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At least 217 records · Page 12Linked to original sources

PubMed1993

Expression of cloned beta-tubulin genes of Haemonchus contortus in Escherichia coli: interaction of recombinant beta-tubulin with native tubulin and mebendazole.

Two distinct beta-tubulin cDNA isotypes (beta 8-9 and beta 12-16) from Haemonchus contortus were expressed for the first time in Escherichia coli and characterised by their specific mebendazole (MBZ) binding and polymerization properties. Beta-tubulin was expressed without translational fusion to an E. coli sequence under the regulation of the tryptophan promoter in the pTrp2 vector. Beta-tubulin was produced in large amounts in insoluble 'inclusion bodies'. The inclusion bodies were purified and solubilised and the beta-tubulin renatured by treatment with urea followed by dilution with alkaline buffer and a shift to physiological pH. The yield was more than 10 mg of beta-tubulin per litre of cell culture. The recombinant tubulin produced was recognized in Western blot by specific anti-beta-tubulin antibodies. Tritiated MBZ binding to the recombinant H. contortus beta-tubulin was measured in the presence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [3H]MBZ high-affinity binding (HB) to 'pure' (no other eukaryotic protein present) beta 8-9 or beta 12-16 was observed. Enhanced high-affinity binding was observed when recombinant beta 8-9 or beta 12-16 were mixed and pre-incubated with whole supernatants or tubulin-enriched extracts from H. contortus. The enhancement was more than additive. Beta 12-16 bound more MBZ and caused a greater enhancement than beta 8-9. Mixing recombinant beta 8-9 or beta 12-16 with whole supernatants or tubulin-enriched fractions from H. contortus promoter polymerization at 37 degrees C. Use of 35S-labelled protein showed that the polymer contained recombinant tubulin. Western blot using specific anti-alpha-tubulin monoclonal antibodies showed that the polymer contained alpha-tubulin. Similarly the recombinant nematode beta-tubulin co-polymerized with tubulin from chicken brain. Our data suggest that the recombinant beta-tubulin can interact and copolymerize with parasite or chicken tubulin. Furthermore the interaction of recombinant nematode beta-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-binding sites. However, interaction of recombinant beta-tubulin with microtubule proteins from chicken brain did not result in the formation of high-affinity MBZ-binding sites.

Animals↗

PubMed1994

Differential expression of two succinate dehydrogenase subunit-B genes and a transition in energy metabolism during the development of the parasitic nematode Haemonchus contortus.

The carbohydrate metabolism of free-living and parasitic stages of the sheep nematode Haemonchus contortus was studied, and it was demonstrated that during development a switch occurred from Krebs-cycle activity towards a more fermentative metabolism. During this switch a transition might take place in complex II of the respiratory chain. In the free-living (L3) and early parasitic (XL3) stages, complex II catalyses the oxidation of succinate to fumarate via the Krebs cycle, whereas in adults complex II functions in the reverse reaction, the reduction of fumarate to succinate. L3 and XL3 were shown to already possess a large anaerobic capacity. They survived well in the absence of oxygen or in the presence of cyanide, which completely blocked respiration. Krebs-cycle activity, however, was only partially inhibited by cyanide; the XL3s in particular produced in the presence of cyanide large amounts of propanol, the production of which probably functions as an alternative electron sink. For further investigation of the observed metabolic switch, complex II of the respiratory chain, a key enzyme involved in this switch, was studied. The B subunit of complex II was cloned and sequenced. These clones all showed sequences similar to the B subunit of succinate dehydrogenase from other species, and included the amino-terminal signal sequence for importation into mitochondria. Two genes were identified, types 1 and 2, based on the DNA and amino acid sequences and on the lack of cross-reaction to each other when used as probes on Southern blots. On Northern blots, the two genes showed a different expression pattern during the development of the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1983

The effects of transfer factor, parasitic antigen and precipitated immunoglobulin treatments in lambs infected with Haemonchus contortus.

Significant reductions in adult worm burdens in lambs less than six months old infected with 5000 third stage Haemonchus contortus larvae were achieved following the combination of a single transfer factor treatment with multiple parenteral treatments of adult parasitic antigen and precipitated immunoglobulin. Specific cellular responses in the abomasa associated with individual treatments were discernible in all the experimental groups and quantitative differences in these responses reflected the varying treatment levels and treatment combinations. The reactions associated with transfer factor were similar to immediate type hypersensitivity responses and their role as possible effector mechanisms in correcting partial immune incompetency in lambs is discussed.

Abomasum↗

PubMed1983

The effects of DL-tetramisole and rafoxanide on tricarboxylic acid cycle enzymes of Haemonchus contortus, in vitro.

Various enzymes of the tricarboxylic acid cycle (TCA) viz., aconitase (E.C. 4.2.1.3), isocitrate dehydrogenase (E.C. 1.1.1.42), succinate dehydrognease (E.C. 1.3.99.1), fumarate reductase (NADH: fumarate oxido-reductase), fumarase (E.C. 4.2.1.2) and maltate dehydrogenase (E.C. 1.1.1.37) were detected in adult Haemonchus contortus (Nematoda: Trichostrongylidae), in vitro. Low activities of aconitase and isocitrate dehydrogenase suggested that the TCA cycle has a minor function and the pathway of CO2 fixation is the major pathway in the energy metabolism of the parasite. In vitro incubation in Tyrode's solution had no significant effect on TCA cycle enzymes and the worm was able to maintain normal metabolism for 12 h. The effects of D L-tetramisole and rafoxanide on various enzymes of the TCA cycle were studied in adult H. contortus. At 50 micrograms ml-1 varying degrees of inhibition of succinate dehydrogenase and fumarate reductase activities were observed. At the same concentration, the activities of other enzymes remained unaltered.

Animals↗

PubMed1983

In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae.

Twenty-five known anthelmintic compounds were evaluated in vitro against the highly motile exsheathed non-feeding third-stage of Haemonchus contortus larvae. Activity was based on lack of motility or death of larvae after 24 h of chemical exposure. Six compounds (avermectins, closantel, levamisole, morantel, phenylhydrazone and ticarbodine) were active at a concentration of 100 micrograms cm-3 or less. The most active compounds were avermectins and levamisole. When higher in vitro concentrations were used, ten compounds (bephenium, coumaphos, dichlorovos, disophenol, hygromycin b, methyridine, parbendazole, phenothiazine, pyrantel and thiabendazole) exhibited activity. Nine compounds were found to be inactive; among these were the new benzimidazoles, i.e., albendazole, fenbendazole, mebendazole and oxibendazole. Because of the inactivity of the new benzimidazoles, this in vitro system is unsuitable as a routine screening tool. Also, the system appears to favor drugs that act quickly through percuticular entry. In an initial group of 5280 untested compounds, 254 (4.8%) exhibited in vitro activity at 100 micrograms cm-3 against the non-feeding larvae stage. The exogenous and in vitro cultivation techniques required for collecting, cleaning and exsheathing the larvae are described.

Animals↗

PubMed1984

Some factors influencing the immunisation of sheep with irradiated Haemonchus contortus larvae.

Over the last 6 years a number of experiments in which sheep were successfully immunised with 2 doses of 10 000 Haemonchus contortus larvae, irradiated at 60 krad in a gamma-source, have been reported by the authors. In this paper, the failure of such a regimen of immunisation, using the same strain of larvae, is reported together with an investigation of the possible causes. Technical errors were eliminated as a cause, on the grounds that the failure occurred in 2 separate laboratories using different 60Co sources, as was increased radiosensitivity of the continuously passaged strain of H. contortus as measured by the percentage development of irradiated larvae in 6 naive lambs. From experiments utilising 18 lambs and using both irradiated and normal larvae, it was postulated that the strain had lost its original degree of immunogenicity. Subsequently, in 2 experiments involving 27 lambs, it was shown that irradiation at 40 krad was completely successful in restoring immunogenicity and producing a degree of protection against challenge similar to that previously reported with 60 krad. It was concluded that, with the current strain of H. contortus, 60 krad is now too high a level of irradiation of larvae to produce a consistently high degree of protection against challenge.

Animals↗

PubMed1984

Experimental infection of sheep by the camel stomach worm, Haemonchus longistipes.

An attempt has been made to infect sheep by Haemonchus longistipes with the objective of developing a relatively cheap and manageable model of camel haemonchosis. A large proportion of inoculated animals (28/34) developed patent infections; the prepatent period was short, lasting for 3 to 4 weeks in the majority of infected animals. Worm burdens were low and so was the total egg output. Most of the recovered worms were reduced in size.

Animals↗

PubMed1984

Amino acid biosynthesis in Haemonchus contortus from C14-labelled precursors, in vitro.

Adult Haemonchus contortus (Nematoda: Trichostrongylidae) was investigated for its ability to utilize various C14-labelled precursors, i.e., glucose, acetate, CO2 and palmitic acid, for amino acid biosynthesis. H. contortus has been demonstrated to be capable of synthesizing essential as well as non-essential amino acids. Label from all the precursors was detected in aspartic acid, lysine, histidine, cystine, cysteine, glutamic acid, proline, arginine, tyrosine, alanine, glycine, serine, valine, methionine, leucine and isoleucine. Glutamic acid, aspartic acid, alanine, glycine and serine were synthesized to a greater extent relative to the other amino acids, regardless of the precursor employed. However, in case of glucose, there was comparatively less incorporation into glycine and serine. Incorporation of C14 into various amino acids is evidence for the operation of tricarboxylic acid cycle. Further, the fact that carbon from palmitic acid appears in amino acids, indicates that adult H. contortus is capable of catabolizing long-chain fatty acids. Possible mechanisms for the involvement of various precursors in amino acids biosynthesis are examined here.

Acetates↗

PubMed1984

Serum and abomasal antibody response of sheep to infections with Haemonchus contortus.

Serum and abomasal IgA, IgG and IgM antibody response against adult worm, L3 and egg antigens of Haemonchus contortus was monitored by the ELISA technique after one or two infections with this nematode. Following the first infection, antibody levels in serum did not change materially. After administration of a challenge dose of infective larvae, antibodies of the three immunoglobulin classes in infected animals rose slightly, but this rise appeared later than the fall in the faecal egg counts. In contrast, in abomasal mucosa, IgA anti-larval antibody levels, which did not increase materially after the primary infection, rose rapidly after a transient inhibition when sheep were challenged. A close temporal relationship was observed between the rise in local anti-worm IgA antibodies and the self-cure reaction, but antibody levels fell rapidly after worm diminution. The local antibody response was thus considered to be related to immunity of sheep to H. contortus.

Abomasum↗

PubMed1984

The pathogenesis of experimental Haemonchus longistipes infection in camels.

The pathogenesis and clinical signs of Haemonchus longistipes infection were studied in four experimentally infected camels two of which were adults and the other two were young. In the former animals, an acute infection developed, characterized by mucoid diarrhoea, anorexia, anaemia, loss of body weight, oedema of the lower parts of the limbs, general malaise and death at 8-10 weeks post-infection. In the two younger camels, a less dramatic disease was encountered with less severe symptoms and no oedema, but also terminating fatally at 19-20 weeks post-infection. Parasitological, haematological and biochemical parameters were determined during the course of the infection and were mostly comparable with those usually encountered in haemonchosis of other animals.

Animals↗

PubMed1986

Haemonchus contortus and other trichostrongylid infections in parturient, lactating and dry ewes.

From autumn (April) to spring (November), groups of pregnant and dry Merino ewes grazed pasture contaminated with infective larvae of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. A periparturient rise in faecal egg counts occurred in the pregnant ewes, following the maturation in early spring of arrested fourth-stage larvae of H. contortus and failure of pregnant ewes to expel the resulting adult worms. Peak egg counts were seen in September, just before lambing. In dry ewes, egg counts were slightly elevated at the time of the rise in the pregnant ewes, but fell to very low levels as adult worms developing from previously arrested larvae were expelled. Lactating ewes acquired greater burdens of O. circumcincta and T. colubriformis than did dry ewes, but were equally refractory to new infections with H. contortus. It is therefore suggested that the impairment of immunity to helminth infection seen in reproductive ewes may be more specific than was previously envisaged.

Animals↗

PubMed1987

Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite.

Florida Native lambs, less than 6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (greater than 30,000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions and secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractionated SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.

Animals↗

PubMed1987

Changes in lipids and free fatty acid fractions in adult Haemonchus contortus during incubation in vitro.

Adult Haemonchus contortus (Rud., 1803) has been investigated for its ability to utilize lipids with regard to the total lipids, sterols, free fatty acids, acylglycerols and phospholipids produced during incubation in vitro. All these components exhibit extensive fluctuations, decreasing at some times and increasing at others, thus indicating both biosynthesis and utilization. Also, changes in fatty acid components of total lipids have been analyzed by gas liquid chromatography. These observations indicate that H. contortus is capable of utilizing 16:0, 18:0, 18:1, 18:2, 20:0, 20:un1, 20:un2, 21:0, 21:un1, 21:1, 21:un2, 22:un1, 22:3, 22:?, 22:un2 and 24:3 fatty acids. At the same time, because of large increases in 15:0, 16:0, 18:0, 18:?, 18:1, 18:2, 20:0, 20:un1, 20:un2, 21:0, 21:un1, 21:1 and 21:un2 acids, it is postulated that these acids are synthesized by adult H. contortus.

Animals↗

PubMed1988

Serial passage of Haemonchus contortus in resistant and susceptible sheep.

A recently isolated field strain of Haemonchus contortus was passaged through resistant (repeatedly reinfected) and susceptible (immunosuppressed) sheep for six and nine generations, respectively. Infectivity of the resulting serially passaged strains was not significantly different when tested in groups of 1-year-old susceptible sheep.

Animals↗

PubMed1989

Comparison of three in vitro techniques to estimate benzimidazole resistance in Haemonchus contortus of sheep.

Three in vitro assays to detect benzimidazole resistance, namely, the egg-hatch assay, tubulin-binding assay, and a larval-development assay, were evaluated by estimating the level of benzimidazole resistance in three field isolates of Haemonchus contortus compared with a susceptible reference strain. Comparisons were also made with estimates of benzimidazole resistance of the three field strains obtained from an in vivo controlled anthelmintic efficacy test. All three in vitro tests showed similar, consistent results which also suggested greater sensitivity than the in vivo assay. These results indicate that selection of an in vitro technique to determine benzimidazole resistance should therefore be based on considerations other than precision, such as technical expertise, availability of equipment, cost and speed in which diagnosis is required.

Analysis of Variance↗

PubMed1989

Motility response of levamisole/benzimidazole-resistant Haemonchus contortus larvae.

Electronic measurements of the motility of third stage larvae of a susceptible and a levamisole/benzimidazole-resistant strain of Haemonchus contortus were made after incubation in solutions of anthelmintics for 24 h. Results confirmed the resistance to benzimidazoles, but failed to show differences in response to levamisole. Visual observations of paralysis of first and third stage larvae in solutions of levamisole also failed to show significant differences between the two strains. The tests as conducted failed to demonstrate levamisole resistance, suggesting that nematode strains may vary in their mechanisms of resistance to levamisole.

Animals↗

PubMed1989

Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus.

The potential of the chitin synthesis inhibitor diflubenzuron (DFB) to alter the development of the parasitic nematodes (Ascaris suum and Haemonchus contortus was investigated. DFB given orally (10 mg kg-1 per day for 30 days) to sheep inoculated with H. contortus infective larvae did not prevent the establishment of adults or affect fecal egg output. However, there was a significant (greater than 90%) decrease in the number of infective larvae recovered from fecal cultures derived from lambs harboring H. contortus adults that were treated with DFB. DFB did not affect egg hatching. Oral administration (10 mg kg-1 per day for 20 days) of DFB to swine harboring adult A. suum adults had no effect on the adult worm burden or on egg morphology, but eggs removed from worms obtained from DFB-treated swine contained less chitin than eggs removed from untreated control swine. DFB also inhibited chitin synthesis in vitro in the isolated reproductive tract of A. suum adults. These results indicate that DFB at high doses can inhibit the subsequent development of H. contortus larvae in the feces. Since H. contortus larvae lack chitin, DFB may act on these larvae by a mechanism independent of a direct effect on chitin synthesis.

Animals↗

PubMed1989

Effects of exogenous progesterone and/or prolactin on Haemonchus contortus infections in ovariectomized ewes.

Reproduction can alter the course of ovine nematodiasis; fecal nematode egg concentrations often increase near lambing and throughout lactation, a phenomenon referred to as the periparturient rise. To identify the host mechanisms that might link these disparate events, i.e. lactation and the fecundity of gastrointestinal trichostrongyles, ovariectomized ewes were injected daily with progesterone and/or prolactin, or saline. Progesterone treatment commenced 20 days before inoculation with Haemonchus contortus and prolactin was administered throughout the 30-day infection period. Ovariectomized ewes receiving prolactin during the experimental infection maintained higher daily fecal egg concentrations than the progesterone, progesterone/prolactin, or the control treatment groups. However, ovariectomized ewes that received 20 days of pre-inoculation exposure to progesterone, as well as prolactin during the infection, had greater numbers of nematodes that were larger than the other treatment groups. Thus, the sequential delivery of these hormones that are associated with the reproductive cycle in ewes produced some of the same results that occur during periparturient rise.

Analysis of Variance↗