Toxocara Excretory-secretory Antigens Research Articles

Deglycosylation of Excretory-Secretory Antigens of the Second-Stage Larvae of Toxocara cati Improves Its Efficacy in the Diagnosis of Human Toxocariasis.

Toxocariasis is an important zoonotic infection, especially in tropical areas. One of the significant challenges in the serodiagnosis of human toxocariasis is the cross-reaction of Toxocara antigens with other parasites due to their relatively similar glycan structures. Removing the glycan structure from Toxocara excretory-secretory (TES) antigens may increase the efficacy of these antigens in the diagnosis of toxocariasis. The current study aimed to assess the efficacy of deglycosylated Toxocara cati excretory-secretory (dTES) antigens for the serodiagnosis of human toxocariasis. Toxocara ES antigens were prepared from T. cati second-stage larvae and deglycosylated using sodium hydroxide (NaOH). The TES antigens, along with the dTES antigens, were used in an ELISA as well as a western blotting system for the detection of anti-Toxocara antibodies. Sera samples collected from 30 confirmed cases of toxocariasis, 30 patients with other diseases, and 30 healthy subjects were evaluated by both systems. The sensitivity of TES and dTES ELISA for the diagnosis of human toxocariasis was 96.67% (95% CI = 82.78-99.92) and 93.33% (95% CI = 77.93-99.18), respectively, while the specificity of dTES (88.33%; 95% CI = 77.43-95.18) increased significantly compared to the TES (80.00%; 95% CI = 67.67-89.22). The sensitivity of both antigens was 100% (95% CI = 88.43-100) by the western blotting system. Moreover, the specificity of TES and dTES antigens was 95% (95% CI = 86.08-98.96) and 98.33% (95% CI = 91.06-99.96), respectively, when using the western blotting system. Results of the current study indicate that the chemical removal of the glycan epitopes of T. cati ES antigens significantly reduces cross-reactivity rates with other parasitic infections. Considering the findings of the present study, the dTES antigens seem to be suitable antigens for the serodiagnosis of human toxocariasis.

Neurotoxocariasis.

Human toxocariasis, caused by larvae of the Toxocara spp., is one of the most prevalent zoonosis with a worldwide distribution. Toxocara larvae can cross the blood-brain barrier, invading the central nervous system (CNS), leading to neurotoxocariasis. The clinical presentation consists of a wide spectrum of neurological manifestations such as meningitis, encephalitis, myelitis and cerebral vasculitis, but asymptomatic CNS infection is common. Despite the high seroprevalence, neurotoxocariasis is thought to be rare, even if in many animal models larvae usually migrate to the brain. Indeed, diagnosis of neurotoxocariasis is rarely considered, and the exact prevalence of CNS involvement is still unknown. Diagnosis of neurotoxocariasis is generally based on the detection of specific antibodies to Toxocara excretory-secretory antigens by ELISA in CSF or serum, and clinical and radiological improvement after anthelmintic therapy, but definitive diagnosis is given by histological confirmation, which is rarely available. Treatment is based on anti-helminthic drugs, commonly albendazole, generally administered with concomitant corticosteroids coverage. At the population level, serological studies suggest an association between Toxocara infections and epilepsy, as well as with other neurodegenerative and psychiatric disorders. Nonetheless the possible role of Toxocara spp. in the pathogenesis of these disorders is still matter of debate.

Toxocara canis mucins among other excretory-secretory antigens induce in vitro secretion of cytokines by mouse splenocytes.

The effect of Toxocara larval antigens on cytokine secretion by mouse splenocytes was studied in vitro. Recombinant mucins were produced in Pichia pastoris yeast, and Toxocara excretory-secretory (TES) antigens were collected from in vitro culture of L2 larvae. Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5 were expressed as glycoproteins and were specifically recognized by Toxocara canis-infected dog serum antibodies. Mouse splenocytes stimulated with recombinant mucins produced IL-5, IL-6, and TGF-β. Cell stimulation with whole TES products was more effective and resulted in secretion of IL-4, IL-5, IL-6, IL-10, and TGF-β and downregulation of TNF-α production. IFN-γ and IL-17 secretion was noted only after ConA treatment. Cells originating from infected animals produced significantly smaller amounts of these two cytokines compared to control cells, which suggests that Th1 and Th17 response in infected mice is strongly inhibited. However, splenocyte stimulation with both TES and ConA upregulated the production of IFN-γ and IL-17. This shows that TES antigens have strong immunomodulatory properties and are able to induce a broad range of effects on murine immune cells.

Immunodiagnosis of Human Toxocariasis and Prospects for Improved Diagnostics

The clinical spectrum of human disease caused by Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert and common toxocariasis. Since the parasite is not typically recovered in affected tissues, detection of parasite-specific antibodies is an important and necessary step in establishing a diagnosis of toxocariasis. Most immunodiagnostic methods have historically used and continue to use the Toxocara excretory-secretory antigens (TES-Ag) in an ELISA format to detect Toxocara-specific antibodies. The TES-Ag ELISA has proven to be specific, robust, and reliable, although questions about specificity and reduced sensitivity leave ample room for improvement in laboratory diagnosis of toxocariasis. Recent advances have focused on development of recombinant protein targets to measure Toxocara-specific antibodies. The potential for development of new diagnostics using recombinant proteins is presented herein, as well as possible roles for antigen and molecular detection methods.

Investigation of anti-Toxocara antibodies in epileptic patients and comparison of two methods: ELISA and Western blotting.

The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA) using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8%) and 3 (3.5%) persons exhibited Toxocara immunoglobulin G (IgG) antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (n = 85), 3 (3.5%) persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy.

Toxocara-induced hepatic inflammation: Immunohistochemical characterization of lymphocyte subpopulations and Bcl-2 expression

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.

Updates in Immunoassays: Parasitology

Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal.

An alternate technique for isolation of Toxocara canis excretory-secretory antigens

The aim of the present study was to test the effectiveness of a sausage-casing membrane for dialysis of Toxocara excretory-secretory antigens (TES). The protein concentrated by the tested membrane was compared with that obtained using a Sigma commercial membrane, as were the protein fractions found by polyacrylamide gel electrophoresis. Standard positive and negative serum samples were evaluated in an ELISA immunoassay, and equivalent data were obtained in all steps, indicating that the sausage-casing membrane is efficient, besides being less expensive to process.

Toxocariasis/cysticercosis seroprevalence in a long-term rural settlement, São Paulo, Brazil

Seroprevalence of Toxocara and Taenia solium and risk factors for infection with these parasites were explored in a long-term rural settlement in São Paulo state, Brazil. An ELISA for the detection of anti-Toxocara IgG and IgE and anti-T. solium cysticerci was standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis and by vesicular fluid antigen from Taenia crassiceps cysticerci (VF). For cysticercosis, the reactive ELISA samples were assayed by Western blot using 18 kDa and 14 kDa proteins purified from VF. Out of 182 subjects, 25 (13.7%) presented anti-Toxocara IgG and a positive correlation between total IgE and the reactive index of specific anti-TES IgE (P=0.0265) was found amongst the subjects found seropositive for anti-Toxocara IgG. In these individuals 38.0% showed ocular manifestations. The frequency of anti-T. solium cysticerci confirmed by Western blot was 0.6%. Seropositivity for Toxocara was correlated with low educational levels and the owning of dogs. Embryonated eggs of Toxocara spp. were found in 43.3% of the analysed areas.

Toxocariasis Might be an Important Cause of Atopic Myelitis in Korea

Atopic myelitis is defined as myelitis with atopic diasthesis but the cause is still unknown. Toxocariasis is one of the common causes of hyperIgEaemia that may lead to neurologic manifestations. The purpose of this study was to evaluate the sero-prevalence of Toxocara specific IgG Ab among the atopic myelitis patients. We evaluated the medical records of 37 patients with atopic myelitis whose conditions were diagnosed between March 2001 and August 2007. Among them, the 33 sera were analyzed for specific serum IgG Ab to Toxocara excretory-secretory antigens (TES). All of 37 patients had hyperIgEaemia. Specific IgE to D. pteronyssinus and D. farinae was detected in 22 (64.7%) and 34 (100%) patients, respectively, of the 34 patients. Thirty-one of 33 patients (93.9%) were found to be positive by TES IgG enzyme-linked immunosorbent assay (ELISA). Based on the image findings of eosinophilic infiltrations in the lung and liver, 8 patients had positive results. These results inferred that the prevalence of toxocariasis was high in patients with atopic myelitis. Our results suggest that toxocariasis might be an important cause of atopic myelitis and Toxocara ELISA is essential for evaluating the causes of atopic myelitis.

Development of a highly sensitive IgG-ELISA based on recombinant arginine kinase of Toxocara canis for serodiagnosis of visceral larva migrans in the murine model

Toxocariasis is a worldwide zoonotic disease caused by the ascarid nematode Toxocara canis. The most common method available for serodiagnosis of toxocariasis is an enzyme-linked immunosorbent assay (ELISA) test using Toxocara excretory-secretory antigen (TES). The present study describes the development of IgG-ELISA based on antiserum prepared against the recombinant arginine kinase of Toxocara canis. Antiserum was prepared against the purified recombinant arginine kinase (AK) using 6-week-old female Japanese white rabbits. Serum samples were collected from experimentally infected BALB/c and C57BL/6 mice at different time periods. The IgG-ELISA was performed using serum samples from mice (infected/uninfected) and TES antigen with antiserum prepared against the recombinant-AK. The optical density (OD450) was measured at 450 nm using a micro-plate ELISA reader. There were significant differences (P<0.01) in the absorbance between infected and control serum samples. Further, we obtained 100% sensitivity for the serum samples from T. canis-infected mice. Therefore, it is suggested that the recombinant-AK based IgG-ELISA could be applied for immunodiagnosis of human toxocariasis. However, it is necessary to evaluate the specificity of this recombinant antigen with similar geohelminth infections.

Sero-epidemiology of toxocariasis in a rural settlement in São Paulo state, Brazil

The seroprevalence of Toxocara canis and risk factors for infection with this parasite were explored in a rural settlement in São Paulo state, Brazil. Total IgA and IgE levels in 79 subjects were determined by turbidimetry and chemiluminescence, respectively. Total counts of leucocytes and erythrocytes and differential counts of leucocytes were made by flow cytometry. ELISA for the detection of anti-Toxocara IgG, IgA and IgE were standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis. Seventeen (21.5%) of the subjects were found positive for anti-Toxocara IgG, with no significant differences in such seropositivity with age or gender. Thirty (38%) of the subjects showed eosinophilia and 70 (89%) had elevated levels of total IgE. Among the 17 subjects found seropositive for anti-Toxocara IgG, the percentage of leucocytes represented by eosinophils (P=0.0069) and total levels of IgE (P=0.0452) were positively correlated with the levels of anti-TES IgE. Although anti-TES IgA was detected in 10 (59%) of the subjects, there was no significant correlation between the levels of total IgA and those of Toxocara-specific IgA. Only one of the 17 subjects found positive for anti-Toxocara IgG had attended a secondary school and all but two belonged to households with monthly incomes of <U.S.$100. In the study community at least, seropositivity may be related to poor living standards and lack of basic sanitary conditions. The presence of anti-Toxocara IgE and IgA may facilitate the diagnosis of toxocariasis and may well be useful for monitoring the success of treatment.

Highlights of human toxocariasis.

Human toxocariasis is a helminthozoonosis due to the migration of Toxocara species larvae through human organism. Humans become infected by ingesting either embryonated eggs from soil (geophagia, pica), dirty hands or raw vegetables, or larvae from undercooked giblets. The diagnosis relies upon sensitive immunological methods (ELISA or western-blot) which use Toxocara excretory-secretory antigens. Seroprevalence is high in developed countries, especially in rural areas, and also in some tropical islands. The clinical spectrum of the disease comprises four syndromes, namely visceral larva migrans, ocular larva migrans, and the more recently recognized "common" (in adults) and "covert" (in children) pictures. Therapy of ocular toxocariasis is primarily based upon corticosteroids use, when visceral larva migrans and few cases of common or covert toxocariasis can be treated by anthelmintics whose the most efficient appeared to be diethylcarbamazine. When diagnosed, all of these syndromes require thorough prevention of recontamination (especially by deworming pets) and sanitary education.

Toxocara canis: Monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae

The major secreted glycoproteins of Toxocara canis larvae appear to be derived from two specialized organs within the nematode organism. Using immunogold electron microscopy, we have analyzed the binding patterns of a panel of monoclonal antibodies (Tcn-1 to Tcn-8) reactive with Toxocara excretory-secretory (TES) antigens. We find, first, that the esophageal gland and lumen are strongly reactive with monoclonals Tcn-4, Tcn-5, and Tcn-8, and because the posterior portion of the gut is closed, we hypothesize that products of this gland are released through the oral aperture. Second, a distinct anti-TES antibody (Tcn-2) localizes solely to the midbody secretory column, which opens onto the cuticle at a secretory pore. Thus, the secretory apparatus is probably functional in this stage of parasite as an important source of TES products. Only one monoclonal, Tcn-7, can bind to both esophageal and secretory structures. In addition, another antibody, Tcn-3, binds both to the epicuticle and to a TES antigen, but our data do not directly determine whether antigens located in the cuticle are subsequently released. Thus there are at least two, and possibly three, independent sources of TES antigens within Toxocara larvae.

Characterization of nematode glycoproteins: the major O-glycans of Toxocara excretory-secretory antigens are O-methylated trisaccharides

Toxocara excretory-secretory antigens (TES) were isolated from the culture media of T.canis and T.cati larvae and their O-glycan content was investigated using fast atom bombardment-mass spectrometry (FAB-MS), gas chromatography and electron impact mass spectrometry. The major oligosaccharides released by reductive elimination of T.canis TES glycoproteins were shown to be two, approximately equi-abundant, trisaccharides: 2-O-Me-Fucp(alpha 1----2)-4-O-Me-Galp(beta 1----3)GalNAcitol and 2-O-Me-Fucp(alpha 1----2)-Galp(beta 1----3)GalNAcitol. In contrast T.cati TES O-glycans are predominantly one component, shown by FAB-MS to be a di-O-methylated trisaccharide, which is probably identical to the di-O-methylated trisaccharide from T.canis. The O-methylated trisaccharides are strong candidates for the carbohydrate epitopes recognized by a panel of monoclonal antibodies which exhibit multiple reactivity against TES antigens. This study constitutes the first rigorous characterization of glycans from a parasitic nematode.

Specificity of Toxocara ELISA in tropical populations

The diagnosis of human infection by Toxocara canis relies heavily upon serological tests, the specificity of which can be inadequate in regions of endemic helminthiasis. When different population groups of tropical Venezuela were evaluated using ELISA based upon Toxocara excretory-secretory antigen (TcESA), solid-phase adsorption of the sera with extracts of a wide variety of non-homologous parasites revealed the existence of significant cross-reactivity. This was effectively and conveniently overcome when the test sera were incubated in the presence of the soluble parasite extracts in a competitive inhibition ELISA. The mean reduction of ELISA values caused by pre-adsorption of the sera tested was 32.2%, and that caused by competitive inhibition was 42.3%, the effects of these two procedures being strongly correlated (r = 0.83). The magnitude of the reduction was inversely proportional to the actual ELISA value (r = -0.55), and ranged from a mean of 68.0% in sera from apparently healthy individuals of medium-high socio-economic level, down to 28.1% in heavily parasitized Amazon indians. Ascaris showed the greatest degree of cross-reactivity in these tests, although under conditions of competitive inhibition even sera with high levels of antibody against this parasite could be negative in Toxocara ELISA. Western blotting revealed a major 81,400 D component that was shared between Ascaris and TcESA. Our results indicate that the competitive inhibition of cross-reactivity by soluble non-homologous parasite extracts provides a convenient and economical means of increasing the specificity of ELISA for the determination of the seroprevalence of toxocariasis in tropical populations.