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Ascaris suum is a helminth parasite of pigs closely related to its human counterpart, A. lumbricoides, which infects almost 1 billion people. Ascaris is thought to modulate host immune and inflammatory responses, which may drive immune hyporesponsiveness during chronic infections. Using transcriptomic analysis, we show here that pigs with a chronic A. suum infection have a substantial suppression of inflammatory pathways in the intestinal mucosa, with a broad downregulation of genes encoding cytokines and antigen-processing and costimulatory molecules. A. suum body fluid (ABF) suppressed similar transcriptional pathways in human dendritic cells (DCs) in vitro. DCs exposed to ABF secreted minimal amounts of cytokines and had impaired production of cyclooxygengase-2, altered glucose metabolism, and reduced capacity to induce interferon-gamma production in T cells. Our in vivo and in vitro data provide an insight into mucosal immune modulation during Ascaris infection, and show that A. suum profoundly suppresses immune and inflammatory pathways., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Albendazole treatment of individuals with human immunodeficiency virus type 1 (HIV-1) and Ascaris lumbricoides co-infection has led to significantly improved CD4(+) cell counts and a trend for lower plasma HIV-1 RNA levels in a previous randomized placebo-controlled trial. To define mechanisms by which deworming contributed to changes in markers of HIV-1 disease progression, plasma cytokine levels were evaluated. Albendazole treatment, compared with placebo, was associated with significantly decreased plasma interleukin (IL) 10 levels (P = .01)ot associated with significant changes in levels of IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12p70, IL-13, interferon gamma, tumor necrosis factor alpha, or thymic stromal lymphopoietin. Treatment of A. lumbricoides co-infection may delay HIV-1 disease progression by reducing helminth-induced, IL-10-mediated immunosuppression.

The 13q33-34 region harbours a susceptibility locus to Ascaris lumbricoides, although the underlying genes are unknown. Immunoglobulin (Ig)E and IgG confer protective immunity and here we sought to investigate in an endemic population whether LIG4, TNFSF13B and IRS2 genes influence IgE and IgG levels against Ascaris and the ABA-1 allergen as a putative resistance marker. Mite-allergic asthmatic patients were analysed for potential relationships between Ascaris predisposition and allergy. One thousand and sixty-four subjects from Cartagena, Colombia, were included. Single nucleotide polymorphisms (SNPs) were genotyped using TaqMan assays. Antibody levels were measured by enzyme-linked immunosorbent assay. Linear and logistic regressions were used to model effects of genotypes on antibody levels. The GG genotype of LIG4 (rs1805388) was associated with higher IgE levels to Ascaris compared with other genotypes. TNFSF13B (rs10508198) was associated positively with IgG levels against Ascaris extract and IgE levels against ABA-1. In asthmatics, IRS2 (rs2289046) was associated with high total IgE levels. Associations held up after correction by population stratification using a set of 52 ancestry markers, age, sex and disease status. There was no association with asthma or mite sensitization. In a tropical population, LIG4 and TNFSF13B polymorphisms are associated with specific IgE and IgG to Ascaris, supporting previous linkage studies implicating the 13q33 region. Our results suggest that genes protecting against parasite infections can be different to those predisposing to asthma and atopy.

Ascaris lumbricoides infections in humans may have important effects on the development of allergy and on susceptibility to infectious diseases that start during early life. To investigate whether sensitization to A. lumbricoides occurs in utero, we measured interferon (IFN)-gamma and interleukin (IL)-4 responses in A. lumbricoides antigen-stimulated cord blood from newborns of infected and noninfected mothers by flow cytometry. There was evidence of higher frequencies of IFN-gamma-expressing and IL-4-expressing CD4+ T cells in newborns of infected mothers than in newborns of noninfected mothers. Our data provide evidence of in utero sensitization to A. lumbricoides and raise the possibility that the immunological effects of infection start in the fetus.

Geohelminth infections are associated with modulation of immunity to parasite antigens and aeroallergens. To investigate the possibility that this modulation is affected by anthelmintic treatment, we compared cytokine responses in children who were treated with repeated doses of albendazole over 1 year versus those in children who had were not treated. Whole-blood samples were cultured with Ascaris antigen and house dust mite and cockroach allergens, and levels of interleukin (IL)-5, IL-13, interferon-gamma, and IL-10 were measured. Anthelmintic treatment was associated with enhanced production of Th2 cytokines in response to parasite antigen but did not affect responses to aeroallergens. The data indicate that long-term treatment may be associated with increased Th2 antiparasite immunity.

Background: In areas where intestinal helminth infections are endemic, infections by these parasites may protect against skin test-measured reactivity to allergens, and it has been suggested that interleukin (IL)-10 may mediate this effect. This study investigated whether IL-10 and populations of IL-10+ T cells may modulate atopy in children living in an area where intestinal helminth infections are endemic., Methods: Ecuadorian schoolchildren from rural communities were assessed for skin test-measured reactivity to Periplaneta americana allergen and Dermatophagoides pteronyssinus allergen. Blood samples were collected from 39 skin test-positive and 41 skin test-negative children, and peripheral-blood leukocytes were cultured in the presence of Ascaris lumbricoides antigen, to measure IL-10 protein and the frequency of T cell populations expressing intracellular IL-10. We also investigated whether these immunological factors affected the association between allergen-specific IgE and skin test-measured reactivity to aeroallergens., Results: There was no evidence of association between the level of A. lumbricoides-induced IL-10 protein or IL-10+ T cells and skin test-measured reactivity to allergens. The association between allergen-specific IgE and skin test-measured reactivity was not affected by the level of IL-10 protein or the frequency of IL-10+ T cells., Conclusions: The results of this study do not support the notion that IL-10 plays a role in modulating atopy in children living in a tropical area where intestinal helminth infections are endemic.

The ratio of Ascaris seropositivity to the presence of eggs in the faeces was 2.44 in children residing near Cape Town, South Africa. Similar and larger ratios have previously been described for children and women living in the city. The new finding strengthens the concept that when helminthic infections occur together with non-helminthic diseases, an analysis of the interaction must include the use of disease-related immunological variables and not be based only on egg excretion status. One of the reasons is that many egg-negative people who live where helminthiasis is highly endemic are immunologically activated as a result of transitory non-patent or intermittent patent infection by Ascaris and/or other worms.

We recently cloned a protective antigen that is commonly expressed in Ascaris species that infect humans and pigs. We evaluated the vaccinal effects of this 16-kilodalton protein (As16) in pigs, the natural host of Ascaris suum, by intranasal immunization. Pigs that received Escherichia coli-expressed recombinant As16 (rAs16) coupled with cholera toxin (CT) had significantly elevated levels of rAs16-specific serum immunoglobulin G (IgG) and mucosal-associated IgA antibodies. rAs16 evoked a type II immune response characterized by elevated levels of interleukin-4 and -10 in the culture supernatants of peripheral blood mononuclear cells of the vaccinated pigs. An increased level of rAs16-specific serum IgG1 was also detected. Pigs vaccinated with rAs16-CT were protected from migration of A. suum larvae through the lungs, as indicated by a 58% reduction in the recovery of lung-stage third-stage larvae (L3), compared with that in nonvaccinated controls. Purified immunoglobulin from rAs16-CT-vaccinated pigs inhibited survival of infective L3 and interrupted the molting of lung-stage L3. Immunofluorescence studies revealed that this immunoglobulin bound to the digestive tracts of L3, suggesting that it might inactivate functions of the gut tissues of Ascaris species. We conclude that rAs16 is a promising mucosal vaccine candidate for pig and human ascariasis.

Some humans are persistently more susceptible to gastrointestinal nematodes than others. Here, for the first time, susceptibility to reinfection has been linked to host cytokine responses. Ascaris lumbricoides and Trichuris trichiura abundance was assessed immediately before and 8-9 months after deworming in a Cameroonian population (starting n=191). Profiles of whole-blood cytokine responses to parasite antigens (for interleukin [IL]-5, IL-13, IL-10, IL-12p40, tumor necrosis factor- alpha , and interferon- gamma), assayed before treatment, were significantly related both to an overall measure of host susceptibility and to susceptibility to reinfection. Significant effects were primarily due to a negative association between IL-13 and IL-5 responses and infection. Persistently susceptible individuals were, therefore, characterized by a weak T helper cell type 2 response. The apparent plasticity of age-specific cytokine response-worm abundance relationships between different populations is also discussed.

Studies have shown a strong inverse relationship between atopy and geohelminth infection, indicating that atopy may protect against geohelminth infection. Resistance to ascariasis in atopic individuals may occur through greater immunoglobulin E-mediated responses and expression of T helper cell type 2 (Th2) cytokines to parasite antigens. To investigate the effect that atopy has on the immune response to Ascaris antigens, school-age children were recruited from rural schools in Ecuador. Immunologic variables were compared between children stratified by atopic and/or A. lumbricoides-infection status; the variables included cytokine expression by peripheral-blood mononuclear cells (PBMCs) and histamine release in response to Ascaris antigens. Atopic children had both greater frequencies of PBMCs expressing interleukin (IL)-4 and IL-5 and enhanced histamine release, compared with those in nonatopic children. Stratification by atopic and A. lumbricoides-infection status revealed the greatest histamine and Th2 cytokine responses in the stratum of atopic, noninfected children. Multivariate regression analyses showed significant effects for atopic status but not for infection status on Th2 cytokine expression and histamine release.

It is well established that malnutrition affects the immune response and increases the susceptibility to parasitic infection. In the present study we evaluated some aspects of the cellular and cytokine network that regulate the IgE responses, which are important components of host defence mechanisms against helminthic parasites in children infected with the intestinal helminth Ascaris lumbricoides, and with differing degrees of malnutrition. We found a defective T cell response in malnourished children, as indicated by diminished levels of circulating total (CD3+), helper (CD4+), IL-2-receptor-bearing (CD4+CD25+) and memory helper T cell responses (CD4+CD45RO+) in keeping with the decreased specific IgE levels against Ascaris lumbricoides. In contrast, the proportions of total B cells (CD20+), and those bearing the low-affinity IgE receptor (CD23+) were increased in the moderated malnourished children. Moreover, serum IL-4 levels and total IgE were also increased in these children. We suggest that malnutrition can cause an imbalance in T cell subpopulations that may lead to a defective T cell maturation and a decreased specific anti-Ascaris IgE response thus increasing the susceptibility to such infections. The high levels of total IgE observed may be related to a non-specific stimulation of the proliferation of activated B cells, probably caused by helminthic parasites and other infectious agents that are frequent in malnourished children.

Because concurrent infections with geohelminth parasites might impair the immune response to oral vaccines, we studied the vibriocidal antibody response to the oral cholera vaccine CVD 103-HgR in children infected with Ascaris lumbricoides and investigated the effect of albendazole pretreatment on the postvaccination response. Children with ascariasis were randomized to receive either 2 sequential doses of 400 mg of albendazole or placebo. After the second dose, CVD 103-HgR was given, and serum vibriocidal antibody levels were measured before and 10 days after vaccination. Postvaccination rates of seroconversion were greater in the treatment group that received albendazole (P=.06). Significantly greater rates of seroconversion and geometric mean titer were observed in the albendazole group in subjects with non-O ABO blood groups. A significant association was observed between vibriocidal seroconversion rates and treatment group, suggesting that A. lumbricoides infections impair the immune response to oral cholera vaccine, particularly in subjects of non-O blood groups.

To define the cytokine response to Ascaris lumbricoides infection, the cellular immune response to adult and larval-stage Ascaris antigens in young adults with moderate infection intensities (n=73) was compared with that of a group of uninfected control subjects (n=40). A. lumbricoides-infected subjects had significantly greater lymphoproliferative responses to adult and larval-stage antigens, compared with uninfected control subjects (P<.01). The frequencies of parasite antigen-stimulated peripheral blood mononuclear cell (PBMC)-expressing interleukin (IL)-4 and IL-5 were significantly greater in the infected group (P<.001), whereas the frequencies of IL-10- and interferon-gamma-expressing PBMC were similar in the 2 groups studied. The ratios of Th2 to Th1 cytokine frequencies were significantly elevated in the infected group, compared with those in uninfected subjects, as was IL-5 protein production by PBMC stimulated with adult (P<.05) and L3/L4 stage (P<.001) antigens. Analysis of these data indicates that A. lumbricoides infections in endemic regions are associated with a highly polarized type 2 cytokine response.

Helminthic infection can stimulate the interleukin-4 (IL-4)-dependent polyclonal synthesis of immunoglobulin E (IgE) in children endemically exposed to these parasites. As such children are also frequently at nutritional risk, in this study we considered the possible influence of malnutrition on serum IL-4 levels and the IgE response in helminthic infection. We evaluated 85 Ascaris-infected children living in an urban slum area of Caracas, Venezuela, and found that the serum levels of IL-4 and total IgE were significantly higher in malnourished children than in their well nourished counterparts. In contrast, the specific anti-Ascaris IgE antibody response was significantly lower in the malnourished group. After anthelmintic treatment of the children, the total serum IgE and IL-4 levels decreased significantly in the well nourished group, while the specific anti-Ascaris IgE antibody response increased. No significant change was detected, however, in the malnourished group. Our results suggested that malnutrition potentiates the polyclonal stimulation of IgE synthesis induced by helminths. As specific IgE antibody has been implicated in the resistance to helminthic infection, and the polyclonal stimulus diminishes this response, these factors may increase the susceptibility of malnourished children to such parasites.

Total and Ascaris-specific serum IgE levels were measured in a group of 98 Ascaris-infected children from a slum area of Caracas, Venezuela, in whom the infections were eliminated by regular treatment for 22 months with the anthelmint Oxantel/Pyrantel ('Quantrel'). The children were re-evaluated at the end of the treatment programme, and then 8 months later, at which time reinfection was assessed. Total IgE levels at the beginning of the study were significantly higher in the children who became reinfected after treatment, compared with those who did not. The anthelmint treatment caused a significant decrease in the total IgE levels in most of the children, and after a period of 8 months without treatment these continued to decrease in the non-reinfected group, but increased again in the reinfected children. The reverse pattern was found for Ascaris-specific IgE antibody levels, and in fact an inverse correlation was found between total and anti-Ascaris IgE levels. Striking associations were found between reinfection and high pretreatment values of total IgE, but low levels of specific IgE antibody. These data support the concept that specific IgE antibody may participate in the protection against helminthic infection, and suggest that the polyclonal stimulation of IgE synthesis caused by these parasites may reduce the effectiveness of such responses. The results also indicate that different individuals have varying propensities to respond polyclonally to the helminths, and this influences their resistance to infection.

We examined a group of urban slum children in Caracas, Venezuela, and demonstrated the relationship that exists between poverty, conditions of hygiene and the prevalence of helminth infection. Concordant with the high prevalence of helminth infection in these children, the total serum levels of immunoglobulin E (IgE) and blood eosinophilia were elevated and were directly related to the degree of poverty and lack of sanitary facilities. However, in contrast, the reverse pattern was observed for the specific allergic response to these parasites. Thus, the immediate hypersensitivity skin test reactivity to Ascaris antigens, and the serum levels of specific anti-Ascaris IgE antibody, were lowest in the poorest children, who had the highest prevalence of helminth infection and the highest total IgE levels. As allergic-type reactions may participate in protective mechanisms against helminths, these results suggest that poverty and poor sanitary conditions may, by influencing the IgE response, compromise the resistance of such children to parasitic infections.

Children from an area of Africa endemic for the large roundworm of humans, Ascaris lumbricoides, were found to vary considerably in the specificity of their serum IgG response to the internal antigens of the parasite. This was particularly noticeable for responses to a 14-kD protein (ABA-1) of the parasite that has previously been shown to be the subject of a strong IgE antibody response in infected animals. The possibility that this heterogeneity in immune repertoire has a genetic basis was explored in inbred mice infected with Ascaris suum. This showed that no strain responded to all the potential antigens, that the recognition profiles of strains bearing independent haplotypes were unique, and only H-2-identical strains had responses of similar specificities. Major histocompatability complex (MHC) restriction was confirmed using H-2-congenic animals on BALB and B10 backgrounds, which responded according to their H-2 haplotype. It is likely, therefore, that it is the MHC which controls the repertoire to Ascaris antigens in infected people. If this is so, then there will be implications for immunopathology associated with ascariasis, and possibly also for resistance and susceptibility to infection.

The sensitivity of in vitro lymphocyte proliferative responsiveness using antigens purified by affinity chromatography, was greater than that of three serological tests and allowed the specific identification of Dirofilaria immitis, Toxocara canis, Angiostrongylus cantonensis and Ascaris lumbricoides in animals sensitized with parasite antigens, and the diagnosis of natural (D. immitis, T. canis and Angiostrongylus cantonensis) and zoonotic nematode infections. Lymphocytes remained viable in whole blood suspensions in RPM 1640 in a polystyrene insulation box at air temperature for at least 12 hours and reacted in the cell-mediated immunity test (CMIT) without loss of response. The CMIT proved useful in the early immunodiagnosis of nematode infections and showed that parasite antigens purified by affinity chromatography retained a high degree of specificity and sensitivity in both the CMIT and serological tests.

Wild-caught non-human primates are naturally sensitive to Ascaris antigen and provide a useful model for studying atopic asthma. The present study was carried out to determine the effect of experimentally infecting home-bred macaques with the nematode Ascaris suum and hence provide an alternative for the naturally occurring model. Following oral infection with the parasite the animals developed a blood eosinophilia and specific antibodies to purified Ascaris antigen. These antibodies appeared to be of the IgE class as they could be detected by a radiometric assay using a radiolabelled antibody to human IgE. However, on further investigation, using the passive cutaneous anaphylaxis test, two classes of antibody were found, a heat labile (56 degrees C) and a heat stable antibody. Lung lavage cells taken from monkeys infected with Ascaris suum were shown to include cells morphologically characteristic of mast cells and released histamine when challenged in vitro with Ascaris antigen. Hence this model of immediate hypersensitivity provides a simple alternative to the less accessible natural model.

The materials released in vitro by the tissue-parasitic larval stages of the large roundworm of man, Ascaris lumbricoides, were analysed by radio-iodination, immunoprecipitation, and SDS-PAGE. The antigens were found to be heterogeneous, ranging in molecular weight from 14 to 410 kD, and were found to alter radically during the parasites' migration to the lungs. The antigens secreted by the infective and lung-stage larvae of the pig homologue, Ascaris suum, were compared with those of the human worms. This revealed a remarkable degree of homology between the products of the two, at both the molecular and immunological levels. The two species could be discriminated, however, on the basis of the SDS-PAGE profiles of the antigens secreted by both developmental stages of the parasites examined. Finally, antiserum to the canine ascarid infective to man, Toxocara canis, was found to precipitate a significant proportion of Ascaris-secreted molecules. These studies, therefore, confirm the potent antigenicity of excretory/secretory materials, and their potential for use in immunodiagnosis, but predict serious difficulties for seroepidemiology and the specific detection of ascariasis in man.

Immunosuppression was examined at 10 to 12 days following oral inoculation of 10,000 to 12,000 embryonated Ascaris suum eggs. Reduced antibody responses to sheep red cells (SRC) following systemic immunization were confirmed in CD-1 and C57Bl/6 mice. Infection alone induced antibody reactive with DNP equivalent to that observed after immunization with DNP--Ficoll. There was a decrease in thymus and spleen size by day 8 of infection, followed by a splenic proliferative response during the second week. In the second week, serum antibodies reactive with SRC, chicken erythrocytes, DNP and bacterial lipopolysaccharide were demonstrated, suggesting polyclonal B-cell stimulation. The cellular basis of immunosuppression was investigated by in vitro culture of splenocytes from C57Bl/6 mice. Differential leucocyte counts of splenocytes before culture demonstrated a relative increase in plasma cells, blastoid cells, complement receptor-bearing lymphocytes and eosinophils, with a relative decrease in small lymphocytes. The splenocytes had reduced responses to T-cell mitogens, as measured by thymidine incorporation in vitro, and reduced antibody responses to SRC and DNP--Ficoll. In vitro, cell mixing experiments did not demonstrate suppressor cells in the spleens of infected mice.

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.

The indirect fluorescent antibody test using frozen sections of infective Toxocara canis and Toxascaris leonina eggs, and the enzyme linked immunosorbent assay using homogenized Toxocara canis embryonated egg extract and T canis excretory-secretory products as adsorbed antigens were used to determine the specificity and development of circulating antibodies in rabbits. Frozen sections were subdivided into four morphologically distinct compartments for analysis of the development of the circulating antibody response. The fluid surrounding the larva was the most reactive up to 21 days after infection, and this material was found to be predominantly excretory-secretory in nature. As the infection progressed antibodies directed against 'somatic' tissue materials increased. Cross reactions between sera from rabbits infected with T. canis eggs and Toxascaris leonina frozen sections, and rabbits infected with T. leonina eggs and Toxocara canis frozen sections occurred between both the excretory-secretory fluid and somatic components of the infective eggs. These results were substantiated using the enzyme linked immunosorbent assay. When T. canis excretory-secretory antigen was used, an earlier response (peak day 21) was detected than when using T. canis embryonated egg extract (peak day 35). However, cross reactions between T. canis excretory-secretory antigen and sera from rabbits infected with Toxascaris leonina occurred, indicating that the serodiagnosis of visceral larva migrans using Toxocara canis excretory-secretory antigen may still prove unsatisfactory when considering the role of Toxascaris as a possible causative agent.

The sera from 660 healthy blood donors from Canberra were tested for antibodies to Toxocara canis by the ELISA test. The results were compared with those from patients with suspected or confirmed visceral larva migrans or ocular toxocariasis. Over 7% of Canberra sera showed elevated levels of antibody reacting with T. canis antigen. Sera from patients resident in Australia with other helminth parasites did not cross-react with T. canis antigen in our tests. However, studies of sera collected in several tropical countries with other parasitic infection, show that cross-reactions with other parasites are possible. The use of purified glycoprotein antigen does not alter the possibility of cross-reaction. Observations and experiments show that people in Canberra may be exposed to the infective eggs of T. canis.