Bactericidal activity in the pig roundworm Ascaris suum

A potent, humoral, bactericidal activity against Micrococcus luteus was discovered in pseudocoelomic fluid of the pig roundworm, Ascaris suum. The activity, which was not bacteriolytic, was not due to lysozyme or to a dietary antibiotic. It was not inactivated by exposure to 100°C, to low or high pH, or to ethanol. Dialysis, electrophoresis and agar-diffusion experiments suggested that the main antibacterial activity in the fluid was associated with a basic substance of molecular weight somewhat less than 14000 Da. Two other Gram-positive organisms, Bacillus megaterium and Staphylococcus aureus , were also killed by the Ascaris fluid, but the Gram-negative Escherichia coli, Proteus vulgaris and Bordetella bronchiseptica were insensitive.     

Ascaris suum: specific antibodies in isolated intestinal loop washings from immunized swine.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.     

Survival and viability of Ascaris suum and Oesophagostomum dentatum in ensiled swine faeces

The survival and viability of eggs from Ascaris suum and Oesophagostomum dentatum and of infective larvae (L3) from O. dentatum were determined in the ensiled solid fraction of swine faeces after 0, 7, 14, 28 and 56 days of ensiling. The experiment had two treatments, un-ensiled and ensiled manure, in a split-plot design. Each of 50 containers was inoculated with 40,000 eggs of both A. suum and O. dentatum, and another 50 containers were inoculated with 32,747 L3 of O. dentatum each. A. suum eggs were not destroyed by the ensiling process, although their viability was diminished. O. dentatum eggs and larvae were destroyed during the first 7-14 days of the ensiling process.     

Phosphofructokinase from Ascaris suum. Purification and properties

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.     

Ascaris suum: development of intestinal immunity to infective second-stage larvae in swine

The development of protective immunity to Ascaris suum was examined in pigs naturally exposed to eggs on a contaminated dirt lot. Pigs became almost totally immune to second-stage larvae migrating from the intestines because few larvae from a challenge inoculum could be found in the lungs, and liver white-spot lesions (an immunopathologic response to migrating larvae) were absent. Blood from these pigs contained lymphocytes that responded blastogenically to larval antigens in vitro, while the serum contained antibody to larval antigens. Immunity was related to parasite exposure and not to the age of the host, and was not affected by the removal of adult A. suum from the intestines. Naturally exposed pigs responded to a variety of A. suum antigens with an immediate-type skin reactivity, and their intestinal mucosa contained relatively large numbers of mast cells and eosinophils. Other pigs were maintained on a dirt lot not contaminated with A. suum eggs and the effects of common environmental conditions on development of resistance to A. suum were studied. Resistance also developed in these pigs because 72% fewer larvae were detected in their lungs following a challenge exposure than in control pigs confined indoors on concrete floors and challenged similarly. This response was not expressed at the intestinal level, however, because their livers had numerous, intense white-spot lesions. To verify that the intestinal immunity that developed in pigs after natural exposure to A. suum was a direct result of homologous infection and not related to other stimuli encountered on a dirt lot, pigs maintained indoors on concrete floors, free from inadvertent helminthic infection, were inoculated orally with A. suum eggs daily for 16 weeks. Intestinal immunity was induced because larvae from a challenge inoculum were not detected in the lungs, and few white-spot lesions appeared on the livers of these pigs. Apparently, continual exposure of the intestinal mucosa to larvae eventually elicits the appropriate effector components necessary to prevent larval migration from the intestines.     

Carboxypeptidase inhibitors from Ascaris suum: the primary structure

The carboxypeptidase A inhibitor from Ascaris suum was isolated from aqueous extracts by affinity chromatography toward immobilized carboxypeptidase A. The amino acid sequence is DQVRKCLSDT10DCTNGEKCVQ20KNKICSTIVE30IQRCEKEHFT40IPCKSNNDCQ50VWAHEKICN K60LPWGL65 . The carboxypeptidase A inhibitor is not homologous with the chymotrypsin/elastase or trypsin inhibitors from Ascaris, but shows homology in a 9-residue internal sequence with the 37/39-residue carboxypeptidase inhibitors from tomato and potato. The carboxy-terminal 5 (4) residues in the three inhibitors are similar, suggesting a common mechanism of inhibition.     

Purification of an anticoagulant from the body fluid of Ascaris suum

An anticoagulant has been purified from the body fluid of Ascaris suum by sequential passage through Sephadex G-50, CM-cellulose and Sephadex G-25 columns then treated with 2 M NaCl, passaged through a Sephadex G-25 column, separated from the phosphate buffer by precipitation of the latter with the CaCl2, then passaged through a Sephadex G-10 column in water. In the body fluid of the worm, the anticoagulant is ionically-bound to a carrier substance. The complex can be split by treatment with 2 M NaCl. The molecular weight of the anticoagulant is slightly less than 1400.     

Purification and characterization of phosphoenolpyruvate carboxykinase from the parasitic helminth Ascaris suum

Phosphoenolpyruvate carboxykinase has been purified from homogenates of Ascaris suum muscle strips to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification is a three-step procedure which yields pure enzyme in milligram quantities with good yield. The subunit molecular weight of the Ascaris enzyme is between 75,000 and 80,000. The native molecular weight is 83,000 as determined by gel filtration. The kinetic constants for substrates of the carboxylation reaction were determined and compared to those measured for the avian liver enzyme. From kinetic studies it appears likely that two separate roles for divalent metal ions exist in the catalytic process. Studies conducted with Mn2+ or with micromolar concentrations of Mn2+, in the presence of millimolar concentrations of Mg2+ suggest that Mn2+ but not Mg2+ binds directly to and activates the enzyme while either Mn2+ or Mg2+ may bind to the nucleotide resulting in the metal-nucleotide complex. The metal-nucleotide is the active form of the substrate for the reaction. In the presence of Mg2+, an increase in the Mn2+ concentration results in a decrease in the Km for P-enolpyruvate suggesting a direct role for Mn2+ stimulation and regulation of activity. The concentrations of Mn2+ and Mg2+ in Ascaris muscle strips were determined by atomic absorption spectroscopy and support the proposed hypothesis of a specific Mn2+ activation of the enzyme. The nucleotides ATP and ITP act as competitive inhibitors against GTP with KI values of 0.50 and 0.75 mM, respectively. ITP is a competitive inhibitor against both IDP and P-enolpyruvate, suggesting overlapping binding sites for the two substrates on the enzyme.     

Isoenzymatic pattern and structure of glutamate dehydrogenase from Ascaris suum.

The isoenzymatic pattern of glutamate dehydrogenase (GDH) has been described for Ascaris suum a parasite of Sus scrofa domestica. Only one band of activity has been revealed, suggesting a monomorphic condition for this enzyme. Also, the structure of GDH has been assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Only one subunit was present with a molecular weight of about 55,000. A hexameric structure for GDH of A. suum is suggested.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.