3) exhibited a sharper decline in recovery rate than Treatment 1 (1800 L3). These findings suggest that the effect of time, from collection to processing, as well as larval concentration on the herbage, must be considered when performing any type of larval recovery technique.

Animals↗

PubMed1993

Detection of Haemonchus contortus surface antigen in faeces from infected sheep.

An antigen capture enzyme-linked immunosorbent assay (ELISA) has been developed that could identify Haemonchus contortus antigens in dilute suspensions of worm homogenates and in faeces from sheep naturally infected with moderate burdens of the parasite. The ELISA is based on a polyclonal (rabbit or sheep) antibody coated solid-phase that captures parasite antigen from a suspension of worms or faeces. The captured antigen is detected by a mouse monoclonal antibody (MAb E2) that is reactive with a cuticle surface antigen of H. contortus. Binding of MAb E2 was shown to be specific for H. contortus and the ELISA was able to detect this antigen in faeces of some infected sheep. Characterisation of the antigen reacting with MAb E2 indicates it to be a collagenase and protease resistant surface protein of H. contortus that under reducing conditions in SDS-polyacrylamide gel electrophoresis exists as polypeptides of molecular weight 122, 56 and 49 kDa.

Animals↗

PubMed1995

Comparison of two enzyme immunoassays for the detection of Haemonchus contortus infections in sheep.

Two enzyme-linked immunosorbent assays (ELISA) using either excretory/secretory (ES) products or crude somatic antigens (CSA) of adult Haemonchus contortus were compared for their ability to detect antibodies against H. contortus in sheep. Serum samples obtained from a group of 32 H. contortus mono-infected sheep were tested in the two ELISAs and the obtained data were compared with the results of the faecal examinations of these sheep. The first sheep became patent 3 weeks post infection (p.i.) and all sheep had positive egg counts at week 5 p.i. The first antibodies against H. contortus were detected 1 week p.i. and all sheep were found positive by both ELISAs at week 4 p.i. Using sera from a large number of H. contortus-infected sheep a difference in sensitivity between the ES ELISA (97.7%) and the CSA ELISA (89.2%) was found. The specificity of each assay was determined by testing sera obtained from sheep with mono-infections of H. contortus, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Ostertagia circumcincta, Nematodirus battus, Cooperia curticei, Fasciola hepatica, Taenia ovis or Eimeria spp. The specificity of the ES ELISA was 87.2%, whereas the specificity of the CSA ELISA was 82.7%.

Animals↗

PubMed1995

Haemonchus placei in calves: effects of dietary protein and multiple experimental infection on worm establishment and pathogenesis.

An experiment was conducted to examine the influence of dietary protein and immunisation on parasite establishment and pathogenesis of Haemonchus placei in calves. Four groups of 4-6-month-old worm-free calves (n = 4) were given a low protein diet (LP) containing 213 g crude protein (CP) per head per day or a high-protein diet (HP) containing 469 g per head per day CP. Five weeks later, calves in one of the two groups of each dietary treatment were given 50,000 H. placei infective larvae (L3). Twenty-five days later, infection in these groups was terminated by dosing with oxfendazole. This immunisation process was repeated 4 days later. Four days after termination of the second immunisation all calves were challenged with 100,000 L3. Five weeks later, all calves were slaughtered for abomasal worm counts. Worm establishment was lower in the immunised groups; however, only the HP-I group showed a significant reduction (P < 0.05). All calves gained weight during the first 13 week period, and after challenge the non-immunised groups lost weight, independent of the level of protein in the diet (P < 0.05). Packed cell volume values for all treatments only dropped after challenge (P < 0.05) and the HP-immunised group presented values significantly higher when compared with the other treatments. All calves were hypoproteinaemic and hypoalbuminaemic at the end of the experiment, regardless of the treatment. Immunised calves showed a normocytic normochromic anaemia, while the non-immunised groups presented a microcytic normochromic anaemia.

Animals↗

PubMed1995

A comparison of the responses to repeated experimental infections with Haemonchus contortus among Scottish Blackface lambs.

Twenty helminth-naive Scottish Blackface lambs were given three infections with 10,000 infective larvae of Haemonchus contortus at 8 week intervals. An additional six lambs served as uninfected controls and eight lambs were infectivity controls. The lambs were 7 months old at the start of the infection. Four of the 20 lambs developed severe haemonchosis and were put down during the experiment. The remaining 16 lambs plus uninfected controls were necropsied 8 weeks after the third infection. The mean faecal egg count peaked 6-8 weeks after the first infection, gave a second smaller peak 6-8 weeks after the second infection but produced no peak after the third infection. Mean red blood cell counts fell rapidly during the first infection, then rose gradually during the second and third infections. The mean values suggested that two infections were sufficient to produce effective immunity in the sheep population but they masked considerable individual variation. Eleven animals appeared relatively resistant following the first infection, while two animals were relatively susceptible to even the third infection. The repeatability of mean faecal egg counts or mean red blood cell counts for each animal during the replicate infections were very high, because the rankings of the individual sheep remained remarkably stable. Faecal egg counts were very strongly correlated with red blood cell counts. Multiple regression analysis showed that four factors--faecal egg counts, red blood cell counts, weight and sex--accounted for essentially all of the observed variation in worm burdens among the lambs. Therefore, under these controlled experimental conditions, additional markers appear unnecessary for the detection of resistance status.

Analysis of Variance↗

PubMed1994

Anthelmintic resistance in Haemonchus contortus from sheep in Malaysia.

Albendazole, oxfendazole, fenbendazole, levamisole, closantel, ivermectin and febantel were administered to sheep on four farms and their efficacy assessed by faecal egg count reduction test. High level of resistance of Haemonchus contortus was found to benzimidazoles (albendazole, oxfendazole, fenbendazole) on all farms and to febantel on the one farm where it was tested. No resistance to closantel and levamisole was observed. Resistance to ivermectin was absent on the three farms examined under this study, but has been reported on the fourth farm in earlier work. It is concluded that anthelmintic resistance to benzimidazoles and the probenzimidazole, febantel, is a serious and widespread problem in H. contortus in sheep in Malaysia.

Animals↗

PubMed1994

Resistance to benzimidazole anthelmintics by Haemonchus contortus in goats in peninsular Malaysia.

A previous study had suggested that local strains of goat trichostrongyles, comprising largely Haemonchus contortus, might have developed resistance to benzimidazole anthelmintics. A trial involving 18 goats was conducted to confirm this. There was a significant (P < 0.01) reduction in worm burdens in goats given levamisole, but this was not so for those animals given albendazole, fenbendazole, oxfendazole and mebendazole (P > 0.05).

Albendazole↗

PubMed1994

Ivermectin resistant Haemonchus contortus in Louisiana lambs.

Fifteen weaned crossbred (Suffolk x Louisiana Native) lambs were treated with albendazole (10 mg kg-1) to remove existing nematode infections. They were inoculated with Haemonchus contortus infective larvae from a residual population surviving treatment with the oral formulation of ivermectin (0.2 mg kg-1). One group of five lambs remained untreated, another group of five was treated with the injectable formulation of ivermectin (0.2 mg kg-1), and a third group of five was treated with the oral formulation of ivermectin (0.2 mg kg-1). Subsequent to the treatments, there was essentially no difference in mean fecal egg count or mean number of H. contortus recovered at necropsy between the three groups.

Albendazole↗

PubMed1994

Development and recovery of Haemonchus contortus first larval stages on experimental plots in Mexico.

The objective of this study was to determine the influence of relative humidity, environmental temperature and precipitation on the development of Haemonchus contortus from egg to infective larvae on experimental plots in a subtropical subhumid climate of Mexico. The study was of 1 year duration, with the highest rates of recovery of first-stage (L1) and second-stage larvae (L2) obtained at the end of the rainy season, and of third-stage larvae (L3) in the post-rainy season during the autumn. The lowest rate of recovery of L1 was in winter, whereas that of L2 and L3 mainly occurred in the dry season. The shortest development period for L1 was 12 h, for L2 18 h, and for L3 36 h. The longest development period for L1 was 66 h, for L2 156 h and L3 216 h. Statistical analysis of the data obtained showed significant differences in the time of development from eggs to infective larvae with respect to the season of the year.

Animals↗

PubMed1995

Multiple anthelmintic resistance in Haemonchus contortus on a sheep farm in India.

Multiple resistance to benzimidazoles (fenbendazole, albendazole and mebendazole) in a strain of Haemonchus contortus in sheep was detected on a farm where fenbendazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the fenbendazole-resistant strain were treated with the recommended doses of various anthelmintics. Compared to the control group, percentage reductions in faecal egg counts of sheep treated with fenbendazole, albendazole, mebendazole, levamisole and morantel varied between 56% and 81% and worm counts between 71% and 86%. The results indicate the presence of multiple anthelmintic resistance in this strain of H. contortus on this farm. Sheep treated with ivermectin and closantel showed 100% reductions in faecal egg and worm counts, suggesting high efficacy of these drugs against the population of H. contortus on this farm.

Albendazole↗

PubMed1996

In vitro screening of Haemonchus contortus third stage larvae for ivermectin resistance.

The indiscriminate repeated use of ivermectin has resulted in the emergence of ivermectin resistant field strains of Haemonchus contortus in sheep in South Africa. There is a need for a rapid and cost effective in vitro test to detect such strains. Infective H contortus larvae known to be resistant to ivermectin, were incubated in vitro in a diluted concentration of the drug. Subsequently their ability to migrate was compared with that of larvae of isolates known to be susceptible to ivermectin. The criterion used to determine resistance was the ability of the larvae to migrate out of gelled agar after exposure to ivermectin using diluted propylene glycol as the control compound. The results obtained by this technique confirmed the results obtained with critical controlled tests; significant differences between the ivermectin sensitive and resistant isolates were demonstrated, thereby confirming that this in vitro technique may be used as a screening method to identify ivermectin resistant isolates of H. contortus.

Animals↗

PubMed1996

Characterization of excretory-secretory products from larval stages of Haemonchus contortus cultured in vitro.

To determine biochemical changes associated with early parasite development, Haemonchus contortus larvae were cultured in vitro to the fourth stage (L4). Infective larvae developed from third to fourth stage in 48-96 h. Metabolic activity increased following stimulus of infective stages by CO2 secretion/excretion of significant amounts of protein into cultures and larval feeding did not occur until larvae had molted to the fourth stage. Larval feeding, as monitored by the ability of larvae to ingest fluorescein-labeled albumin, correlated with molting to the fourth stage and only fourth stage larvae were observed to feed. Fourth stage larvae secreted/excreted several enzymes into culture media including a metalloprotease, an acid phosphohydrolase, a cathepsin C-like enzyme, a phospholipase C-like enzyme and an N-acetyl-beta-D-glucosaminidase. Excretory-secretory (ES) products produced by L4 had antigenic homologies with parasite products produced during the second molt and with proteins and glycoproteins extracted from third and fourth stage larvae. ES products were recognized by sera from sheep infected with H. contortus. The enzymes identified here serve as markers for maturation to the fourth larval stage as well as the initiation of feeding and are likely to be involved in extracorporeal digestion. Further, they might serve as potential targets for immune or chemical control of trichostrongyle infections.

Acetylglucosaminidase↗

PubMed1996

Use of free living stages to study the effects of thiabendazole, levamisole, pyrantel and ivermectin on the fine structure of Haemonchus contortus and Heligmosomoides polygyrus.

Ultrastructural changes induced in vitro by thiabendazole, levamisole, pyrantel and ivermectin in the free living larval stages of two trichostrongyles (Heligmosomoides polygyrus and Haemonchus contortus) were analysed. The observed damage for each anthelmintic is related to the known mode of action and compared to the damage commonly described in adults. The advantage of using larvae to study the effects of anthelmintics on the fine structure of nematodes rather than adults is described. Thiabendazole induced alteration of the cellular organization especially epithelial cells of the digestive tract. Changes in mitochondria were also seen. Levamisole caused contraction of muscle fibres whereas no specific lesions were observed with pyrantel. Ivermectin caused an hypertrophy of muscular groups. The interest of such a technique in research on the modes of action of anthelmintics is emphasized.

Animals↗

PubMed1996

The role of the sheath in resistance of Haemonchus contortus infective-stage larvae to proteolytic digestion.

Surface iodinated larvae of Haemonchus contortus were incubated in the presence of the fungal protease, proteinase K, and proteolysis quantified by scintillation counting of released radioactivity. No radioactivity was released from live ensheathed infective-stage larvae (L3(2M)). In contrast, 58% of the radioactivity was released from ecdysed, second molt (2M) cuticles (sheaths) of L3(2M) and 48% from live exsheathed third-stage larvae (L3). When L3(2M) larvae were killed by heat (80 degrees C for 10 min) prior to proteinase K incubation, 61% of the radioactivity was released, whereas less than 7% was released from larvae killed by the metabolic inhibitors NaN3 or KCN. Proteinase K released 44% of the radioactivity from live L3(2M) larvae which had been preincubated with 1% sodium dodecylsulfate (SDS), whereas no radioactivity was released from L3(2M) larvae preincubated with either 1% Triton X-100, 0.2% CTAB, 50% methanol, 50% ethanol, or water. Following incubation with proteinase K, only L3(2M) larvae which had been heat-killed or preincubated with SDS showed visible damage to the sheath. Material released from L3(2M) larvae by exposure to either heat or SDS contained a 98,000 M(r) protein by SDS-PAGE autoradiography. These results indicate that viable L3(2M) larvae are resistant to attack by proteinase K and that this resistance is dependent on structural properties of the sheath.

Animals↗

PubMed1989

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization.

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.

Amino Acids↗

PubMed1990

Biochemical characterization of cuticle polypeptides from the infective larvae of Haemonchus contortus.

1. Ecdysis of infective Haemonchus contortus larvae is effected by the enzymatic degradation of a specialized region of the second molt cuticle containing a biochemically unique polypeptide (mol. wt = 160,000). 2. The 160,000 mol. wt polypeptide and related polypeptides are synthesized at approximately 6 days of larval development. Antigenically similar polypeptides occur in other ruminant trichostrongyles. 3. Cuticle polypeptides digested during ecdysis differ from second molt cuticle collagens in amino acid composition and collagenase sensitivity. However, some antigenic homology between the 160,000 mol. wt polypeptide and cuticle collagens suggests structurally similar regions.

Aging↗

PubMed1993

Ecdysteroids during development in the ovine parasitic nematode, Haemonchus contortus.

1. Samples of embryonating eggs, infective larvae, fourth-stage larvae, young adults, and male and female mature adults of Haemonchus contortus were collected for steroids analysis; lipids were extracted, partially purified, fractionated with HPLC, and analyzed for ecdysteroids by radioimmunoassay. 2. Free ecdysone and 20-hydroxyecdysone were detected in all samples; maximal concentrations occurred in the fourth-stage larvae and young adults. 3. Conjugated ecdysone and 20-hydroxyecdysone were detected in all samples but the infected larvae; maximal concentrations occurred in the fourth-stage larvae and young adults. 4. Patterns of ecdysteroid occurrence support regulatory roles in embryogenesis, cuticular deposition, and gonadogenesis. 5. Relative concentrations of ecdysteroids are comparable to those previously reported in eggs and adults of Ascaris suum.

Animals↗