A phosphorylcholine-containing glycolipid-like antigen present on the surface of infective stage larvae of Ascaris spp. is a major antibody target in infected pigs and humans

BACKGROUND: The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential. METHODOLOGY/PRINCIPAL FINDINGS: Pigs were immunized by trickle infection for 30 weeks, challenged with 2,000 eggs at week 32 and euthanized two weeks after challenge. At necropsy, there was a 100% reduction in worms recovered from the intestine and a 97.2% reduction in liver white spots in comparison with challenged non-immune control animals. Antibodies purified from the intestinal mucus or from the supernatant of cultured antibody secreting cells from mesenteric lymph nodes of immune pigs were used to probe L3 extracts to identify antibody targets. This resulted in the recognition of a 12kDa antigen (As12) that is actively shed from infective Ascaris L3. As12 was characterized as a phosphorylcholine-containing glycolipid-like antigen that is highly resistant to different enzymatic and chemical treatments. Vaccinating pigs with an As12 fraction did not induce protective immunity to challenge infection. However, serological analysis using sera or plasma from experimentally infected pigs or naturally infected humans demonstrated that the As12 ELISA was able to detect long-term exposure to Ascaris with a high diagnostic sensitivity (98.4% and 92%, respectively) and specificity (95.5% and 90.0%) in pigs and humans, respectively. CONCLUSIONS/SIGNIFICANCE: These findings show the presence of a highly stage specific, glycolipid-like component (As12) that is actively secreted by infectious Ascaris larvae and which acts as a major antibody target in infected humans and pigs

A role for eosinophils in the intestinal immunity against infective Ascaris suum larvae

The aim of this study was to explore the mechanisms of resistance against invading Ascaris suum larvae in pigs. Pigs received a low dose of 100 A. suum eggs daily for 14 weeks. This resulted in a .99% reduction in the number of larvae that could migrate through the host after a challenge infection of 5000 A. suum eggs, compared to naı¨ve pigs. Histological analysis at the site of parasite entry, i.e. the caecum, identified eosinophilia, mastocytosis and goblet cell hyperplasia. Increased local transcription levels of genes for IL5, IL13, eosinophil peroxidase and eotaxin further supported the observed eosinophil influx. Further analysis showed that eosinophils degranulated in vitro in response to contact with infective Ascaris larvae in the presence of serum from both immune and naı¨ve animals. This effect was diminished with heat-inactivated serum, indicating a complement dependent mechanism. Furthermore, eosinophils were efficient in killing the larvae in vitro when incubated together with serum from immune animals, suggesting that A. suum specific antibodies are required for efficient elimination of the larvae. Together, these results indicate an important role for eosinophils in the intestinal defense against invading A. suum larvae

The intestinal expulsion of the roundworm Ascaris suum is associated with eosinophils, intra-epithelial T cells and decreased intestinal transit time

Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections

The detection of a 12kDa <i>A</i>. <i>suum</i> L3 antigen in the L3 PBS extract by intestinal antibodies.

<p>IgG and IgA antibodies purified from the supernatant of purified mononuclear cells (MNC) from mesenteric lymph nodes or the intestinal mucus were used to screen the L3 PBS extract on Western blot. Antibodies from immune pigs (Group B) strongly react to an antigen migrating at 12 kDa. Pigs from the challenged control group (Group A) also had antibodies against the 12kDA antigen, but the reactivity on Western blot was lower than in immune pigs. Conjugate alone (cc) did not react with the antigen.</p

Long term trickle infection of pigs with <i>A</i>. <i>suum</i> results in reduced liver white spots and L4 recovered from the small intestine 14 days post challenge infection with 2,000 infective eggs.

<p>Long term trickle infection of pigs with <i>A</i>. <i>suum</i> results in reduced liver white spots and L4 recovered from the small intestine 14 days post challenge infection with 2,000 infective eggs.</p

Mucosal antibodies from immune pigs recognize antigens on the surface of <i>A</i>. <i>suum</i> L3 that likely contain phosphorylcholine groups and are actively secreted by living larvae.

<p>(A) UV-microscopy images (400x) of <i>A</i>. <i>suum</i> L3 larvae stained by mucosal antibodies from naïve pigs, challenge control pigs (Group A) or immunized pigs (Group B) and incubated with FITC labeled anti-pig IgG. (B) UV-microscopy image (400x) of <i>A</i>. <i>suum</i> L3 larva partially stained by mucosal antibodies from immune pigs and incubated with FITC labeled anti-pig IgG. (C) A UV-microscopy image (400x) of an <i>A</i>. <i>suum</i> L3 larva stained by anti-PC antibodies and (D) a stained larva in the process of shedding these antigen-antibody complexes. (E) The percentage of <i>A</i>. <i>suum</i> L3 stained by mucosal antibodies from immune pigs decreased over time when L3 were kept alive in culture media (dotted line) but not when L3 were killed after staining (full line) (P < 0.01).</p

Exploring the properties and composition of the As12 antigen.

<p>(A) Coomassie staining, carbohydrate staining and Western blot developed with mucosal IgG antibodies from immune pigs (Group B) of the complete L3PBS extract (1), the pellet after Folch extraction (2), the upper ‘methanol phase’ (3) and the lower ‘chloroform phase’ (4). (B) Recognition of the As12 antigen by mucosal IgG antibodies from immune pigs after chemical or enzymatic treatments of the <i>A</i>. <i>suum</i> L3 PBS extract: untreated (1&17); overnight digestion at 37°C with pronase (2); lipase (3); trypsin (4); pepsin (5); overnight incubation at 37°C (6), 60°C (7) and 90°C (8); overnight incubation with 20mM periodic acid at 37°C (9) and 60°C (10), 1M NaOH at 37°C (11) and 60°C (12), 1M trifluoracetic acid at 37°C (13) and 60°C (14); 1M HCl at 37°C (15) or 60°C (16); 48 hrs incubation in 48% HF at 4°C (18); overnight incubation at 37°C without (19) or with PNGase-F (20) and 48 hrs. incubation at 37°C with (21) or without (22) Jack Bean ß-N-Acetylglucosaminidase. (C) Recognition of <i>A</i>. <i>suum</i> L3 PBS antigens by purified mucosal IgG antibodies from immune pigs (1) and specific anti-phosphorylcholine antibodies (TEPC-15) (2).</p