Bile and bile salts and exsheathment of the intestinal nematodes Trichostrongylus colubriformis and Nematodirus battus

1972. Bile and bile salts and exsheathment of the intestinal nematodes Trichostrongylus colubriformis and Nematodirus battus. International Journal for Parasitology, 2: 433–438. Exsheathment of T. colubriformis was potentiated at physiological levels of CO₂ by bile and bile salts. Lamb bile and crude sodium taurocholate preparations produced the greatest increase in exsheathment while rabbit bile, sodium glycocholate and deoxycholate had less pronounced effects. Exsheathment in bile and taurocholate occurred at a pH above 4 and it is therefore suggested that infective larvae which fail to exsheath in the abomasum could well do so in the proximal part of the small intestine. Pure sodium taurocholate was found to greatly potentiate exsheathment of N. battus in vitro but this occurred at a pH below 3 and thus the action of this bile salt could not affect exsheathment in the host.     

Organic and inorganic acids as the stimulus for exsheathment of infective juveniles of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1986. Organic and inorganic acids as the stimulus for exsheathment of infective juveniles of nematodes. International Journal for Parasitology 16: 163–168. A variety of organic and inorganic acids stimulated exsheathment of infective juveniles of Nematospiroides dubius over the range pH 2–7. Activity, in relation to pK values, suggests that the undissociated form of the acid was the active agent. Under normal conditions of exposure, exsheathment of infective juveniles of Haemonchus contortus was not obtained below pH 6, and at higher pH values activity was low except in the presence of CO₂ at pressures >9-5 kPa in the gas phase. Brief exposure to HCl at concentrations up to 2N did not induce exsheathment but in the presence of CO₂ in the gas phase, 95.3 kPa, activity was obtained in 0-O1N HCl when the times of exposure were 3–25 min.The differences in the responses of N. dubius and H. contortus to a stimulus at low pH values may be attributed to the stability of the exsheathing enzyme of N. dubius in acid media as well as to the relative sensitivity of the two species to stimuli generally.     

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.     

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.     

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.     

Leucine aminopeptidase in exsheathing fluid of North American and Australian Haemonchus contortus

Rogers W. P. and Brooks F. 1978. Leucine aminopeptidase in exsheathing fluid of north American and Australian Haemonchus contortus. International Journal for Parasitology8: 55–58. Juveniles of Haemonchus contortus from north America and Australia produced exsheathing fluid containing leucine aminopeptidase when stimulated in tetraborate-carbon dioxide medium. Exsheathment in this medium was inhibited by 1, 10-phenanthroline, 10^?3M, and this inhibition was largely reversed by Zn²⁺, 10^?3M. This supports the view that the enzyme is produced by the juveniles and that it is concerned in exsheathment.     

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.     

Skin penetration by ensheathed third-stage infective larvae of Necator americanus, and the host's immune response to larval antigens.

In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator americanus. The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.     

Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus

Davey K. G. and Sommerville R. I. 1982. Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus. International Journal for Parasitology12: 503–507. Changes in the optical path difference (opd) between various parts of the worm and the medium in which the worms were immersed were determined by quantitative interference microscopy. The opd of the oesophagus and the excretory cells both increased upon stimulation of the worms with CO₂ at 38.5°C, suggesting a decrease in volume of those structures. The oesophagus decreased markedly in length and slightly in diameter, yielding a decrease in volume of approximately 15 to 17 pl. Desheathing the worms with NaOCl produced changes in the oesophagus but not the excretory cells. This confirms previous findings that exsheathment involves at least two parallel processes, both of which are initiated by CO₂ and only one of which is stimulated by exposure to NaOCl.     

Nematospiroides dubius: exsheathment of infective juveniles

When infective juveniles of Nematospiroides dubius exsheath, the cuticle fractures 25 μm from the anterior end. Most infective juveniles exsheath within 5 min in the stomach of the mouse. Juveniles exsheath as readily in vitro in 0.85% sodium chloride at pH 2–5, 36–37 C, under a gas phase of 40% carbon dioxide in air. Exsheathment also takes place under air after passage through potassium hydroxide, or under nitrogen, but is enhanced when carbon dioxide is added. Reducing agents have no effect.     

Undissociated bases as the stimulus for the development of early parasitic stages of nematodes

Petronijevic T. and Rogers W. P. 1987. Undissociated bases as the stimulus for the development of early parasitic stages of nematodes. International Journal for Parasitology 17 :911–915. The effects of NH₄Cl and NH₂CH₃ on infective stages of Haemonchus contortus, Nematospiroides dubius and Ascaris suum have been compared with the action of H₂CO₃. Detailed experiments were carried out with H. contortus. NH₄Cl at pH 8 under air was less toxic to infective stages than it was to free-living juveniles of Panagrellus redivivus. The induction of exsheathment or hatching by bases was slow (20–30 h) though the time of development of 4th stage H. contortus was not proportionally increased. Activity at pH 6 was less than that at pH 8. In contrast to the action of H₂CO₃ as the stimulus, NH₄Cl was not Ca²⁺-dependent. Prolonged exposure to anoxia at pH 8 was toxic, but in the presence of NH₄Cl or H₂CO₃ toxicity was decreased. The inhibition of exsheathment due to Dnp at pH 7 was greater when NH₄Cl was the stimulus.The process whereby host signals induce development of parasitic stages is discussed.     

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.     

Cultivation of Haemonchus contortus (Nematoda: Trichostrongylidae) from infective larvae to the adult male and the egg-laying female

The parasitic nematode Haemonchus contortus was cultured in vitro to the adult male and the egg-laying female. Gastric contents from 2 cannulated calves and 2 sheep as well as extracts of stomach mucosa from both hosts were added to a mixture of API-1 culture medium and Fildes' reagent (a pepsin digest of defibrinated bovine blood) adjusted to pH 6.4 for the first week and pH 6.8 thereafter. The gas phase was 85% N2:5% O2:10% CO2. Best results were obtained when API-1 culture medium plus Fildes' reagent was supplemented with ovine gastric contents. Adult males up to 10 mm were obtained, and they produced and stored sperm at about 28 days in culture. Spermatozoa were never seen in the uteri of females grown in vitro. After 36 days in culture, female worms up to 16 mm were obtained which produced and layed eggs. Some of the eggs underwent division up to the 8-cell stage but did not develop further. No live larvae were observed at a stage earlier than the fourth. Presumably, this was because no matings were observed, although large clumps of worms did occur in the culture medium and mating might have occurred. The growth of adults from young to mature phases of this stage included: for the female, oogenesis and laying of eggs; for the male, development of paired sclerotized spicules, and spermatogenesis with the production of spermatozoa.     

Response to ivermectin treatment of parasitic stages of Haemonchus contortus resistant or susceptible to ivermectin.

Two groups of 33 helminth-naive lambs were infected with 5,000 L3 of an ivermectin-resistant or -susceptible strain of Haemonchus contortus (groups R and S). On days 6, 10, 16, and 21 postinfection, 5 animals from each group were chosen at random and orally treated with 0.2 mg/kg of ivermectin. On each occasion, 2 randomly selected lambs from each group were also killed to determine the number and stage of development of the worms present at the time of treatment. These necropsies revealed that by day 6 early and late fourth-stage larvae were present, whereas on day 10 the early fifth stage had been reached; by days 16 and 21 all worms had reached the adult stage. Necropsies on day 28 postinfection revealed that although animals treated at day 6 had 26.3% fewer worms than the controls, there was no significant difference (P greater than 0.05) between worm burdens from any of the animals infected with the R strain and treated at different times after infection when compared with the untreated controls. With ivermectin significant reductions were obtained in the worm burdens of the animals infected with the susceptible strain; these were reduced by 96% when treatment was given on day 6 against fourth-stage larvae and 98.9% when the drug was given on day 21 against adult stages. From these results it is clear that resistance to ivermectin in this strain of H. contortus is present as early as the fourth larval stage.     

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.     

几种不同因子对哥伦比亚食道口线虫离体发育的影响

本文研究了二氧化碳、温度、犊血清和鸡胚提取物等对哥伦比亚食道口线虫(O.columbianum)感染性幼虫(L_3)发育至第四期幼虫(L_4)的影响;对培养前后虫体的形态学(着重在头部、口、排泄系统、肠和尾部)变化进行了详细的观察和描述。     

Immunity to Litomosoides carinii in Mastomys natalensis. II. Effects of chemotherapeutically abbreviated and postpatent primary infections on challenges with various stages of the parasite

Naive Mastomys natalensis, Litomosoides carinii-infected M. natalensis at a postpatent stage of the infection and L. carinii-infected M. natalensis treated chemotherapeutically with furazolidone (FUR), FUR and diethylcarbamazine (FUR/DEC) or amoscanate (AMOS) were challenged by either injection or implantation of 40 third stage larvae (L3, s.c.), 40 fourth stage larvae (L4, 16 days old, i.p.), 20 male and 20 female preadult worms (36 days old, i.p.), 12 adult female worms (i.p.) or 6 X 10(6) microfilariae/kg (i.v.). Microfilaraemia in animals challenged at a postpatent stage (independent of the kind of challenge), was either totally suppressed or at least greatly reduced. Necropsy of L3-challenged animals showed that neither the length of the worms nor their content of morphologically intact, intrauterine stages was affected. Infected, treated animals challenged with developing stages (L3, L4 and preadult worms) showed reduced levels of microfilaraemia (by up to 75%). Dissection of AMOS-treated, L3-challenged animals showed that both the developmental rate and the fertility of the worms were affected. Microfilaraemia was also reduced after implantation of adult worms into treated animals. This was independent of the interval between treatment and challenge (44-150 days) except in animals challenged 10 days after AMOS-treatment, which showed no difference from naive controls. However, infected, treated M. natalensis, cotton rats and gerbils did not develop immunity against intravenously injected blood microfilariae.     

Adult worms as a factor in the inhibition of development of Ostertagia ostertagi in the host

The removal, by anthelmintic treatment, of adult worms from calves that had been infected daily for 204 days with larvae of Ostertagia ostertagi, stimulated the resumed development of inhibited early fourth stage larvae. The calves remained susceptible to the establishment of worms and the continued administration of infective larvae after the 204th day reduced the rate at chich the burden of inhibited forms decreased. The relevance of these findings to the regulation of worm burdens is discussed.     

The changes of histopathology and serum anti-sparganum IgG in experimental sparganosis of mice

The present study is intended to observe the chronologic changes of experimental sparganosis by histopathological observation and detection of circulating anti-sparganum IgG antibody using ELISA. Each of 25 mice was infected with five spargana, and they were examined after 1, 2, 4, 10 weeks or 6 months from infection. The followings are summarized results. 1. The plerocercoids were detected in the subcutaneous tissue of the trunk, neck or axilla, but a few often extended into the skeletal muscle. The recovery rates were 72% at the first week, 80% at the second week, 95% at the fourth week, 92% at the tenth week and 100% at the sixth month. The larvae grew slowly in both length and weight until 6 months. 2. Histopathologically, most of the larvae were observed alive in the soft tissue or skeletal muscle. Numerous eosinophils, neutrophils, lymphocytes and plasma cells were infiltrated focally around the worms by the second week, but they surrounded the worms to form a layer of inflammatory reaction after 4 weeks of infection. Also histiocytes and fibroblasts began to appear around the inflammatory cells at 4 weeks. After 10 weeks, the worms encircled by a thin fibrous layer were found. After 6 months, the worms were surrounded by either fibrous tissue or active inflammatory cells. The inflammation looked more severe in the tracks left by the worms, rather than around the worms. 3. The level of anti-sparganum IgG antibody in the serum showed an increase by the fourth week, and a rapid and continuous increase was observed thereafter by the tenth week after infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.