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Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. I. Biochemical characterization and electron transport of Ascaris microsomes.

Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.

Animals

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. II. Purification and characterization of b-type cytochrome and NADH-ferricyanide reductase from Ascaris muscle microsomes.

A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography. The purified b-type cytochrome displayed absorption bands at 560 (alpha-peak), 525 (beta-peak), and 424 nm (gamma-peak), with a marked shoulder at 555 nm in the reduced from, 415 nm (gamma-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5. The molecular weight was estimated to be about 100,000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme. The molecular weight of the purified NADH-FC reductase was estimated to be about 55,000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the membrane-bound reductase, was not inhibited by thiol reagents.

Animals

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.

Adenosine Triphosphatases

Effect of ascaris extract applied intravenously, on segment bronchus and the influence of ipsilateral vagus blockade.

Respiratory hypersensitivity and systemic effects after local application of Ascaris suum extract on the segmental bronchus and after its intravenous infusion are studied on four boxer dogs. Influence of ipsilateral vagus blockade on the respiratory hyperreactivity after intrabronchial application of ascaris was also investigated on the same dogs. Ascaris extract was applied in liquid form directly through a catheter into the right segmental bronchus. Egg albumin was applied in the same way for control. Local application of ascaris was repeated after ipsilateral central vagus blockade and once more after recovering vagus effect by lavage. At the end of the experiments ascaris extract was infused intravenously. The parameters studied were: deltaP(oes) mm Hg/100 ml TV (as a measurement of flow resistance in the airways), respiratory rates, arterial blood gases and pH, heart frequency, systemic blood pressure and histamine concentration per milliliter of plasma. All the animals presented a remarkable respiratory distress with local ascaris application, which could be clearly avoided with ipsilateral central vagus blockade. This respiratory hyperreactivity was not observed after intravenous infusion of ascaris extract, but a significant systemic effect, which was not the case after local application, could be observed.

Airway Resistance

Ascaris lumbricoides and allergic asthma: A new perspective.

Infestation of humans with the parasite Ascaris lumbricoides may induce high total serum IgE levels, but the influence of this immunogenic response on allergic asthma has not been defined. In this study, the specific antiparasitic IgE-mediated response as determined by skin-prick testing was related to the incidence of allergic asthma in Ascaris-infested patients. A limited number--17% of the non-allergic controls and 51% of the allergic asthmatics--had a clinically detectable immunogenic response to the parasite. The predicted incidence of asthma was significantly higher than the observed incidence in the subjects in whom the Ascaris skin test was positive. This was not found in subjects in whom the Ascaris skin test was negative. Inhalation of Ascaris antigen induced asthmatic reactions in 7 of 8 patients who were Ascaris-positive on skin testing, but not in the negative controls. The groups of patients who respond immunogenically to parasite infestation need to be defined, as they may be predisposed to allergic diseases such as asthma.

Adolescent

Elevated levels of IgE antibodies to ascaris and mite antigens in Papua New Guinea.

Levels of IgE as well as specific IgE antibodies to ascaris and mite have been studied in two groups in Papua New Guinea (PNG), patients with primary liver carcinoma (PLC) and normal blood donors, and in two groups in Japan, normal subjects and asthmatic patients. A radioallergosorbent test was used to measure IgE antibodies to ascaris and mite antigens. Much higher levels of IgE were found in the Papua New Guinean subjects than the normal Japanese subjects. Also levels of IgE antibodies to ascaris were much higher in Papua New Guineans than in the Japanese subjects. Though similar results were observed in IgE antibodies to mite, difference between normal Papua New Guineans and normal Japanese was small, whereas difference between the patients with PLC in PNG and the normal Japanese subjects was very large. Highest levels of IgE antibodies to mite were detected in Japanese with asthma. No correlations were observed among IgE levels, levels of IgE antibodies to ascaris and levels of IgE antibodies to mite. It is concluded that both ascaris and mite IgE antibodies contribute the very high levels of IgE seen in Papua New Guineans, but many other factors may be operative.

Antibodies

Haemagglutination tests in the study of Ascaris epidemiology.

Haemagglutination tests using adult Ascaris suum antigen were performed on sera from 810 people from three areas in Papua New Guinea and East Timor. In the area with the highest childhood Ascaris infection rates and egg-counts (Kaul), titres were highest and remained high throughout life. There was a significant negative correlation between titre and eosinophil count, and it is suggested that high titres in adult life result from continuous usually unsuccessful reinfection, and that the declining egg-counts and infection rates with increasing age may be the result of an immune mechanism, and not decreased exposure. In the two other populations the less intense Ascaris infection was reflected in the lower titres, with no correlation between titres, infection or eosinophilia. Although the haemagglutination test as used here is unsuitable as a diagnostic aid, it is valuable in studying the epidemiology of Ascaris infections.

Animals

IgE-type antibodies to Ascaris antigens in man.

IgE antibodies to Ascaris antigens were detected by radioallergosorbent test (RAST) in the serum obtained from a person experimentally sensitized with Ascaris suum antigens prepared from the body fluid of worms. This serum passively sensitized human skin in vitro and histamine release from the sensitized skin was observed by challenge with antigens. The allergenicity of A. suum antigens was present over a very broad spectrum of molecular size. The present results suggest that specific IgE antibodies to Ascaris antigens might be in Ascaris-infected patients and allergic symptoms which belong to type 1 reaction might be mediated by IgE in these patients.

Antibody Formation

Airway sensitivity to slow-reacting substance of anaphylaxis, histamine, and antigen in Ascaris sensitive monkeys.

The effects of Ascaris suum antigen, histamine, and slow-reacting substance of anaphylaxis (SRS-A) on the respiratory system were compared in 3 anesthetized rhesus monkeys. The agents were administered by instillation into the trachea, and the animals were studied in a volume displacement body plethysmograph. Two of the animals showed skin and bronchial sensitivity to Ascaris suum antigen and responded to it with increased pulmonary resistance and decreased dynamic compliance. A similar response was seen in all 3 animals after instillation of histamine, but SRS-A at 2 concentrations produced a predominant effect of decreased dynamic compliance with lesser alterations in pulmonary resistance. The effects of SRS-A were slow in onset and prolonged, as compared to the abrupt and short-lived effects of Ascaris suum antigen and histamine. The predominant effect of SRS-A on dynamic compliance suggests a more peripheral site of action of this mediator. In 5 monkeys allergic to Ascaris, no SRS-A could be detected in the blood at one and 5 min after antigen challenge, using the bioassay techniques.

Administration, Topical

Specificity and sensitivity of skin test reactions to extracts of Toxocara canis and Ascaris suum II. Homologous 48-hour passive cutaneous anaphylaxis tests with sera from infected guinea pigs.

The specificity and sensitivity of adult and larval somatic antigens and perienteric fluid of Toxocara canis and Ascaris suum were investigated by using a modified passive cutaneous anaphylaxis procedure in guinea pigs. Pooled sera from animals infected with low doses (0.01, 0.1, or 1.0 egg/g) were most reactive with the homologous larval antigen preparation. However, the Toxocara antisera were highly reactive with this antigen only, whereas the Ascaris antisera reactions could not be interpreted as being clearly positive with any of the antigen preparations. Sera from hyperinfected animals were also reactive with the homologous larval antigen. In addition, Ascaris larval antigen was reactive with Toxocara antiserum. The reciprocal relationship, i.e., reactivity of Toxocara larval antigen with Ascaris antiserum, was no apparent. In no instance did adult antigens induce reactions that could be interpreted as specific or sensitive indicators of antibody.

Animals

Scalable assembly of Ascaris mitogenomes from whole-genome data reveals a novel clade.

The genus Ascaris is an important group of giant parasitic roundworms, infecting over 700 million people globally and causing substantial economic losses in domestic pigs. Whilst species of Ascaris are morphologically indistinguishable, analysis of mitochondrial loci has revealed three clades (A, B, C) broadly associated with host species and geographic distribution. The diversity within these lineages may expand with the addition of further genomic data. Here, we present a bioinformatic framework for de novo assembly of complete mitochondrial genomes (mitogenomes) from low-coverage whole-genome data through host-read depletion or mtDNA read enrichment, followed by mtDNA-specific assembly. Our approach yielded 149 high-quality Ascaris mitogenome assemblies, enabling the study of population-level diversity, including the identification of a novel clade (Clade D, designated here) associated with human samples from Ethiopia. Our analysis further revealed Clade C to comprise of pig-derived samples from Europe based on characterisation of worms isolated in Germany. The methods described here provide a scalable framework for mitogenome reconstruction with insights into roundworm population-genomic and phylogenetic studies.

Animals

In vivo porcine multi-omics integration identifies microbiome-driven histamine elevation and lasting gut perturbations following Ascaris suum infection and fenbendazole treatment.

Ascaris roundworms impair human and swine health. While treatments using anthelmintic drugs are generally effective in eliminating worms, their effects on the gut microenvironment remain poorly understood. Here we applied integrated multi-omics to characterize infection- and treatment-associated alterations in the pig-Ascaris system. In vitro anaerobic cultures were conducted as supportive validation of selected observations. Ascaris suum infection altered microbial composition and dysregulated 182 serum and fecal metabolites, including histamine and p-cresol sulfate. Compared with time-matched uninfected controls, infected pigs treated with fenbendazole showed marked differences in gut microbial composition 13&#x2009;days after confirmed worm clearance. Eleven microbial pathways were enriched in successfully treated pigs, including peptidoglycan biosynthesis and histidine metabolism, indicating that infection-associated alterations may persist after treatment. In vitro co-exposure of Lactobacillus reuteri to fenbendazole and A. suum proteins increased histamine production by approximately 79% at 48&#x2009;h (p&#x2009;<&#x2009;0.05), serving as supportive evidence of a microbiome contribution. Collectively, our in vivo findings support that host-microbiota-parasite interactions are multifaceted. Microbiota-derived metabolites were associated with regulation of host gene expression, such as TFF2 and IL8. Microbiota plasticity allows the exploitation of the niche differentiated upon infection, resulting in the proliferation of certain Lactobacillus strains in treated animals. Nevertheless, interpretations of treatment effects are made cautiously given the absence of an uninfected drug-only group and the cross-sectional design. Understanding these complex interactions will be important for the design of next-generation functional anthelmintics.

Animals

Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development.

The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.

Animals

Investigation of the role of histamine in antigen-induced bronchoconstriction in the ascaris-hypersensitive dog.

1 Aerosol administration of ascaris antigen to the airways of ascaris-hypersensitive dogs provoked increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn). These changes in pulmonary mechanics were not inhibited by the histamine H1-receptor antagonists, diphenhydramine or mepyramine. 2 Increases in Rp and decreases in Cdyn induced by a histamine aerosol were markedly or totally inhibited by comparable doses of these H1-antihistamines. 3 Doses of antigen which produced pathophysiological pulmonary responses failed to produce a detectable histamine release from the cardiopulmonary system in vivo. Aerosol antigen provocation, equivalent to 5 to 9 times greater than that which produced substantial pathophysiological pulmonary responses, did cause histamine release in vivo. 4 The canine cardiopulmonary system showed only a modest ability to remove and/or degrade circulating histamine. 5 It is concluded that histamine may not play a major role in mediating the acute antigen-induced bronchoconstriction in the ascaris-hypersensitive dog.

Anaphylaxis

[Experimental studies on anthelmintics (XXVI). Biochemical and pharmacological studies of 4-iodothymol on Ascaris lumbricoides suum].

We have shown previously that 4-iodothymol (IT) produces a contraction in Ascaris muscle, probably due to the myogenic action. In this paper, the effects of IT on the carbohydrate metabolism in Ascaris muscle have been investigated in comparison with those of hexylresorcinol (Hex), santonin (S) and piperazine (Pip). (1) Hex (200 approximately 400 mug/ml) showed a strong nonspecific inhibition on the formation of succinate from fumarate in muscle homogenate, the phosphofructokinase (PFK) activity in cytoplasm, and the electron transfer activity in mitochondria. (2) S(100 approximately 400 mug/ml) and Pip (100 approximately 400 mug/ml) were inneffective on these activities. (3) IT inhibited the formation of succinate from glucose and fumarate in muscle homogenate (100 approximately 400 mug/ml), the PFK activity in cytoplasm (400 mug/ml), and the mitochondrial succinate oxidase system (25 approximately 400 mug/ml). These results suggest that IT elicits the wormcidal action by inhibiting the energy metabolism of Ascaris muscle mitochondria.

Animals

A comparative study of the effect of oxantel-pyrantel suspension and mebendazole in mixed infections with Ascaris and Trichuris.

A comparative trial was carried out in 4 orphanges in Seoul, to compare the anthelminthic efficacy of oxantel-pyrantel suspension and mebendazole tablets against mixed infections of Ascaris lumbricoides and Trichuris trichiura. 40 patients were divided into 4 groups and received oxantel-pyrantel suspension at 15 or 20mg/kg once only or on 2 consecutive days. Another group of 20 patients was treated with 100mg of mebendazole twice a day for 3 days. Both drugs achieved a 100% cure rate for Ascaris infections. Against Trichuris infections, the average cure rate and egg reduction rate of oxantel-pyrantel treated groups was 87.5% and 92.3% respectively, and that for mebendazole was 65% and 89.1%. There were no side-effects. The results of the present study suggest that oxantel-pyrantel is more effective in the treatment of mixed infections of Ascaris and Trichuris, and was more acceptable to the patients, than mebendazole.

Ascariasis

The role of complement in Ascaris suum induced histopathology.

The role of complement in the histopathology of primary and challenge re-infection by Ascaris suum was assessed. Guinea-pigs deficient in C4 or depleted of the terminal components C3 to C9 by cobra venom factor (CVF) were employed. Pathological changes in the livers of complement-deficient guinea-pigs differed slightly from controls. The most striking findings were in the lungs of CVF-treated infected animals, where marked eosinophilic abscesses were observed following either a primary or repeat infection with Ascaris suum. Additionally, greater numbers of larvae were harvested from the lungs of both CVF-treated and C4-deficient infected guinea-pigs as compared with controls. Serum C4 levels varied over the first 7 days after primary infection, declining in several of the normal and CVF-treated animals, but remaining stable in others. The C4 levels remained unchanged in re-infected animals. The C3 to C9 levels remained within the normal range in both primary and re-infected guinea-pigs. The data suggests that complement is involved in the host responses to infection with Ascaris suum. In the absence of complement (C3 to C9), enhanced pulmonary eosinophilic infiltration and eosinophilic granuloma formation occur in both primary and re-infected animals.

Animals