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The intestinal helminth Ascaris lumbricoides infects over 800 million people. Infections are often chronic and immunity is not sterilizing due to host-immune modulation, therefore reinfection is common after antihelmintic treatment. We have previously demonstrated a role for Ascaris spp. extracellular vesicles (EVs) in host immune modulation but whether EVs are recognized by the adaptive immune system and are present systemically in the host remains unknown. Therefore, we employed a well-established trickle infection model in pigs to mimic natural Ascaris infection in humans. EVs were isolated from adult Ascaris suum followed by immunoblotting of EV and EV-depleted secretory fractions using plasma from infected and uninfected pigs. Next, EVs were isolated from pig plasma at day 56 post first infection and subjected to deep small RNAseq analysis. RNAs were aligned to A. suum and Sus scrofa miRNA complements to detect A. suum EVs and elucidate the host EV micro RNA (miRNA) response to infection, respectively. Infection generates robust antibody responses against A. suum EVs that is distinct from EV-depleted fractions. However, A. suum miRNAs were not detectable in EVs from the peripheral blood. Notably, host plasma-derived EV miRNA profiles showed significant changes between infected and uninfected pigs, indicating that Ascaris infection drives systemic changes in host EV composition., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The aim of this work was to elucidate the immunopathological mechanisms of how helminths may influence the course of a viral infection, using a murine model. Severe virulence, a relevant increase in the virus titres in the lung and a higher mortality rate were observed in Ascaris and Vaccinia virus (VACV) co-infected mice, compared with VACV mono-infected mice. Immunopathological analysis suggested that the ablation of CD8 + T cells, the marked reduction of circulating CD4 + T cells producing IFN-γ, and the robust pulmonary inflammation were associated with the increase of morbidity/mortality in co-infection and subsequently with the negative impact of concomitant pulmonary ascariasis and respiratory VACV infection for the host. On the other hand, when evaluating the impact of the co-infection on the parasitic burden, co-infected mice presented a marked decrease in the total number of migrating Ascaris lung-stage larvae in comparison with Ascaris mono-infection. Taken together, our major findings suggest that Ascaris and VACV co-infection may potentiate the virus-associated pathology by the downmodulation of the VACV-specific immune response. Moreover, this study provides new evidence of how helminth parasites may influence the course of a coincident viral infection., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Studies related to the immunobiological aspects of an Ascaris spp. infection are still scarce, especially those that aim to elucidate the early events of the immune response. In this study, we demonstrated a novel standardized method for early experimental Ascaris infection, providing additional information about the infectivity of eggs embryonated in vitro as well as the influence of host age on development of the infection. Finally, we characterised the immunopathology of early infection, focusing on the tissue and systemic cytokine profiles and the histopathology of infection in the lungs of BALB/c mice. Our results demonstrated that the highest egg infectivity occurred on the 100th and 200th days of in vitro embryonation and that 8 week-old BALB/c mice were more susceptible to infection than 16 week-old mice. Ascaris-infected mice showed an early, significant level of IL-5 production in the lungs 4 days p.i., followed by an increase in the level of neutrophils in the inflammatory infiltrate at 8 days p.i, which was correlated with the peak of larval migration in the tissue and a significant level of IL-6 production. The inflammatory infiltrate in the lungs was gradually replaced by mononuclear cells and eosinophils on the 10th and 12th days p.i., respectively, and an increase in TNF levels was observed. The downmodulation of systemic TCD4(+) cell numbers might suggest that T cell hyporesponsiveness was induced by the Ascaris spp. larvae, contributing to safeguarding parasite survival during larval migration. Taken together, the novel aspects of Ascaris infection presented here enabled a better understanding of the immunopathological events during larval migration, providing insight for further studies focused on immunisation and immunoprophylatic assays., (Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Helminths almost invariably have an over-dispersed distribution in the host population. Human and animal studies have provided evidence suggesting that a large part of this variation is due to host genetic factors. Recently, the heritability for roundworm (Ascaris suum) infection levels in pigs was estimated to be 0.45. We used single nucleotide polymorphism markers to perform a whole-genome scan on 195 pigs experimentally infected with A. suum. A putative quantitative trait locus for worm burden on chromosome 4 covering 2.5 Mbp was identified by measured genotype analysis, although none of the SNPs reached genome-wide significance. To validate the putative quantitative trait locus, we genotyped two of the SNPs within the region in unrelated, informative animals exposed to experimental or natural infections and from which we had worm counts and/or faecal egg counts; the validation studies showed that one of the SNPs (TXNIP) was associated with total worm burden (P < 0.001) and adult worm burden(P < 0.0001), whereas the other SNP (ARNT) was associated with adult worm burden (P < 0.025) in these populations. We were thus able to confirm the existence of the quantitative trait locus on chromosome 4.This is to our knowledge the first report of a quantitative trait locus associated with helminth burden in pigs.

Previous studies have shown that antigens from various life-cycle stages of Ascaris suum can induce host-protective immunity against challenge infections with infective eggs of A. suum. This study evaluated whether Escherichia coli-expressed recombinant 24-kDa antigen from A. suum (rAs24) was a suitable vaccine candidate for the control of Ascaris infections by examining its performance in a mouse model. Immunization of BALB/c mice in three consecutive doses with rAs24 in Freund's Complete Adjuvant (FCA) results in protection against challenge infections as manifested by a 58% reduction (P<0.001) in recovery and stunted development of A. suum lung-stage larvae at day 7 post-challenge. Sera obtained from immune protected mice had a significantly increased level of immunoglobulin G (IgG) (P<0.0001) but had no IgE response. Analysis of IgG-subclass profiles revealed that IgG1 (P<0.0001) showed the greatest increase followed by IgG2b (P<0.005), IgG2a (P<0.006) and IgG3 (P<0.04). Splenic T cells from rAs24-FCA immunized mice secreted significantly high levels of both Th1 cytokine gamma-interferon (P<0.005) and Th2 cytokine interleukin-10 (P<0.001) after stimulation with rAs24 in vitro. Interestingly, affinity purified anti-rAs24 IgG was shown to inhibit moulting of A. suum lung-stage L3 to L4 in vitro by 26%, indicating an in vivo function of the endogenous As24 in the moulting processes. An intense expression of endogenous As24 in the hypodermis and gut epithelium of A. suum lung-stage L3 by immunofluorescence supports a function for endogenous As24. These findings may contribute to the understanding of rAs24-induced Th1/Th2-mediated effector mechanisms required for the protection of A. suum lung-stage larval infection.

This study investigated associations between pre-treatment cytokine expression and infection patterns, before and after de-worming, in humans exposed to two gastrointestinal nematode species. Quantitative measures of Ascaris lumbricoides and Trichuris trichiura infection (based on faecal egg counts) were estimated immediately before and 8-9 months after treatment in a Cameroonian population. Whole blood cytokine responses to parasite-derived antigens were assayed immediately pre-treatment. An overall measure of the tendency towards species-specific infection (increasing with A. lumbricoides faecal egg counts and decreasing with T. trichiura faecal egg counts) was significantly positively related to IL-10 levels in older (14-57 year) hosts. There was a significant negative influence of IL-5 on reinfection probability in T. trichiura but not A. lumbricoides. This effect coincided with reduced reinfection success in T. trichiura compared to A. lumbricoides. T(H)2 cytokine expression by younger hosts (4-13 year) was negatively associated with contemporary A. lumbricoides faecal egg counts before treatment. Following treatment, the pre-treatment T(H)2 cytokine expression data for younger hosts (now reflecting responsiveness 8-9 months in the past) were negatively associated with T. trichiura faecal egg counts. Taken together, these observations suggest a successional interaction between T(H)2-driven immune responses and species infection over time. However, any differential effects of the measured immune responses on species-specific recruitment, maturation and mortality were superimposed upon (and outweighed by) the effects of other factors favouring coinfection.

Antigens from larval stages of Ascaris suum have been shown to induce protection against challenge infection with infective A. suum eggs. We previously identified several antigens that reacted strongly with serum from pigs inoculated with infective eggs containing L3. In this study, we isolated an antigen with a molecular mass of 37 kDa and a pI of 4.8 (As37) from A. suum infective eggs using two-dimensional electrophoresis, and obtained a full-length cDNA by reverse transcription-polymerase chain reaction using primers designed based on the internal amino acid sequence of As37. The cDNA sequence consisted of 1,540 bp coding for a protein of 321 amino acids with a complex domain organisation. Simple modular architecture research tool (SMART) analysis indicated that As37 contains three immunoglobulin domains, indicating that it is a member of immunoglobulin superfamily (IgSF). A homology search of GenBank showed that As37 has significant similarity to Caenorhabditis elegans DIM-1 protein and has low similarity to part of the multi-repeat Ig domain from nematode twitchin and mammalian skeleton muscle titin, and to members of the IgSF at the amino acid sequence level. Localisation analysis revealed that antibodies to Escherichia coli-expressed recombinant As37 (rAs37) bound to muscle cells and the hypodermis. The antibodies identified a 37 kDa native antigen in human and dog roundworms, suggesting that there are As37 homologues in ascarid nematodes. Sera from mice, rabbits and pigs immunised with A. suum infective eggs reacted with rAs37 in immunoblot analyses. The potential use of rAs37 for protection against A. suum infection is discussed.

A metalloaminopeptidase was identified in culture fluids collected during in vitro cultivation of adult Ascaris suum. The enzyme was purified by anion-exchange and size-exclusion HPLC. The M(r) of the enzyme was estimated at 293 kDa and consisted of subunits with M(r)s of 153 and 142kDa. The isoelectric point of the aminopeptidase was 4.7. The aminopeptidase displayed a substrate preference for terminal arginyl residues. Aminopeptidase activity was also present in muscle, female reproductive tissue, pharynx, pseudocoelomic fluid and intestine. Among the various tissues, aminopeptidase activity was highest in the intestines; the highest activity was found in culture fluids (three-fold higher than intestinal tissue). The aminopeptidase released by adult A. suum was enzymatically and biochemically identical to an aminopeptidase released during in vitro development of A. suum third- to fourth-stage larvae.

The ELISA method using larval ES products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worms extract, was used to determine the possible cross-reactions in BALB/c and C57BL/10 mice inoculated with embryonated eggs or adult worms extract of T. leonina of T. leonina or A. suum in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. leonina against different heterologous Ag, no cross-reactions against T. canis ES and A. suum ES Ag were observed using a single dose. Similarly, in multiple doses no response against T. canis ES Ag was observed. In mice inoculated with adult worms extract of T. leonina cross-reactions with T. canis ES and A. suum ES Ag did not occur. Sera from BALB/c mice infected with embryonated eggs of A. suum, was tested using ES Ag from both A. suum and T. canis and no reactions were observed. This fact confirmed the resistance of this murine strain to A. suum embryonated eggs. When we used sera of susceptible C57BL/10 mice infected against different heterologous Ag, we observed no cross-reactions against T. canis ES Ag. In the case of both BALB/c and C57BL/10 and C57BL/10 mice immunized with a single dose of A. suum adult crude extract no cross-reactions were seen against ES T. canis Ag and with sera from C57BL/10 mice against ES T. leonina. These facts confirmed the specificity of the ES T. canis Ag.

Fifty-four crossbred, 4-week-old pigs divided into nine equal groups were used to test whether multiple inoculations with high numbers of A. suum eggs with or without anthelmintic would result in patent infections. All pigs exposed to multiple prechallenge inoculations of 500, 1000, 2000, 5000, 10,000 and 20,000 and challenged orally 2 weeks later with 10,000 eggs harboured adult worms. When prechallenge infections were removed by pyrantel tartrate treatment the animals were more susceptible to challenge than controls not previously exposed to infections. The same drug used from 2 days before until 10 days after the last prechallenge infection eliminated that effect. Pigs subjected to the same multiple egg dosing regimen but given feed containing fenbendazole immediately before, during and for 10 days after multiple dosing developed significantly more adult intestinal worms after challenge than any other group. These worms were, however, significantly shorter than those that developed in any group of pigs. Adult worms from all these groups produced eggs that after embryonation were infective to mice.

Crossbred young pigs were used to test whether abbreviated infections with eggs of Ascaris suum can stimulate the acquisition of resistance to challenge. Weanling pigs from an Ascaris-free colony were kept free of A. suum until they were divided into groups at the age of 7-8 weeks. The experimental animals received pyrantel tartrate during the period when they were being exposed to increasing numbers of infective A. suum eggs and challenged 10 days after the last infective dose. Liver milk-spot counts and larval recoveries from the lungs indicated that the strongest resistance was acquired by the animals that received the drug continuously for 6 weeks while being exposed to six weekly infective egg doses. The data do not suggest any drug-related suppression of the resistance response to A. suum infection.

Mice were evaluated for immunological responses and resistance to challenge infections after single infections. When given infections simultaneously with thiabendazole, larval migration to the liver and lungs was blocked but not when mice were given infections only. Enlargement of Peyer's patches was observed after infection followed by increased numbers of IgA-containing cells in the lamina propria of the intestines and increased levels of hepatobiliary IgA. Ig-containing cells were variable in the spleens during the early phase of infection. The observed changes quickly returned to normal control levels and did not change throughout the rest of the experiment. Serum Ig levels did not change dramatically. Challenge with high doses of eggs indicated that mice given single very low-dose infections became highly resistant.