Adult Ascaris Suum Research Articles

Biochemical characterisation of the enzyme responsible for “activated l-serine sulphydrase” activity in nematodes

The biochemical properties of the enzyme responsible for nematode “activated l-serine sulphydrase” activity ( l-cysteine + R-SH → cysteine thioether + H 2S) have been investigated using primarily the gastro-intestinal nematodes Nippostrongylus brasiliensis and Haemonchus contortus. The activated l-serine sulphydrase enzyme was found to be cytosolic in origin and exhibited maximal activity at pH 9.0. Enzyme activity was widely distributed amongst the major tissues of adult female Ascaris suum but was particularly abundant in longitudinal muscle. The enzyme appeared to have a rigid specificity for l-cysteine as the primary thiol substrate, but was capable of utilising a number of sulphur amino acids (and derivatives) and nonphysiological thiols as second substrates. The best second thiol substrates were nonphysiological, hydroxyl-containing thiols that showed some structural similarity to the standard second substrate, 2-mercaptoethanol. Kinetic analyses revealed that the enzyme operates by a sequential catalytic mechanism, and the absolute Michaelis constants were: K l-cysteine = 0.21 ± 0.02 m M and K 2-mercaptoethanol = 5.58 ± 0.59 m M. The enzyme was relatively insensitive to inhibition by a variety of substrate analogues and known inhibitors of pyridoxal 5′-phosphate dependent enzymes, whilst plant phenols caused significant levels of inhibition. The most potent inhibitors discovered were the anthelmintics bithionol, dichlorophene and hexachlorophene. Further characterisation revealed that hexachlorophene was a parabolic competitive inhibitor of the activated l-serine sulphydrase enzyme.

The onset and recovery from airway hyperresponsiveness: relationship with inflammatory cell infiltrates and release of cytotoxic granule proteins.

Previous studies from our laboratory have demonstrated a temporal relationship between eosinophil influx into the airways and the onset of airway hyperresponsiveness to inhaled methacholine. The purpose of the present study was to extend this observation by evaluating changes in airway cellular composition and measuring the levels of granulocyte-derived mediators recovered in BAL fluid during the onset and recovery from antigen-induced airway hyperresponsiveness. Airway cellular composition, airway responsiveness to inhaled methacholine and the levels of BAL fluid EPO and MPO were monitored over a 32 day study in eight adult male Ascaris suum sensitive cynomolgus monkeys. Repeated Ascaris suum inhalation (nine challenges during days 0-21) resulted in a selective, sustained airway eosinophilia that was temporally related with the onset and maintenance of airway hyperresponsiveness (r = 0.67, P less than 0.001). The level of BAL eosinophil-derived EPO was increased and remained elevated concurrent with the increase in airway eosinophils and airway responsiveness. During the recovery phase (days 22-32) the actual number of eosinophils remained elevated, while BAL EPO levels were significantly decreased. The recovery phase was also associated with a transient increase in the number of BAL neutrophils and MPO concentration. We conclude that the number and state of activation of airway eosinophils directly correlate with the onset and maintenance of airway hyperresponsiveness. Recovery from airway hyperresponsiveness is associated with a decrease in eosinophil activation and a transient increase in the number of activated neutrophils.

Isolation of Ascaris suum components which suppress IgE antibody responses.

An extract from adult Ascaris suum was fractionated on a Sephadex G-200 column and individual elution fractions assessed for their ability to induce or suppress an IgE antibody response in mice immunized simultaneously with a heterologous antigen (ovalbumin). The results showed that, whilst fractions eluted in the first peak suppressed the anti-ovalbumin IgE antibody production, those eluted in the third peak induced a significant anti-Ascaris IgE response. The mixture of the two kinds of fractions resulted in suppression of anti-Ascaris IgE antibodies. The apparent molecular weight (MW) of the first and third peaks was 530,000 and 29,000 daltons, respectively. After delipidation, the extract still retained most of its biological activities. SDS-PAGE of this material showed selective loss of high-MW components when compared to the whole extract. No bands were observed in SDS-PAGE of peak I in contrast to peak III, which displayed several bands. Immunoblots of all these samples showed at least 3 bands above MW 150,000, which reacted with anti-peak I antiserum, in the unfractionated extracts and in peak I, but not in peak III. When the extract was fractionated by affinity chromatography using anti-peak I antibodies as ligands, the immunogenic components were present in the effluent, whereas the suppressive components were recovered in the eluate.

Distribution of a fluorescent ivermectin probe, bodipy ivermectin, in tissues of the nematode parasite Ascaris suum.

A fluorescent derivative of the anthelmintic ivermectin (4''-5,7-dimethyl bodipy proprionylivermectin, referred to hereafter as bodipy ivermectin) was synthesized for an investigation of the distribution of avermectins. Injected into adult Ascaris suum at doses greater than 0.1 micron per worm, it produced a dose-dependent immobilization. Fluorescent microscopy of frozen sections revealed the distribution of the probe in the whole nematode. Staining of collagenase-isolated muscle cells was studied using bath application of bodipy ivermectin. The trypan-blue quenching technique showed that the ivermectin probe was located in the outer monolayer of the muscle membrane. The cytoplasm was not stained. The interpretation of these observations is discussed in view of the known lipophilic nature of avermectins. Staining of the muscle membrane and nerve cord is consistent with the view that avermectins act at these sites. The significance of the hypodermal and lateral line staining is also discussed.

Analysis of the cuticular collagens of Ascaris suum

The nematode cuticle is an extracellular structure composed mainly of collagens, with an insoluble epicuticle on the surface. The extracted collagens from adult Ascaris suum can be separated by SDS-PAGE into three major groups of polypeptides with apparent molecular masses of 34, 60–70 and 120–140 kDa. Densitometric evaluation of the polypeptide bands indicated that the three groups are present in the ratio of 1:2:6. Rotary shadowing of reduced, extracted molecules showed fibers 45 nm in length. This length is in excellent agreement with the calculated total length of amino acids in (Gly-X-Y) regions deduced from the collagen gene sequence of Caenorhabditis elegans and A. suum. It is proposed that the three groups of collagen polypeptides found in SDS-PAGE correspond to collagen monomers, dimers and trimers, and that the molecules in the dimeric and trimeric forms are cross-linked via non-reducible bonds.

Antibodies against the cuticlin of Ascaris suum cross-react with epicuticular structures of filarial parasites

The insoluble cuticlin from the cortical zone of the cuticle of adult Ascaris suum was purified and used to raise antibodies in C57/bl mice. The specificity of the antibodies for the external cortical layer was shown in the indirect immunofluorescence antibody test and by immunoelectronmicroscopy. A very high specificity for the external cortical layer was found. Some cross-reactions with cuticular collagens occurred, and increased after booster immunizations. The anti-cuticlin antibody cross-reacted with the electron-dense layers of the cortical zones of adult Acanthocheilonema viteae and Brugia pahangi. A very weak reaction was found in the cortical zone of adult Onchocerca volvulus. In the cuticles of third stage larvae of all three species mainly epitopes in the cortical zones were labelled. In no case did the anti-cuticlin antibody interact with the outermost surface of the cuticle.

Biotin as a probe of the surface of Ascaris suum developmental stages

Sulfo-NHS-biotin (aqueous soluble) and NHS-biotin (organic soluble) labeled similar SDS-2ME (sodium dodecyl sulfate/β-mercaptoethanol) soluble cuticular proteins of second stage larvae (L2) and third stage Ascaris suum larvae (L3). Comparable analysis of biotin-labeled fourth stage larvae (L4), young adults, and mature adult Ascaris suum revealed strong labeling of several SDS-2ME soluble cuticular proteins with NHS-biotin, while sulfo-NHS-biotin appeared to strongly label a single SDS-2ME soluble cuticular protein. Both biotin probes labeled only cuticular proteins, since no evidence of internal labeling was observed in any developmental stage examined by either electroblot analysis or by electron microscopy. Our data suggest a greater cuticular permeability to the organic soluble biotin reagent in the later developmental stages (>L3) of A. suum than to the aqueous soluble biotin reagent, and may indicate the presence of a hydrophobic barrier in the cuticle of the later stages of the parasite.

Identification of the major Ascaris allergen and its purification to homogeneity by high-performance liquid chromatography

The major allergen from the body fluid of adult Ascaris suum (ABF) has been identified and purified to homogeneity using gel permeation high-performance liquid chromatography (HPLC). The purity of the Ascaris body fluid allergen (ABA-1) was confirmed by HPLC and SDS-PAGE. ABA-1 appears to be a 25-kDa dimer in its native form, which runs as a 10-kDa monomer on SDS-PAGE under reducing conditions. The allergenicity of the HPLC-purified protein was confirmed using isolated in vivo-sensitised mast cells from infected rats in an in vitro histamine release assay. ABA-1 is responsible for >80% of the allergenicity of ABF in this sensitive and specific system. Amino acid composition analysis and N-terminal amino acid sequencing were performed on ABA-1. Comparisons are made between ABA-1 and some of the heterogeneous Ascaris allergen preparations previously published. It is suggested that Asc-1 and allergen A both contain ABA-1 in large quantities and that discrepancies in the literature result from contaminating proteins in these preparations and technical differences in characterization of the predominant molecules present in the preparations. Compositional data suggests that the ABA-1 monomer is a molecule of 94 amino acids (based on a molecular weight estimate of 10 kDa) with a composition resembling that previously published for allergen A. The first 10 amino acids are identical to those of a protein affinity purified from the body fluid of Ascaris suum at the Wellcome Laboratories for Experimental Parasitology (WLEP-14K). The similarity between ABA-1 and WLEP-14k is also apparent on SDS-PAGE. ABA-1 appears also to be the major protein component in the body fluid of a viable Ascaris lumbricoides isolated from a hospital patient.

Serotonin receptors in the tissues of adult Ascaris suum

Serotonin (5-hydroxytryptamine, 5-HT) receptors in the muscle and intestinal tissues of adult Ascaris suum have been investigated. [ 3H] lysergic acid diethylamide (LSD) exhibited specific and saturable binding to membranes prepared from both intestine and muscle. The intestinal tissue membranes had an equilibrium dissociation constant ( K d) of 2.70 nM for LSD and a K d of 2.50 μM for 5-HT. As compared to the intestine, the muscle membranes had comparatively higher affinity for both LSD ( K d = 1.80 nM) and 5-HT ( K d = 0.68 μM). The muscle membranes also had a high binding affinity for ketanserin, a 5-HT 2 antagonist, ( K d = 16.7 nM) whereas intestinal membranes exhibited no specific binding of ketanserin. Serotonin significantly inhibited the binding of LSD to the intestinal and muscle tissue membranes while adrenergic and cholinergic drugs and histamine did not. This suggested that the binding of LSD, 5-HT and ketanserin to the parasite membranes was specific. Collectively, the data demonstrated the presence of a serotonin receptor in the muscle and intestinal tissues of the adult A. suum. The receptor in the muscle was pharmacologically similar to the mammalian serotonin type 2 receptor.

Monoclonal antibodies specific for Onchocerca volvulus as determined by immunofluorescence

Nogami S., Hayashi Y., Korenaga M., Tada I. and tanaka H. 1988. Monoclonal antibodies specific for Onchocerca volvulus as determined by immunofluorescence. International Journal for Parasitology 18: 503–507. A total of four murine monoclonal antibodies (mAb) was determined to be specific for Onchocerca volvulus when examined by immunofluorescence. Adult Ascaris suum as antigen was used to eliminate cross-reactive mAb during screening of clonal products from mice immunized with O. volvulus extracts. The selected mAbs were further examined for specificity using 13 species of filarial and non-filarial helminths. Four mAbs, non-reactive to A. suum, reacted to O. volvulus alone but did not react to the other 12 helminths, suggesting that A. suum may be an effective tool for selecting O. volvulus-specific mAb. The isotypes of these four mAbs were all IgG; i.e. three were of the IgG1 subclass and one was IgG2a. Worm antigen sites bound by these specific mAbs were all in the intestinal tissue of adult O. volvulus. Analysis with polyacrylamide slab gel electrophoresis in sodium dodecylsulfate (SDS-PAGE) and immunoblotting revealed that an epitope specific for O. volvulus exists on a M r 27,000 polypeptide.

Tryptophan hydroxylase and aromatic l-amino acid decarboxylase activities in the tissues of adult Ascaris suum

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to modulate carbohydrate metabolism in adult female Ascaris suum. Two enzymes, l-tryptophan hydroxylase (TOH) and aromatic l-amino acid decarboxylase (AADC), which are responsible for the biosynthesis of 5-HT in mammalian systems, were demonstrated in isolated muscle and intestinal tissue of adult female A. suum. The specific activities of TOH in muscle and intestine were 0.22 nmol min −1 g −1 and 1.48 nmol min −1 g −1, respectively. The apparent Michaelis-Menten constant ( Km) and maximum initial velocity ( V max) of TOH for l-tryptophan (TRP) in the intestine were found to be 21.50 μm and 2.30 nmol min −1 g −1, respectively. The specific activities of AADC in the isolated muscle and intestinal tissues were 1.64 nmol mm −1 g −1 and 5.72 nmol min −1 g −1, respectively. The apparent Km and V max of the muscle AADC for l-5-hydroxy-tryptophan (5-HTP) were 10 μM and 3.40 nmol min −1 g −1. The apparent Km and V max of the intestinal AADC for 5-HTP were 1.08 μ M and 14.50 nmol min −1 g −1, respectively. The apparent inhibitor constant ( Ki) of TOH for p-chlorophenylalanine, an inhibitor of mammalian TOH activity, in isolated intestine of A. suum was 1.72 mM. The TOH activity from A. suum intestine was inactivated upon exposure to oxygen and this inactivation could be partially reversed by anaerobic incubation in the presence of dithiothreitol and ferrous sulfide at 25 ° C. Collectively, the data demonstrated the presence of TOH and AADC activities in adult A. suum isolated muscle and intestinal tissues.

Evidence for the absorption and synthesis of 5-hydroxytryptamine in perfused muscle and intestinal tissue and whole worms of adult Ascaris suum

The metabolites of 5-hydroxytryptamine (5-HT, serotonin) namely, L-tryptophan, 5-hydroxytryptophan, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were measured in perfused tissue and whole worms from adult female Ascaris suum using reversed-phase liquid chromatography with electrochemical detection. The intracellular levels of each metabolite were quantitated in response to several physiological effectors but only L-tryptophan (TRP) caused dose-dependent changes in these metabolites. Serotonin itself could also be absorbed by perfused A. suum muscle and intestinal tissue. When live A. suum were tied at the anterior and posterior regions to restrict TRP absorption by the intestine, TRP was absorbed through the cuticle and converted into 5-HT by the muscle tissue. In united live parasites TRP absorption was observed in both muscle and intestinal tissue. Collectively, the data indicated that 5-HT may be either absorbed directly or synthesized de novo from absorbed TRP in the isolated tissue of A. suum. The 5-HT, in the adult female A. suum, can be synthesized de novo from TRP, or 5-HT can be absorbed from the environment both through the cuticle and by the intestine of living parasites. Data also indicated that there was preferential sequestering of 5-HT and the metabolites of 5-HT in the anterior tissues of the worms.

Serotonin (5-hydroxytryptamine) turnover in adult female Ascaris suum tissue

1. 1. The 5-hydroxytryptamine (5-HT, serotonin) turnover was examined in the tissues of adult female Ascaris suum. The 5-HT turnover was highest in the intestine at 34.7 ng 5-HT produced/mg protein/hr and 13.8 ng 5-HT produced/mg protein/hr in muscle tissue. 2. 2. The levels of 5-HT metabolites namely tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were measured in muscle and intestinal tissue of adult A. suum. 3. 3. Parachlorophenylalanine inhibited 5-HT production in muscle and intestinal tissue providing in situ evidence for the presence of tryptophan hydroxylase in this tissue. 4. 4. Pargyline increased 5-HT production in muscle and intestinal tissue providing in situ evidence for the presence of monoamine oxidase in this tissue.

Ascaris suum: Partial isolation and characterization of hypodermis from the adult female

A method was developed to remove the muscle from body wall strips of adult female Ascaris suum resulting in a hypodermis cuticle preparation. Optimum treatment for obtaining the hypodermis cuticle was a 15 min incubation with trypsin (2.0 mg/ml) at room temperature, followed by mechanical removal of the muscle. The hypodermis cuticle prepared in this manner incorporated radiolabeled amino acids into cuticular and hypodermal proteins; incorporation was inhibited by protein synthesis inhibitors. Characterization of the hypodermal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the hypodermis apparently contains proteins that differ from those of the cuticle and that the hypodermis of adult A. suum appears to lack cuticle protein precursors. This result will now allow detailed biochemical and physiological investigations of the hypodermis, a tissue which is critical for cuticle synthesis.

Immunochemical characterization of the pyruvate dehydrogenase complex in adult Ascaris suum and its developing larvae

Polyclonal antibody was prepared against the pyruvate dehydrogenase complex purified from adult Ascaris suum body wall muscle. The antibody reacted with the E2, X, αE1 and βE1 subunits of the complex in immunoblots of mitochondrial supernatant fractions and homogenates of adult muscle. In addition, the same subunits were observed in immunoblots of homogenates of L3 and L4 ascarid larvae, suggesting that a similar enzyme complex was present in all developmental stages despite their marked differences in energy metabolism. The phosphorylated and dephosphorylated αE1 peptides migrated differently during sodium dodecylsulfate polyacrylamide gel electrophoresis and both forms of the enzyme were recognized by the antibody. These results and those obtained with ELISA suggest that both phosphorylated and dephosphorylated forms of the αE1 subunit react equally well with the antibody. In immunoblots of adult body wall muscle, the phosphorylated αE1 peptide predominated, while immunoblots of L3 larvae contained predominantly the dephosphorylated form. These results reflect the in vivo activity state of the pyruvate dehydrogenase complex in these two stages and suggest that this technique may be useful for determining the activity state of enzyme complex directly from immunoblots of homogenates A. suum and other helminths.

Ascaris suum: Role of ecdysteroids in molting

Three studies were conducted to examine the function of ecdysteroids in the development of parasitic nematodes. Ecdysone and 20-hydroxyecdysone were extracted, separated chromatographically, and measured in the reproductive tracts of adult female Ascaris suum. Perienteric fluid and the body wall did not contain measurable levels of these steroids. Levels of 20-hydroxyecdysone were correlated with the third and fourth molts of larvae grown in vitro from the third stage. In a bioassay, addition of ecdysteroid extracted from the female reproductive tract or synthetic ecdysteroid increased the proportion of third-stage larvae that molted after 4 days in culture. This evidence supports the role of ecdysteroids in molting in A. suum, as well as suggesting a function in gametogenesis and embryogenesis.

Ascaris suum: Biosynthesis and isoinhibitor profile of chymotrypsin/elastase isoinhibitors

Chymotrypsin/elastase isoinhibitors were radiolabeled when live, adult Ascaris suum were incubated in tissue culture medium (NCTC-135) supplemented with l-[ 35S]cysteine. This is the first demonstration that the synthesis of these proteins occurs in A. suum; the isoinhibitors are not host products utilized by the parasite against its host. Of five chymotrypsin/ elastase isoinhibitors demonstrable in A. suum, only isoinhibitors 1, 4, and 5 were found in each worm. The amino acid sequences of these three isoinhibitors indicate that they are gene products and are not obtained by modification after translation. The two inhibitors that are not observed could arise by limited proteolysis. The same chymotrypsin/elastase isoinhibitor profile present in each nematode eliminates any speculation that the multiple forms arise from an adaptation between A. suum and its host. A new chymotrypsin/elastase isoinhibitor nomenclature is proposed, so that isoinhibitor 1 is now Isoinhibitor A, and isoinhibitors 4 and 5 are now Isoinhibitors B and C, respectively.

Studies on the comparative migration patterns of Ascaris suum larvae between primary and re-infected mice

In the present study, the effect of primary infection to reinfection with Ascaris suum larvae was experimented in mouse model. Mice were challenged with l,000 infective stage eggs of Ascaris suum. The embryonated eggs were directly introduced into stomach of mice. Reinfection was performed at 50 days after the primary infection with same method as primary infection. Mice were sacrificed 3, 5, 7, 10, 15 and 20 days after infection in both groups respectively. Larvae collected from livers and lungs with Baermann's apparatus were enumerated and measured after sacrifice. Sera of mice were also collected at same time. The results of the experiment were as follows: With antigen prepared from coelomic fluid of adult Ascaris suum and sera collected from mice before reinfection, the production of antibody in experimental mice was confirmed by the gel-diffusion technique. In the livers of reinfected mice, the larvae were recovered up to 10 days after challenge, otherwhile in the primary infected mice, the larvae were observed up to 7 days. The maximum number of larvae were observed in the lungs of primary infected mice on 10 days after inoculation. In the lungs of reinfected mice, maximum number of larvae were recovered on 7 days after, only few larvae were recovered on 10 days after reinfection. As regards the growth of the larvae, the third stage larvae, over 500 micrometer in length, appeared in livers at 5 days after reinfection, but it couldn't be found on 7 days and 10 days after challenge. The third stage larvae continuously developed were observed in lungs of mice from 5 days after reinfection. In conclusion, it was found that development of larvae in livers of immune mice were probably repressed by the immune mechanisms being rised in livers and defence mechanism is also acting by interfering with the process of larval penetration into the lung from the liver.

Ascaris suum: Continuous perfusion of the pseudocoelom and nutrient absorption

A double-cannulation apparatus was constructed for continuous perfusion of the pseudocoelom of adult Ascaris suum while maintaining the intact parasite in a controlled incubation chamber. Peristaltic pumps maintained a constant flow rate of artificial perienteric fluid through the incubation chamber (1 ml/min) and through the parasite (100 μl/min). Based on protein determinations, perienteric fluid was removed from the pseudocoelom within 35 min of initiation of perfusion (3.5 ml). A nonabsorbable dye, Blue Dextran, was detected first in the perfusate 4 min (400 μl) after initiation of infusion into the pseudocoelom, and was maintained at a constant concentration in the perfusate by 8 min after initiation of dye infusion. Removal of the dye from the pseudocoelom was accomplished within 8 min (800 μl) after the cessation of dye infusion. Occlusion of the digestive tract had no effect ( P < 0.05) on the short-term (3 hr) absorption of 3H-labeled cholesterol, [ 14C]-3- o-methylglucose or [ 14C]glucose from the incubation medium into the perienteric cavity. Concentrations of isotopes in the pseudocoelom reached steady-state levels within 60 min of the initiation of incubation, but remained low (>0.5%) when compared to medium concentration. Similarly, the time course of the accumulation of [ 14C]glucose into individual tissue components did not differ in intact worms with or without patent intestinal tracts. Thus, the cuticular/muscle tissue largely appears to be the primary route of absorption of cholesterol and glucose in adult A. suum. The cannulation and perfusion procedure for A. suum is an adaptable system for continual or intermittent measurements of internal physiological processes, in particular, the absorption and metabolism of nutrients. Furthermore, this system permits simple modification for “closed-loop” perfusion or internal delivery of select compounds.

Antigens in perienteric fluid of Ascaris suum as detected through antibodies in pigs orally inoculated with fully embryonated eggs

Perienteric fluid (Pf) of adult Ascaris suum was fractionated by ammonium sulfate precipitation, gel filtration or DEAE-cellulose chromatography, and sucrose density gradient centrifugation. Anti-sera (anti-EE) from pigs which were inoculated orally with fully embryonated eggs (EE) of A. suum were used in an indirect radioimmunoassay (IRIA) to determine which fractions of Pf reacted positively in the analyses at the lowest protein concentration. These fractions were considered to contain more potent antigens. Comparative IRIA were performed employing antisera (anti-Pf) produced by injecting Pf into pigs. Six out of 35 fractions reacted positively at ⩽ 0.2 μg protein when anti-EE was used in the IRIA. Twenty-two out of 35 fractions reacted positively at ⩽ 0.2 μg protein when anti-Pf was used. Five of the 6 fractions reacting positively with anti-EE also reacted with anti-Pf. A 76 000 dalton component appears to be one of the major proteins in the 6 fractions which react positively with anti-EE while components from 10 000–138 000 daltons were present in the various fractions reacting positively with anti-Pf at ⩽ 0.2 μg protein.