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Toxocara cati is one of the causative agents of human toxocariasis. Serological methods are used for diagnosis in paratenic hosts like humans but the humoral immune response triggered by this parasite is unknown. We characterized the humoral immune response to T. cati excretory-secretory antigens (TES) in pigs as animal model during the acute and chronic stages of infection. ELISA and Western Blot techniques were used to determine antibody response. Pigs were experimentally inoculated with 100,000 infective Toxocara cati eggs. Blood was collected at 7, 14, 21 and 28 days post-inoculation (d.p.i.) to assess the acute stage of infection and 90, 120 and 180 d. p.i. for chronic stage analysis. ELISA showed values higher than the cut-off of specific IgM and IgG at 7 d. p.i. with significant differences at 0 and 7 d. p.i. for IgM and at 14, 21 and 28 d. p.i. for IgG in the acute stage. Higher and stable levels were detected in the chronic stage. Western Blot showed bands from 102 to 38 kDa detected by specific IgM and IgG. More immunogenic bands were identified by specific IgG. In the chronic stage of infection a band near 31 kDa was the only band detected by IgM until 150 d. p.i. Specific IgG recognized bands between 102 and 31 kDa. This study demonstrates how the humoral immune response evolves in the acute and chronic stages of infection and provides evidence on the role of the pig as a paratenic host of T. cati., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Detection of ascarid excreted or secreted (E/S) molecules is an alternative approach to the identification of infection by egg flotation. E/S molecules serve as direct markers for the ascarid nematode commonly found in cats and dogs (Toxocara spp., Toxascaris leonina and Baylisascaris procyonis). The nematode derived E/S material mixes with the intestinal contents of the host animal and is available for detection as a coproantigen in the host's faeces. Antigen capture immunological techniques allow sensitive coproantigen detection. Different patterns of antigen to egg agreement are demonstrated in an experimental Toxocara canis infection throughout the prepatent, patent, and post-treatment phases. Examination of faecal samples from a large field population of dogs and cats tested for both egg shedding and antigen indicates that more infections were identified by antigen. Host age influences the agreement of antigen and Toxocara egg results. Older dogs and cats were less likely to have a patent infection (egg positive and antigen positive) result pattern. An egg observation in the absence of antigen detection may indicate a spurious egg. The impact of spurious eggs was further examined by comparisons of cohorts of dogs separated by presence or absence of a pseudoparasite observation or by egg semi-quantification bin. Lastly, the antigen to egg agreement was calculated for other ascarid species., (© 2020 Elsevier Ltd All rights reserved.)

Toxocariasis is a geohelminth zoonosis with worldwide distribution, mainly transmitted through the ingestion of embryonated eggs of nematodes of the Toxocara genus. The disease can also be transmitted to humans as a result of eating raw or undercooked meat of paratenic hosts, such as chickens. Here, we standardized an enzyme-linked immunosorbent assay (ELISA) for evaluating experimentally the kinetic and avidity index (AI) of IgY in broiler chickens infected with different doses of Toxocara canis eggs (G1:100; G2: 1000; and G3: 5000; n = 12 per group). The test showed 91.7% sensitivity (CI 95%: 77.5-98.3) and 100% specificity (CI 95%: 92.6-100), and highest efficiency (97.0%) at 60 days post infection. Infection was characterized by the presence of high avidity antibodies in the chronic phase. Our results support that the ELISA can be a highly useful tool for the detection of anti-Toxocara antibodies in chickens., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human toxocarosis is an important zoonosis caused by larvae of Toxocara canis/cati. The objective was to evaluate the role of IgG anti-Toxocara antibody detection and the specific IgG avidity in diagnostics of human toxocarosis. Anti-Toxocara IgG antibodies and IgG avidity were evaluated by excretory-secretory (ES)-enzyme-linked immunosorbent assay (ELISA). The IgG anti-Toxocara seroprevalence in people (n = 7678) from western Slovakia was 15.3% and found to be highest in the oldest age groups. The presence of low- IgG avidity in 179 suspected patients for toxocarosis was evaluated in relation to sex, age, IgG antibody levels, eosinophilia, increased total IgE, domicile, geophagia, dog/cat ownership, anamnesis. Low- IgG avidity index was found in 30.7% of the patients. The low- IgG avidity in eosinophilic group (42.1%) was significantly higher than in non-eosinophilic group (22.0%; P = 0.043). Substantially higher eosinophilia was detected in children (under 10 years old; 55.6%) than in adults (aged ≥ 41 years; 17.6%; P = 0.009). Significant difference between seroprevalence of total IgE in patients coming from towns (48.8%) and patients from villages (21.3%) was established (P = 0.007). Mild negative correlation (r = -0.477, P = 0.043) was observed between the amounts of eosinophils and the values of IgG avidity. The sensitivity and specificity of IgG avidity assay were 43.8% and 83.3%, respectively. Our results suggest that besides anti-Toxocara IgG, measurement of IgG avidity may be useful for the determination of acute toxocarosis. Moreover, these tests should be accompanied by other immunological markers and determinants of examined patients such as eosinophilia, increased total IgE and age., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Toxocariasis is a soil-transmitted helminthic disease due to infection of humans by larvae of Toxocara canis (T. canis). It is one of the most commonly reported zoonotic infections in the world. The aim of this study was to characterize the key immune cells and activity of Bcl-2 in hepatic inflammation during the course of experimental infection by T. canis. Mice experimentally infected with T. canis were divided into two groups: mice with primary infection by Toxocara, and those infected after sensitization by Toxocara excretory-secretory antigen. CD4+, CD8+, and Bcl-2-expressing T lymphocytes were identified in the liver by immunohistochemistry at different durations post-infection. Recruitment of both CD4+ and CD8+ T lymphocytes within the inflammatory reaction in the liver was observed, with difference in count and localization. These cells were detected within and around Toxocara-induced granulomas as well as in isolated inflammatory foci in the portal tracts or within the hepatic parenchyma. The antiapoptotic protein Bcl-2 showed no significant change at different periods post-infection. On the other hand, immunization of mice with Toxocara excretory-secretory antigen prior to experimental infection caused earlier and more pronounced recruitment of CD4+ and CD8+ T cells to the liver and enhanced expression of Bcl-2. Moreover, CD8+ cells became more diffuse within the inflammatory infiltrate. These results suggest a dynamic change in key immune cells according to the duration of infection as well as the immune status of the host., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Toxocara canis is a widespread intestinal nematode parasite of dogs, which can also cause disease in humans. We employed an expressed sequence tag (EST) strategy in order to study gene-expression including development, digestion and reproduction of T. canis. ESTs provided a rapid way to identify genes, particularly in organisms for which we have very little molecular information. In this study, a cDNA library was constructed from a female adult of T. canis and 215 high-quality ESTs from 5'-ends of the cDNA clones representing 79 unigenes were obtained. The titer of the primary cDNA library was 1.83×10(6)pfu/mL with a recombination rate of 99.33%. Most of the sequences ranged from 300 to 900bp with an average length of 656bp. Cluster analysis of these ESTs allowed identification of 79 unique sequences containing 28 contigs and 51 singletons. BLASTX searches revealed that 18 unigenes (22.78% of the total) or 70 ESTs (32.56% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest of the 61 unigenes (77.22% of the total) or 145 ESTs (67.44% of the total) were closely matched to the known genes or sequences deposited in the public databases. These genes were classified into seven groups based on their known or putative biological functions. We also confirmed the gene expression patterns of several immune-related genes using RT-PCR examination. This work will provide a valuable resource for the further investigations in the stage-, sex- and tissue-specific gene transcription or expression., (Copyright © 2011. Published by Elsevier Inc.)

In this study, Mongolian gerbils were used to analyse features of Toxocara infection that included larval migration, humoral immune responses to Toxocara canis excretory-secretory antigens (TES) and aspects of host physiology. At day 10 post-infection (p.i.) most larvae were in the intestine and the lungs while later the total number of larvae was higher in the carcass tissue; the number of larvae per gram of tissue was lower elsewhere other than in the brain. Infected animals showed several neurological abnormalities, an early increase in leukocyte and neutrophil levels, two peaks of peripheral eosinophilia (5 and 40 d.p.i.) and high antibody levels against TES in the circulation and in the vitreous humor. A sequential recognition of eight T.canis larval antigens with MW from 24 to 200 kDa was detected by Western blot. The results obtained in this study further support the use of gerbils as an experimental model for systemic, ocular and cerebral toxocariasis.

Infection of mice with Toxocara canis results in pulmonary inflammation and the induction of a Th2 type of immune response. The aim of this study was to determine whether the effect of infection with this nematode depends on the inoculum size. Results indicate that mice infected with either a high or a low inoculum size showed, in a dose-dependent manner, pulmonary inflammation with eosinophil infiltration, increased levels of total IgE, and Toxocara-specific IgG1 that persisted up to 60 days p.i. Relative quantification of cytokine expression in lungs of mice infected with different doses showed proportional increased expression of the IL-4, IL-5, and IL-10 transcripts, whereas the expression of the IFN-gamma transcript was not different from that of uninfected controls. These results indicate that infection of BALB/c mice with T. canis results in chronic pulmonary inflammation and a dominant Th2 type of immune response, independent of the inoculum size.

The present study was conducted to better understand the immure response to Toxocara canis pneumonia in mice with preweaning nutritional deprivation. Breast-fed Swiss mice, undernourished due to large litter size (up to 15 pups) and paired controls with only 5-8 pups were used. At 21 days old, both groups were infected with T. canis larvae. Liver retinol, retinyl palmitate, and inflammatory infiltrate in lungs were compared in both groups. Significantly lower levels of retinol and retinyl palmitate in liver tissue confirmed the hypovitaminosis A (P < 0.0001 for both comparisons) in the nutritionally deprived animals. Histological analysis showed similar eosinophilic infiltration in both groups at day 3 but was significantly more severe in undernourished mice at day 20 post-infection (P = 0.01). The present findings indicate that preweaning undernourishment is associated with a more severe inflammation in response to T. canis pneumonia. It suggests that vitamin A deficiency that persists after nutritional rehabilitation, may contribute to the severity of T. canis infection. The authors suggest that nutritional status should be carefully investigated in patients with more severe clinical findings.

We examined eosinophil chemotactic activity (ECA) in bronchoalveolar lavage fluid (BALF) obtained from rats infected with Toxocara canis. For 4 weeks after infection, the number of eosinophils was determined in peripheral blood and BALF. ECA was assayed using a microchemotaxis chamber. Eosinophils in peripheral blood and BALF increased markedly after infection, peaking at 12 days and 2 weeks, respectively. ECA in BALF also increased significantly and peaked 2 weeks after infection. Partial characterization revealed that ECA was heat labile, lipid soluble, and resistant to trypsin digestion. Two ECA peaks were identified by molecular sieve column chromatography: one near the egg albumin marker (MW 45,000) and the other observed after elution with quinacrine (MW 472.9). Treatment with a specific leukotriene (LT) B4 receptor antagonist (ONO-4057), a platelet activating factor (PAF) receptor antagonist (TCV-309), and an anti-interleukin (IL)-5 monoclonal antibody (TB13) significantly reduced the ECA, suggesting that LTB4, PAF, and IL-5 contribute to the accumulation of eosinophils in the lungs of rats infected with T. canis.

Eosinophils obtained by bronchoalveolar lavage (BAL) from the lungs of mice infected with Toxocara canis were characterized by flow cytometry with respect to cytophilic antibodies and surface Fc receptors. Freshly harvested BAL eosinophils were negative for sIgM, sIgA, sIgE, and Fc epsilon RII. These eosinophils were positive for sIgG1 and Fc gamma RII, although not all Fc gamma RIIs contained bound ligand. Culturing eosinophils for 24 or 48 hr with exogenous IgE and/or IL-4 did not induce IgE binding capacity or Fc epsilon RII expression. IL-4 did not decrease Fc gamma RII expression but did decrease ligand binding capacity by Fc gamma RII. These findings are in marked contrast to the results of studies characterizing the surface of both human and rat eosinophils and may indicate different functional activities for mouse BAL eosinophils in helminth infections.

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.

Infection of CBA/J mice with 250 ova of Toxocara canis results in development of splenic lymphocytes hyperergic to nonspecific stimuli. These studies were performed to see if the granulomatous response to a nonspecific stimulus was exaggerated. Using eosinophil counts and MBSA ear thickening it was demonstrated that concurrent infection with the parasite and immunization with methylated bovine serum albumin (MBSA) elicit no immunologic cross reactions. Eosinophil counts were proportional to the worm burden and unaffected by concurrent MBSA immunization. Embolization of Sepharose 4B beads conjugated to MBSA into the pulmonary vasculature of MBSA-immune mice, elicited large, florid macrophage-rich granulomata while in MBSA-naive mice infected with 10 T. canis ova, the beads elicited small, foreign body reactions. In MBSA-naive mice infected with 250 T. canis ova MBSA-coated beads elicited eosinophil-rich granulomata that were as large or larger than the granulomata in MBSA-immune mice. In mice immune and infected, lesions contained eosinophils in proportion to larval worm burdens. Because MBSA beads elicited large, eosinophilic granulomata in T. canis infected, MBSA-naive mice it is concluded that the lung is hyperergic and the disease process appears similar to other kinds of eosinophilic pneumonitis such as bronchial asthma or Loeffler's pneumonia.

This study was carried out to investigate the nature of the immunological responses which took place in a child who had recently recovered from toxocariasis. She had developed a marked eosinophilia and had high titers of toxocara antibodies. Experiments were performed to examine whether Toxocara canis infective larvae could be killed in the presence of her serum and human eosinophils. Eosinophils with human complement, or this patient's serum, adhered to the surface of the larvae within 10 min. By 40 min, using both light and electron microscopy, it was shown that the cells had flattened against the cuticle and degranulated. However, by 3 hr, eosinophils had begun to detach, and the larvae remained alive for at least 1 week afterward. Further addition of serum or of eosinophils, which were shown to be able to immobilize T. spiralis infective larvae, failed to kill the T. canis larvae. It was concluded that, in this patient, the development of an inflammatory response to a T. canis infection was not associated with the appearance of antibodies capable of inducing eosinophil dependent toxicity to the larvae in vitro. Eosinophil dependent killing mechanisms may be less important than other components of the immune response, in immunity to this parasite in humans.