Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus contortus

The activity of concentrated exsheathing fluid of Haemonchus contortus against isolated sheaths was not inhibited by ethylenediamine tetra-acetic acid (EDTA), 10^?2 M, even when the concentrations of Mg and Mn were < 4 × 10^?4 M and < 0·9 × 10^?6 M respectively. Purified or diluted solutions of exsheathing fluid, even in the presence of Mg²⁺, 10^?3 M, were inhibited. Leucine aminopeptidase (LAP) in exsheathing fluid was active even at concentrations of Mg < 1·3 × 10^?5M. Concentrated solutions were partially inhibited by EDTA, 10^?2 M, at low concentrations of Mg; inhibition was increased in diluted and purified preparations.1,10-phenanthroline (Ophen) strongly inhibited exsheathing activity (Zn < 1 × 10^?6 M). When Zn²⁺, 10^?3 M was added, the inhibition was abolished. The hydrolysis of l-leucinamide was greatly increased in the presence of Ophen, 10^?4 M; this effect was abolished by adding Zn²⁺, 10^?3 M.It is suggested that exsheathing fluid from at least some ‘strains’ of H. contortus contains a Zn metallo-enzyme, probably LAP, which is involved in the process of exsheathment.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.     

Some analyses of exsheathing fluid from infective Haemonchus contortus larvae from Ontario

Infective Haemonchus contortus larvae from Ontario were exsheathed, and the exsheathing fluid was prepared, using several procedures some of which duplicated those of other researchers. Infective larvae were exsheathed successfully using the rapid (20-min) tetraborate system. Second-stage sheaths were dissected from infective larvae and were incubated with various preparations of exsheathing fluid. Up to 30% of the sheaths incubated with dilute exsheathing fluid for 1 h had refractile rings. When the fluid was concentrated by dialysis or lyophilization the exsheathing activity was not lost. Heat destroyed the ability of concentrated exsheathing fluid to cause refractile rings in dissected sheaths, but Cu²⁺, Hg²⁺ or diaminoethanetetra-acelic acid did not. The enzyme leucine aminopeptidase was not found in concentrated exsheathing fluid.     

Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus

Rogers W.P. and Brooks F. 1978. Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus. International Journal for Parasitology 8: 449–452. Exsheathing activity relative to leucine aminopeptidase activity (LAP) was greater in exsheathing fluid of infective juveniles of Haemonchus contortus than extracts of homogenates of the same organism. In both preparations the biological and enzyme activities were precipitated with acetone 20 v/v and ammonium sulphate, 40% saturation. Broad peaks of exsheathing and LAP activities obtained by sucrose density-gradient centrifugation and on Sephadex G150 overlapped but the peak of biological activity was always found on the low mol. wt. side of the LAP peak. LAP in exsheathing fluid was separated into two sharp peaks in polyacrylamide gradient-pore electrophoresis. In four experiments the major peak gave a mol. wt. within the limits 345,000–354,500. A minor peak was obtained at 1,800,000. Exsheathing activity remained broadly distributed but fell mostly on the low mol. wt. side of the major LAP peak.It is concluded that LAP cannot be the sole agent involved in exsheathment a lipase may be necessary to expose the substrate attacked by LAP.     

Haemonchus contortus: Local and serum antibodies in sheep immunised with irradiated larvae

Smith W. D. and Christie M. G. 1978. Haemonchus contortus: lccal and serum antibodies in sheep immunised with irradiated larvae. International Journal for Parasitology8: 219–223. Two doses of Co⁶⁰ irradiated Haemonchus contortus protected 9-month-old sheep against a challenge infection of 10,000 normal larvae. This resistance was associated with increased concentrations of IgG antibodies in the serum as well as IgA and IgG antibodies in the abomasal mucosa.     

The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics

Prichard R. K. 1973. The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics. International Journal for Parasitology3: 409–417. Fumarate reductase activities in both thiabendazole susceptible and tolerant strains of Haemonchus contortus have been determined using spectrophotometric and radioisotopic methods. The effects of the anthelmintics, thiabendazole, cambendazole, 1-tetramisole, morantel tartrate, mebendazole and disophenol on the fumarate reductase system of these strains were assayed. Thiabendazole inhibited the fumarate reductase system of the susceptible strain, but no effect could be observed in the tolerant strain with a concentration of thiabendazole of 2 × 10^?3 M. Cambendazole inhibited the system in both strains, but the thiabendazole tolerant strain was significantly less susceptible to cambendazole than the thiabendazole susceptible strain. 1-tetramisole, morantel tartrate and disophenol inhibited the fumarate reductase system in both strains. Mebendazole was not found to affect the fumarate reductase system in either strain. The significance of the fumarate reductase system and its associated phosphorylation in the metabolism of helminths and in the susceptibility of helminths to anthelmintics is discussed.     

The effect of ethoxyzolamide, an analogue of insect juvenile hormone, nor-adrenaline and iodine on changes in the optical path difference in the excretory cells and oesophagus during exsheathment in Haemonchus contortus

Davey K. G., Sommerville R. I. and Rogers W. P. 1982. The effect of ethoxyzolamide, an analogue of insect juvenile hormone, nor-adrenaline and iodine on changes in the optical path difference in the excretory cells and oesophagus during exsheathment in Haemonchus contortus. International Journal for Parasitology12: 509–513. Ethoxyzolamide, an inhibitor of carbonic anhydrase, markedly inhibits exsheathment of Haemonchus when the larvae are subsequently exposed to an exsheathing stimulus of CO₂ at 38.5°C. Ethoxyzolamide at 2 × 10^?5M does not prevent the increase in optical path difference in the oesophageal region which normally accompanies exsheathment, but markedly inhibits the increase in optical path difference in the excretory cells. An analogue of juvenile hormone (JHA; the methyl ester of 3,7,11 trimethyl-7,11-dichloro-2-dodecenic acid) does not affect the optical path difference in either the oesophagus or the excretory cells of ensheathed worms. When worms are artificially desheathed by exposure to NaOCl, a procedure which mimics the effect of CO₂ upon the oesophagus, but which does not affect the excretory cells, subsequent exposure to JHA at room temperature increases the optical path difference in the excretory cells. This increase is enhanced by subsequent incubation of the worms at 38.5°C at 30–60 min and further enhanced when CO₂ is present during the incubation at 38.5°C. The stimulation of the excretory cells by JHA is inhibited by ethoxyzolamide at 2 × 10^?5M. Noradrenaline at 10^?3M has no effect on ensheathed larvae, but causes an increase in optical path difference in the excretory cells of larvae desheathed with NaOCl. This increase is inhibited by ethoxyzolamide. A brief exposure to I₂ blocks the response of the excretory cells of both CO₂ and JHA, but does not significantly reduce the effect of nor-adrenaline. On the basis of these and previous results, it is proposed that both CO₂ and JHA stimulate a hypothetical CO₂ receptor which leads to the release of nor-adrenaline. The noradrenaline in turn stimulates, either directly or indirectly, the excretory cells.     

Meiotic abnormalities in backcross lines of hybrid Haemonchus

Le Jambre L. F. and Royal W. M. 1979. Meiotic abnormalities in backcross lines of hybrid Haemonchus. International Journal for Parasitology10: 281–286. Hybrid worms from the reciprocal matings between Haemonchus contortus and H. placei were backcrossed to the parent species. Six of the possible eight backcrosses produced fertile eggs. The six backcross lines were passaged through sheep for four generations. The adults of this generation had various levels of meiotic abnormality as evidenced by abnormal variations in chromosome structure and aneuploidy. The eggs from backcross lines with a degree of meiotic abnormality had the lowest percentage hatch. Four of the six backcross lines required a higher temperature for larval development than did H. contortus. The size of the third-stage larvae varied from one with larvae larger than H. placei to one line with larvae smaller than H. contortus.     

The response of sheep to parenteral vaccination and immunizing infections against the abomasal nematode, Haemonchus contortus

Adams D. B., Beh K. J. and Davies H. I. 1982. The response of sheep to parenteral vaccination and immunizing infections against the abomasal nematode, Haemonchus contortus. International Journal for Parasitology12: 445–449. Subcutaneous injection of relatively large amounts of unfractionated homogenates of adults plus infective larvae of Haemonchus contortus emulsified in complete Freund's adjuvant produced a degree of protective immunity when challenge infection was given eight weeks after the first or only dose of vaccine. In an attempt to improve acquired immunity, parenteral vaccination was either followed or preceded by a short immunizing infection with H. contortus, which was terminated by anthelmintic before patency. This treatment aimed at stimulating general responsiveness to worm antigens and invoking mucosal immunity in the abomasum. Disparate results were obtained; immunizing infections either increased immunity or made sheep more susceptible to challenge infection. In this latter situation, the unresponsiveness associated with primary infection with H. contortus may have been increased.     

Genetics of vulvar morph types in Haemonchus contortus: Haemonchus contortus from the Northern Tablelands of New South Wales

Le Jambre L. F. and Royal W. M. 1977. Genetics of vulvar morph types in Haemonchus contortus: Haemonchus contortus from the Northern Tablelands of New South Wales. International Journal for Parasitology7: 481–487. The inheritance of the vulvar morph types, smooth, knobbed, linguiform A-right, linguiform A-left and linguiform C-type was studied in Haemonchus contortus from the Northern Tablelands of New South Wales. The dominance hierarchy of these characters was found to be smooth > knobbed > linguiform. The linguiform phenotype was the most common in the wild type population. The modifications on the linguiform phenotype were found to be highly heritable with A-right responding most to selection. Realised heritabilities for these characters was A-right 0.37, A-left 0.15, and C-type 0.21. It appeared that the dominance of a phenotype increased following selection.     

Time of onset and target of immune reactions in sheep with acquired immunity against Haemonchus contortus

Adams D. B. 1982. Time of onset and target of immune reactions in sheep with acquired immunity against Haemonchus contortus. International Journal for Parasitology12: 439–443. Nonimmune sheep and sheep rendered immune by infection with the abomasal nematode, Haemonchus contortus, were infected with the parasite and treated at various times with the glucocorticosteroid, dexamethasone. The results show that this immunosuppressant drug abolished acquired but not innate immunity to H. contortus and that acquired responses were not important in restraining the fecundity of adult worms during primary infection. By treating immune sheep with dexamethasone during reinfection, it was shown that the responses acting against the establishment of infection commence later than the fourth day after larval administration and are complete by the seventh day. H. contortus more advanced in development than the fourth-larval stage were relatively insusceptible to this manifestation of acquired immunity.     

Enzymes in the exsheathing fluid of nematodes and their biological significance

Rogers W. P. 1982. Enzymes in the exsheathing fluid of nematodes and their biological significance. International Journal for Parasitology12: 495–502. The characteristics of an enzyme which hydrolysed denatured collagen and a lipase in exsheathing (ecdysal) fluid are described. A highly purified collagenase from Clostridium histolytica attacked isolated sheaths and reacted to additives in the same way as exsheathing fluid. However, relative to their activities with Azocoll or $\text{p-}\text{phenylazobenzyloxycarbonyl}\text{-}\text{L}\text{-}\text{Pro}\text{-}\text{L}\text{-}\text{Leu}\text{-}\text{Gly}\text{-}\text{L}\text{-}\text{Pro}\text{-}\text{D}\text{-}\text{Arg}$ as substrates, the enzyme of exsheathing fluid was >400 times as active as the bacterial collagenase in its action on isolated sheaths.It is suggested that the lipase in exsheathing and hatching fluids may, in association with the pseudocollagenase (and sometimes with chitinase also) have a function in the hatching of eggs. The pseudocollagenase alone may serve as the exsheathing enzyme. The leucine aminopeptidase in hatching and exsheathing fluids may be concerned in the breakdown of the excretory cell and the release of the fluids.     

Glutathione-S-transferase,a possible drug-metabolizing enzyme,in Haemonchus contortus: Comparative activity of a cambendazole-resistant and a susceptible strain

Kawalek J. C., Rew R. S. and Heavner J. 1984. Glutathione-S-transferase, a possible drug-metabolizing enzyme in Haemonchus contortus: comparative activity of a cambendazole-resistant and a susceptible strain. International Journal for Parasitology14: 173–175. A drug metabolizing enzyme (DME), glutathione-S-transferase, was detected in homogenates of a cambendazole-susceptible and a cambendazole-resistant strain of Haemonchus contortus. The activity was 1.5–1.8 times higher in the resistant strain. DME activation is a possible mechanism for anthelmintic resistance in H. contortus.     

Carbonic acid as the host signal for the development of parasitic stages of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1985. Carbonic acid as the host signal for the development of parasitic stages of nematodes. International Journal for Parasitology15: 661–667. This paper gives results on which may be based an identification of the component of the system CO₂ + H₂O ai H₂CO₃ ai H⁺ HCO₃^? which acts as the stimulus from the animal host for some nematodes. Using infective juveniles of Nematospiroides dubius and Haemonchus contortus, the effects on exsheathment of (1) low pCO₂ values, (2) the presence of carbonic anhydrase in the stimulating medium, and (3) the inhibition of carbonic anhydrase within the juveniles have been examined. The results lead to the suggestion that it is the “readily available” undissociated H₂CO₃, or H₂CO₃ + HCO₃^? which is the critical factor in the stimulus for development. The wide range of [H⁺]s over which “readily available” H₂CO₃ is present in physiological environments suggests that this host signal may be important for infection with many species.     

Gene activity and the development of early parasitic stages of nematodes

Actinomycin-D, 8 × 10^?6m, blocked the action of the stimulus from the host which normally initiates the development of the infective stage of Haemonchus contortus to the first parasitic stage. However, the action of the stimulus in initiating exaheathment was not affected by the antibiotic.The significance of these results in relation to the activity of the gene set of the first parasitic stage and the control of exsheathment of the infective stage is discussed.     

Immunity acquired by sheep from an experimental infection with haemonchus contortus

Adams D.B. and Beh K.J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology11: 381–386. A primary infection of sheep with a single dose of Haemonchus contortus larvae was traced by faecal egg counts until it had substantially declined after 55 weeks. These primed sheep were then given a sequence of two reinfections with the parasite. Comparison of faecal egg counts in primed sheep and in two separate groups of previously worm-free sheep showed that primary infection conferred significant immunity. This, however, was not sufficiently protective to prevent the development of further anaemia and faecal egg counts indicative of clinical haemonchosis. It is suggested that an adaptation in the host-parasite relationship which promotes the longevity of primary infection with H. contortus may also moderate the induction of acquired immunity.The titre of haemagglutinating antibody specific for H. contortus rose in serum during the course of primary infection, but the two reinfections did not stimulate a rise in titre. Titres of haemagglutinating antibody before reinfection did not correlate with subsequent faecal egg counts.     

Respiratory metabolism and thiabendazole susceptibility in developing eggs of Haemonchus contortus

Weston K. M., O'Brien R. W. and Prichard R. K. 1984. Respiratory metabolism and thiabendazole susceptibility in developing eggs of Haemonchus contortus. International Journal for Parasitology14: 159–164. The respiratory metabolism of and uptake of thiabendazole (TBZ) by unembryonated (8–16 cells) and embryonated eggs of Haemonchus contortus have been compared. Lipid, which forms the greatest energy reserve in the eggs, decreases during embryonation and seems to be the sole source of respiratory energy. Trehalose increases to the same extent as glycogen decreases during this development. Isocitrate dehydrogenase (NADP⁺) and lactate dehydrogenase were not detected in the unembryonated eggs, but were present after embryonation. In addition, the activities of citrate synthase and malate dehydrogenase significantly increased during embryonation. Respiratory enzymes involving cytochrome c oxidation and reduction were detected in eggs at both stages of development. However, in line with other results indicating an increased capacity for and utilization of aerobic metabolism, the rate of oxygen uptake more than tripled during development of the eggs. Although both unembryonated and embryonated eggs took up palmitate, its metabolism to CO₂ only occurred in the embryonated eggs.The unembryonated eggs, exposed to TBZ for 4 h, concentrated it 5.9 times and the embryonated eggs 2.1 times, which are in proportion to their respective lipid contents. Uptake of TBZ was dependent on the concentration in the incubation medium and appears to be a passive process.The studies indicate that the embryonated eggs have a greater capacity for, and do utilize aerobic metabolism to a greater extent than do unembryonated eggs. The reduced susceptibility of embryonated eggs to TBZ could be associated with this metabolic difference and/or with their reduced uptake of TBZ.     

Observations on crystals found in the intestinal cells of Haemonchus contortus and in the intestinal lumen of Ostertagia ostertagi.

Bird A. F., Waller P. J., Dash K. M. and Major G. 1978. Observations on crystals found in the intestinal cells of Haemonchus contortus and in the intestinal lumen of Ostertagia ostertagi. International Journal for Parasitology8: 69–74. Crystals are described which are located in the distal intestinal cells of the fourth stage larva of Haemonchus contortus and in the distal intestinal lumen of both larvae and adults of Ostertagia ostertagi.The crystals are thought to be by-products of degenerative processes in these nematodes. They are irregular hexagonal rod-shaped structures which measure approx. 2.5 × 15 um in H. contortus. In O. ostertagi they are also irregular hexagonal structures commonly reaching dimensions of 25 × 50 μm, but they vary considerably in size and are not rod-shaped.Histochemical tests. X-ray analysis and electron microscope studies reveal that both types of crystal contain protein and the element sulphur and are of uniform composition, being neither hollow nor having a core of different refractive index.They differ in that the crystals of O. ostertagi contain more sulphur, exhibit birefringence under polarized light, stain with methylene blue, toluidine blue and osmium tetroxide. The crystals from H. contortus on the other hand are not birefringent and do not stain with the dyes mentioned above.     

Trypanosomatidae: isoleucine requirement and threonine deaminase in species with and without endosymbionts

Abomasal and duodenal concentrations of Haemonchus contortus eggs were measured in four lambs fitted with permanent abomasal and duodenal cannulas and infected with 25,000 Haemonchus contortus larvae. During the period of maximal egg laying, i.e., when the abomasal H. contortus egg concentration was above 2000 eggs/ml, the animals received cimetidine (20 mg/kg) intravenously or pentagastrin (5 μg·kg^?1·h^?1) for 90 min and the changes in abomasal and duodenal egg concentrations were followed for 2.5 hr. Pentagastrin infusion reduced the abomasal and duodenal pH significantly and in less than 15 min decreased the abomasal and duodenal egg concentrations which represented only 21.4 and 12.0% of the control values at the end (90 min) of infusion. During pentagastrin infusion, both the abomasal (r = 0.56, P ? 0.01) and the duodenal (r = 0.72, P ? 0.01) egg concentrations correlated positively with the corresponding pH values. After cimetidine injection, the abomasal and duodenal pH had increased 150 min later to, respectively, pH 6.16 and 6.27. During the first 30 min for an abomasal pH lower than 4.5–5.0, egg production increased by 106%; then the abomasal and duodenal egg concentrations decreased progressively, representing, respectively, only 39.3 and 16.4% of the control values 120 min later. It is concluded that the level of egg laying of adult H. contortus was related to the abomasal acidity, the maximal egg production occurring when the abomasal pH was between pH 4 and 4.5.     

Developmental arrest of Haemonchus contortus in sheep treated with a corticosteroid

Adams D. B. 1986. Developmental arrest of Haemonchus contortus in sheep treated with a corticosteroid. International Journal for Parasitology16: 659–664. Developmental arrest of the nematode, Haemonchus contortus, at the fourth larval stage within sheep appears to be controlled by a complex of signals and events in which both host-associated and parasite-associated factors are essential participants. Treatment of worm-free sheep with the corticosteroid, dexamethasone, during infection demonstrated the existence of an arrest-prone state in infective larvae and a host-associated influence actuating the phenomenon of arrest. Not all subpopulations of the parasite responded to the influence of dexamethasone and a selection experiment in which the parasite was passaged through immune and non-immune sheep showed that the arrest-prone state was largely determined by genetic factors. The host-associated factor initiating or actuating arrest and identified by dexamethasone-treatment could be ascribed more satisfactorily to a manifestation of immune responsiveness than to a direct effect of the corticosteroid.