Excretory-secretory products of Toxocara canis larvae have been considered as a major functional antigen in immune responses against toxocariasis. We studied ultrastructural localization of T. canis second-stage larval antigen using a seropositive human serum under immunogold electron microscopy. High-density gold particles were observed in the secretory cells, excretory duct, intestinal epithelium, and cuticle of the larval worm sections. The distribution of the positive reactions in the larval worms suggests that the nature of the antigen is excretory-secretory antigen including waste metabolites and secretory enzymes.
Animals ; Antigens, Helminth/*immunology/ultrastructure ; Humans ; Larva/*immunology/ultrastructure ; Toxocara canis/*immunology/ultrastructure ; Toxocariasis/*immunology

The third-stage larvae (L3) of the parasitic nematode, Anisakis simplex, have been implicated in the induction of hyperimmune allergic reactions in orally infected humans. In this work, we have conducted a review of an investigation into immune reactions occurring in animals experimentally infected with A. simplex L3. The patterns of serum antibody productions in the experimental animals against excretory-secretory products (ESP) of A. simplex L3 contributed to our current knowledge regarding specific humoral immune reactions in humans. In our review, we were able to determine that L3 infection of experimental animals may constitute a good model system for further exploration of immune mechanisms and allergy in anisakiasis of humans.
Rats ; Mice ; Larva/immunology ; Immunoglobulin E/blood ; Hypersensitivity/*etiology/immunology ; Humans ; Enzyme-Linked Immunosorbent Assay ; Disease Models, Animal ; Antigens, Helminth/immunology ; Anisakis/growth & development/*immunology ; Anisakiasis/immunology ; Animals

The furcocercus cercariae of Neodiplostomum seoulense (Digenea: Neodiplostomidae) penetrate the skins of tadpoles and shed their tails. The speculated mechanism of this tail loss was physical efforts required to produce a vigorous zigzag motion during skin penetration; no other mechanism has been proposed. We examined the relationship between the host serum and cercarial tail loss. Cercariae of N. seoulense were collected from experimentally infected Segmentina hemisphaerula, and lots of 300 cercariae were cultured in medium 199 contained several types of sera. Cercarial tail degradation was induced in all media, but all the cercariae cultured except those cultured in media containing fetal bovine serum (FBS) died within 48 hr. After 72 hr cultivation in media containing FBS, cercarial tail degradation was induced in 67.0%; in continuous cultivation 13.3% of larvae survived for 7 days. Tail degradation did not occur in the absence of serum and when serum was heat inactivated at 56 degrees C for 30 min. The addition of 20 mM ethylenediaminetetraacetic acid (EDTA) blocked cercarial tail degradation completely. Moreover, the addition of 20 mM MgCl2 restored tail degradation blocked by EDTA. These results suggest that the alternative complement pathway is related with the N. seoulense cercarial tail degradation induced by serum.
Trematoda/*physiology ; Tail/*physiology ; Larva/parasitology ; Complement System Proteins/immunology/*physiology ; Anura/parasitology ; Animals

OBJECTIVETo analyze the diagnostic value of larval excretory-secretory antigen in Angiostrongylus cantonensis (LESA) infection.

METHODSA.cantonensis larvae harvested from mice brain were cultured in vitro. The LESA and the adult worm antigens of A.cantonensis (AWA) were collected and analyzed using SDS-PAGE and Western blotting. Two ELISA systems were established using the two antigens (LESA-ELISA and AWA-ELISA) to detect the serum spectra from different sources.

RESULTSSDS-PAGE and Western blotting displayed fewer protein and antigen bands for LESA than for the adult antigen. Two distinct bands of LESA (with relative molecular masses of 40 000 and 26 000) showed reactivity with the sera from patients with A. cantonensis infection. The serum levels of IgG and IgM antibodies to LESA increased at the beginning of infection in mice, reaching the peak on day 5 after infection and decreased on day 10. Compared with AWA-ELISA, LESA-ELISA showed a lower seropositive ratio in suspected patients with A.cantonensis, with also a lower cross-positive ratio in patients with schistosomiasis and clonorchis sinensis.

CONCLUSIONLESA possesses fewer antigen reaction bands than AWA. Although with a slightly lower positive ratio than AWA, LESA has a higher specificity for detecting serum antibodies in suspected cases of A.cantonensis infection, and therefore shows a potential for the diagnosis of angiostrongyliasis especially in the early stage and in current infection.

Angiostrongylus cantonensis ; immunology ; Animals ; Antigens, Helminth ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Larva ; immunology ; Mice ; Mice, Inbred BALB C ; Rats ; Rats, Sprague-Dawley ; Strongylida Infections ; diagnosis ; parasitology

A 15-year-old boy, who had recently arrived back from a trip to Cambodia for a missionary camp, presented with several serpiginous thread-like skin lesions that began as small papules on the left upper extremities 2 weeks before his visit to Hospital. The skin lesions were pruritic and erythematous, and had migrated to the chest and abdomen. The histopathological findings showed only lymphocytic and eosinophilic infiltrations in the dermis of the biopsied skin lesion. The patient's serum reacted strongly to the Ancylostoma caninum antigen by an ELISA method. Therefore, he was diagnosed with cutaneous larva migrans by A. caninum. After the oral administration of albendazole and ivermectin, the skin lesions resolved without recurrence. This is the first reported case of a cutaneous larva migrans caused by Ancylostoma canimum diagnosed serologically using ELISA in Korea.
Adolescent ; Ancylostoma/*immunology ; Animals ; Antibodies, Helminth/*blood ; Enzyme-Linked Immunosorbent Assay ; Humans ; Larva Migrans/*diagnosis/parasitology ; Male

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.
Adolescent ; Adult ; Aged ; Animals ; Antigens, Helminth/chemistry/*diagnostic use/immunology/isolation & purification ; Cats ; Dogs ; Enzyme-Linked Immunosorbent Assay/*methods ; Female ; Humans ; Larva/chemistry/immunology ; Male ; Middle Aged ; Serologic Tests ; Toxocara canis/chemistry/*immunology ; Toxocariasis/*diagnosis/immunology/parasitology ; Young Adult

Roundworms of Toxocara canis and Toxascaris leonina are common gastrointestinal helminths of canids over the world. Humans are infected with T. canis larvae through ingestion of infective eggs in contaminated environments or larvae by consumption of raw or uncooked meat or livers. Recently, patients of clinically diagnosed toxocariasis are increasing and require correct diagnosis in Korea. The present study investigated serological cross-reactivity between crude antigens of T. canis (TCLA) and T. leonina (TLLA) larvae. We collected serum specimens from 177 toxocariasis patients who were clinically suspected in the Seoul National University Hospital and 115 healthy controls. An ELISA method for toxocariasis was used to evaluate diagnostic efficacy of TLLA for serodiagnosis of human toxocariasis. The IgG ELISA using TLLA gave 14 (14.3%) positives of 98 TCLA positive specimens among 177 suspected toxocariasis patients. Most of them showed high absorbances with TCLA. In conclusion, there is a partial cross reaction between serum specimens of toxocariasis and TLLA.
Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/*immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Larva/immunology/metabolism ; Toxascaris/growth & development/*immunology/isolation & purification ; Toxocara canis/growth & development/*immunology/isolation & purification ; Toxocariasis/*diagnosis/parasitology

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-alpha (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.
Administration, Intranasal ; Animals ; Anisakiasis/*immunology/parasitology ; Anisakis/*immunology/metabolism ; Bronchoalveolar Lavage Fluid ; Chemokines/metabolism ; Cytokines/analysis/*metabolism ; Eosinophils/metabolism ; Female ; Gene Expression Regulation/*immunology ; Helminth Proteins/*immunology ; Hypersensitivity/*immunology/parasitology ; Immunoglobulin E/immunology ; Immunoglobulin G/immunology ; Larva/immunology/metabolism ; Lung/metabolism ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins/immunology ; Th17 Cells/metabolism ; Th2 Cells/metabolism

OBJECTIVETo study the inducible antibacterial activity of the hemolymph from housefly larva and analyze the antibacterial molecules.

METHODSThe hemolymph was collected from the third instar housefly larvae 48 h after pricking treatment. Nine standard bacterial strains were used for determination of the antibaterial activity of the collected hemolymph and its combination with ampicillin. The anti-yeast activity of the hemolymph and its mixture with fluconazol was also assayed. The antibacterial molecules in the hemolymph was analyzed by SDS-PAGE.

RESULTSThe growth of E.coli, Staphylococcus aureus, Staphylococcus albus, subserotypes of Shigeila flexneri, Bacillus proteus, Bacillus subtilis, Bacillus typhi, Bacillus paratyphosus, and Micrococcus lysodeikticus could be inhibited by the hemolymph collected from housefly larva, and the effect differed significantly between the groups (Plt;0.001). The hemolymph produced the strongest antibacterial activity against Micrococcus lysodeikticus, and the combination of the hemolymph with ampicillin most conspicuously inhibited the growth of Staphylococcus albus. The hemolyph and fluconazol exhibited obvious synergistic effect against yeast. SDS-PAGE identified some specific antibacterial molecules in the hemolymph.

CONCLUSIONThe induced hemolymph from housefly larva possesses strong antibacterial and antifungal activities especially against Micrococcus lysodeikticus. The hemolymph in combination with ampicillin produces the strongest effect against Staphylococcus albus, and fluconazol can significantly enhance the anti-yeast activity of the hemolymph through a synergistic mechanism.

Ampicillin ; pharmacology ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antifungal Agents ; pharmacology ; Bacteria ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Fluconazole ; pharmacology ; Hemolymph ; immunology ; Houseflies ; immunology ; Larva ; immunology ; Microbial Sensitivity Tests ; Yeasts ; drug effects

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.
Animals ; Anthelmintics/therapeutic use ; Antibodies, Helminth/blood ; Biological Markers/blood ; Diethylcarbamazine/therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Human ; Immunoglobulin E/blood ; *Larva Migrans, Visceral/diagnosis/epidemiology ; Toxocara/immunology