An agent-based model of exposure to human toxocariasis: a multi-country validation.

Seroprevalence data illustrate that human exposure to Toxocara is frequent. Environmental contamination with Toxocara spp. eggs is assumed to be the best indicator of human exposure, but increased risk of exposure has also been associated with many other factors. Reported associations are inconsistent, however, and there is still ambiguity regarding the factors driving the onset of Toxocara antibody positivity. The objective of this work was to assess the validity of our current conceptual understanding of the key processes driving human exposure to Toxocara. We constructed an agent-based model predicting Toxocara antibody positivity (as a measure of exposure) in children. Exposure was assumed to depend on the joint probability of 3 parameters: (1) environmental contamination with Toxocara spp. eggs, (2) larvation of these eggs and (3) the age-related contact with these eggs. This joint probability was linked to processes of acquired humoral immunity, influencing the rate of antibody seroreversion. The results of the simulation were validated against published data from 5 different geographical settings. Using simple rules and a stochastic approach with parameter estimates derived from the respective contexts, plausible serological patterns emerged from the model in nearly all settings. Our approach leads to novel insights in the transmission dynamics of Toxocara.

Toxocara seropositivity, atopy and asthma: a study in Cuban schoolchildren.

INTRODUCTION: Evidence suggests that human toxocariasis (HT) could stimulate the onset of allergic diseases such as asthma. More specifically, in subjects having a hypothetical 'atopic genotype', HT could boost preexistent allergy symptoms. We tested the latter hypothesis in Cuba, a country where both asthma and HT are prevalent. MATERIAL AND METHODS: In a group of Cuban school-aged children (n = 958), we investigated the association of Toxocara seropositivity and atopic status with asthma. Toxocara seropositivity was diagnosed with ELISA and atopy by allergen skin prick test. Both physician-diagnosed asthma and current wheeze, as determined by International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire, were considered. Associations were assessed using multivariable logistic regression analyses, with either 'physician-diagnosed asthma' or 'current wheeze' as outcome variable. RESULTS: 40.1% of the children were Toxocara seropositive. Prevalences were 21.7% for current wheeze and 32.7% for physician-diagnosed asthma. The odds of having asthma were almost two times higher in atopic children, but only reached borderline significance (OR=1.90, CI 95%: 0.95-3.80 for physician-diagnosed asthma and OR=1.94, CI 95%: 0.98-3.85 for current wheeze). Toxocara seropositivity and physician-diagnosed asthma were associated (OR=1.51, CI 95%: 1.01-2.26). Moreover, in children without antibodies to Toxocara, being atopic was significantly associated with having physician-diagnosed asthma (OR=2.53, CI 95%: 1.63-3.90), while this association was not present in Toxocara positives (OR=1.38, CI 95%: 0.82-2.37). CONCLUSION: Our data confirm previous observations of higher Toxocara seropositivity rates in asthmatic children. Toxocara seropositivity appeared to abrogate the apparent association between atopy and asthma in Cuban children. Although this observation was limited to physician-diagnosed asthma, it challenges the hypothesis that HT stimulates the onset of allergic diseases such as asthma in atopic individuals.

Frequency of antibodies to Toxocara in Cuban schoolchildren.

OBJECTIVE: The aim of the study was to determine the frequency of antibodies to Toxocara in Cuban schoolchildren. METHODS: The frequency of antibodies to Toxocara canis was assessed with a commercial enzyme-linked immunosorbent assays kit in school-aged children from two municipalities of Cuba. Univariate analysis and a multivariable logistic regression analysis adjusted for age, sex, municipality and co-infection with helminth and/or protozoa were conducted. RESULTS: The percentage of children with antibodies to Toxocara was 38.8% (392/1011; 95% CI = 36.8-42.8). Antibody positivity was significantly associated with gender and co-infections with intestinal parasites, but not with age or municipality. CONCLUSION: Cuban children are highly exposed to the Toxocara parasite, corresponding well with reported environmental contamination with Toxocara parasite eggs and T. canis prevalences in dogs in Cuba. Relevant policy makers and the Cuban population need to be better informed about this preventable infection.

Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis.

Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.

Use of ProteinChip technology for identifying biomarkers of parasitic diseases: the example of porcine cysticercosis (Taenia solium).

Taenia solium cysticercosis is a significant public health problem in endemic countries. The current serodiagnostic techniques are not able to differentiate between infections with viable cysts and infections with degenerated cysts. The objectives of this study were to identify specific novel biomarkers of these different disease stages in the serum of experimentally infected pigs using ProteinChip technology (Bio-Rad) and to validate these biomarkers by analyzing serum samples from naturally infected pigs. In the experimental sample set 30 discriminating biomarkers (p<0.05) were found, 13 specific for the viable phenotype, 9 specific for the degenerated phenotype and 8 specific for the infected phenotype (either viable or degenerated cysts). Only 3 of these biomarkers were also significant in the field samples; however, the peak profiles were not consistent among the two sample sets. Five biomarkers discovered in the sera from experimentally infected pigs were identified as clusterin, lecithin-cholesterol acyltransferase, vitronectin, haptoglobin and apolipoprotein A-I.

Serological responses in porcine cysticercosis: a link with the parasitological outcome of infection.

An oral infection model with Taenia solium whole proglottids was used to study host-parasite relationships and the mechanisms underlying resistance to infection in pigs. In addition, an attempt was made to link the parasitological findings to serological data. Groups of six piglets aged 1, 3 and 5 months were infected and slaughtered 3 months p.i. Circulating antibody and antigen levels were monitored weekly. At autopsy total cyst counts were performed. Although the detailed carcass dissection at necropsy revealed a high variation in the number of cysts, the trend was that the number of viable cysts decreased with the age at which the animals were infected. The kinetics of the antigen levels throughout the course of the infection differed markedly between the three age groups of the experimental infection model. In the younger animals, a fast increase in titres of circulating antigen was observed in most animals, reaching a plateau as early as 2 weeks p.i. Besides its faster increase, antigen levels in pigs infected at younger ages also reached higher levels than in older animals and were associated with weaker antibody responses. Results also demonstrated that a relationship exists between the number of cysts and the titre of circulating antigen. This is promising in view of the development of an assay to quantify the progress of an active T. solium infection and would be a useful tool in epidemiological studies to assess the infection burden and the risk of transmission of the disease. The use of specific antibody-detection assays combined with circulating antigen detection could improve our understanding of this relationship.

Combined use of an antigen and antibody detection enzyme-linked immunosorbent assay for cysticercosis as tools in an epidemiological study of epilepsy in Burundi.

OBJECTIVE: To evaluate the benefits of the detection of both circulating antibodies (Ab) and antigens (Ag) for the diagnosis of cysticercosis in people with epilepsy. Neurocysticercosis is a cause of neurological diseases world-wide, especially epilepsy. The clinical symptoms of neurocysticercosis are non-specific and diagnosis is often difficult. METHODS: Serum samples were collected from subjects in a matched case-control study for epilepsy in the Kiremba area, Burundi, between March and April 2001 (epileptic cases=303; controls without epilepsy=606). The enzyme-linked immunosorbent assay (ELISA) was used for the detection of antibodies (Ab-ELISA) and circulating Ag (Ag-ELISA). RESULTS: The Ab-ELISA revealed 58.7% positivity in epilepsy cases and 31.4% in healthy controls; and Ag-ELISA revealed 38.3% positivity in epilepsy cases and 20.0% in controls. The matched odds ratios were 3.6 (95% CI: 2.5-4.9) for Ab-ELISA, and 2.9 (95% CI: 2.1-4.3) for Ag-ELISA. CONCLUSION: Both Ag- and Ab-ELISA detected a significantly higher number of seropositives among people with epilepsy than among controls. The risk of epilepsy was high in cases with a positive Ag-ELISA, although less important than in cases with positivity for Ab-ELISA. Dead or degenerating cysticerci appear to be more frequently associated with epilepsy than living cysts. The high number of people with circulating Ag of Taenia solium suggests that the study area is a focus of active transmission of the parasite.

Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its evaluation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis.

A fraction with a major band of 14kDa was obtained from crude cyst fluid of Taenia solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration (Sephacryl S-300 high resolution) was purified using an anion exchange column (Mono Q HR 5/5) on high performance liquid chromatography. Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody enzyme linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense and Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with metacestode cyst fluid of T. hydatigena and T. solium. Antibody titres of lightly or heavily infected pigs differed in their kinetics. However, the increase in F3-specific antibodies could not be related to the infection level. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using cyst fluid as antigen, but the F3 antigen fraction was not recognized by rabbit hyper-immune serum samples to T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric receiver operating characteristic (ROC) analysis using 66 serum samples from Zambian village pigs. The total number of cysticerci of these pigs was determined by dissection (28 pigs harboured T. solium cysticerci and 38 were negative at dissection). In addition, 58 serum samples from Cameroonian pigs (28 pigs from cysticercosis-free farms and 30 pigs with cysticerci at tongue inspection) were used in a separate ROC analysis. The results from the ROC analysis yielded a low diagnostic value (area under ROC curve=0.48) with the sera from the Zambian pigs while a relatively high diagnostic value was obtained with the sera from Cameroonian pigs (area under ROC curve=0.78). The main factor contributing to a low diagnostic value based on the Zambian serum samples seemed to be the false-positive reactions that were likely caused by the occurrence of transient antibodies in the non-infected animals.

Individual variation and effect of priming dose level on establishment, growth and fecundity of Cooperia oncophora in re-infected calves.

We investigated the effect of bovine anamnestic immunity on a range of parasitological variables. To this end, calves were primed with a single oral dose of 30000 or 100000 infective larvae (L3) of Cooperia oncophora, drenched with anthelmintic, maintained worm free in the following 2.5 months and subsequently re-infected with 100000 L3. Parasitological profiles of low, intermediate, and high responders were compared. The reduction in establishment of the worms was shown by a lower worm burden and increased percentage of fourth-stage (L4) larvae. Worm length and fecundity were similarly reduced by both priming doses but, the speed by which the effect occurred differed between animals primed with 30000 or 100000 L3. The difference in establishment between the responder types demonstrates that the ability of intermediate responders to mount a more effective and faster immune response compared to low responders is sustained after secondary infection.

Vaccination against the nematode Haemonchus contortus with a thiol-binding fraction from the excretory/secretory products (ES).

Fractionated excretory/secretory products (ES) of adult Haemonchus contortus were evaluated as protective antigens. The proteins were successively eluted from a Thiol Sepharose column using 25 mM cysteine followed by 25 mM Dl-dithiothreitol (DTT). Sheep were vaccinated three times and challenged with 5000 third stage infective larvae (L3) of H. contortus. Highest level of protection was found in sheep vaccinated with the DTT-eluted fraction in which egg output and worm burden were reduced by 52 and 50%, respectively, compared to the adjuvant control group. There was a positive correlation between fecundity (number of eggs per female) and the cumulative EPG or worm burden. Serum and mucus antibody levels of ES-specific immunoglobulins increased after immunizations and after challenge for IgG, IgA and IgE. The harvesting of H. contortus from animals clustered per group revealed the presence of cysteine protease activity in the ES of all groups but in addition to that, metalloprotease activity was also detected in the groups vaccinated with the DTT-eluted fraction, total ES and adjuvant only, in contrast to previous batches of ES (completely inhibited by E64) obtained from non vaccinated animals.

B cells and antibody response in calves primary-infected or re-infected with Cooperia oncophora: influence of priming dose and host responder types.

We investigated whether the generation of protective memory humoral immunity in Cooperia oncophora infected calves occurs in a dose-dependent way and whether it depends on the animal responder types. To this end, serum and mucus antibody responses were measured in animals primary-infected with 30000 or 100000 L3, treated with anthelmintics and subsequently challenged with 100000 L3. A detailed phenotypic and functional analysis of B cells was done in animals infected once or twice with 100,000 L3. Based on the similarity in parasitological variables of animals primed with 30000 or 100000 L3, we concluded that with these doses priming conferred protection in a dose-independent way. Upon challenge significant increases in Cooperia-specific serum and mucus IgG1 and IgA and total serum IgE titres were induced in primed animals in a dose-independent way. In contrast, intermediate and low responders differed in the onset of the production of Cooperia-specific serum IgG1. Furthermore, not only the onset but also the level of total serum IgE significantly differed between intermediate and low responders. Phenotypic and functional analysis of B lymphocytes revealed that (i). priming induced the generation of memory B cells which upon challenge readily differentiated into antibody secreting cells; (ii). sensitised B cells were more efficiently recruited to the intestinal effector sites; (iii). based on the expression of CD62L and CD86 two distinct B cell subpopulation could be differentiated. CD62L(+)CD86(-) B cells that were likely lymphocytes not yet activated and with an enhanced recirculation capacity, and CD62L(-)CD86(+) B cells that were activated B cells with a reduced recirculation ability; and finally (iv). the increased expression of CD86 and subsequent correlations with parameters of the T helper 2 immune response induced by C. oncophora, suggested that CD86- interactions are involved in the generation of protective immunity against Cooperia.

T-cell mediated immune responses in calves primary-infected or re-infected with Cooperia oncophora: similar effector cells but different timing.

Cooperia oncophora is the most prevalent intestinal nematode of cattle occurring in Western Europe. Primary infection with 100000 third stage infective larvae (L3) induces acquired immunity in a high proportion of the animals but there is little information on immunity against re-infection. In the current experiment, the contribution of the T-cell mediated immunity in protection against re-infection with C. oncophora was investigated in detail. Priming elicited long-lasting protective immunity that was evidenced by a significantly decreased worm burden and egg excretion in primed animals compared to challenge control animals. Lymphocyte proliferation tests with excretory/secretory products (ESP) of C. oncophora and with three distinct ESP fractions indicated an enhanced reactivity in primed animals and suggested that by fractionating of ESP we selected for proteins involved in protective immunity against re-infection with C. oncophora. Phenotypic analysis of T cell subsets at diverse anatomical locations revealed that the enhanced reactivity of lymphocytes from peripheral blood and lymph nodes of the infected animals coincided with a significantly increased frequency of CD4(+) cells at these locations but a deceased frequency of CD4(+) cells in the lamina propria. These findings were independent of the immune status of the animals but more pronounced in the primed animals than in the challenge control animals. In addition we demonstrated that primary and secondary infections with C. oncophora were associated with two waves of eosinophils and that the kinetics of this cell population differed as a result of priming. Based on the observed correlations we propose that the early increase of eosinophils is T cell independent and merely a consequence of inflammation in the parasitised gut. In contrast, the second wave of eosinophils depends upon CD4(+) cells and correlations with parasitological parameters at this time point support a role of eosinophils as effector cells against adult stages of C. oncophora.

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA.

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.

Characterization of host responder types after a single Cooperia oncophora infection: kinetics of the systemic immune response.

After primary infection with 100,000 third stage larvae of the intestinal nematode Cooperia oncophora in 3-month-old calves, a high variability in egg output and worm counts is observed. Based on this variability, infected animals can be divided in different responder types. The three major phenotypes can be classified as high, intermediate and low responder animals. We investigated whether calves classified into different responder types show different immune responses during infection. Peripheral blood eosinophil counts and flow cytometric analysis of different lymphocyte subsets of the blood did not reveal major differences between infected and control animals, nor between responder types. However, the levels of Cooperia-specific immunoglobulin (Ig)G1 and IgA during primary infection were significantly higher in intermediate responders than in low responders. In the intermediate responders, isotype specific responses were negatively correlated with parasitological parameters expressing worm expulsion and influence on worm fecundity. Total serum IgE levels were elevated in most of the infected animals. A quantitative positive relationship between worm counts and total serum IgE levels was observed. Based on the observed correlations, we propose a role for the humoral response against the maintenance of the infection in the gut.