Cross-reactivity of Toxocariasis with Crude Antigen of Toxascaris leonina Larvae by ELISA

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.

Meningitis by Toxocara canis after Ingestion of Raw Ostrich Liver

Recently reports on toxocariasis are increasing by serodiagnosis in Korea. A previously healthy 17-yr-old boy complained of headache, fever, dyspnea, and anorexia. He showed symptoms and signs of eosinophilic meningitis with involvement of the lungs and liver. Specific IgG antibody to Toxocara canis larval antigen was positive in serum and cerebrospinal fluid by ELISA. He took raw ostrich liver with his parents 4 weeks before the symptom onset. His parents were seropositive for T. canis antigen but had no symptoms or signs suggesting toxocariasis. This is the first report of toxocariasis in a family due to ingestion of raw ostrich liver in Korea.

Seroprevalence of Antibodies against Anisakis simplex Larvae among Health-Examined Residents in Three Hospitals of Southern Parts of Korea

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.

A 24 kDa Excretory-Secretory Protein of Anisakis simplex Larvae Could Elicit Allergic Airway Inflammation in Mice

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.

Ultrastructural Localization of Toxocara canis Larval Antigen Reacted with a Seropositive Human Serum

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.

Avaliação da resposta imune humoral frente a antígenos de Strongyloides venezuelensis / Evaluation of the humoral immune response to Strongyloides venezuelensis antigens

A estrongiloidíase afeta 30 milhões de pessoas em 70 países. Usualmente, o diagnóstico dessa enteroparasitose é realizado por testes parasitológicos baseados no hidro termotropismo das larvas eliminadas nas fezes, porém esses têm se mostrado pouco sensíveis. Neste trabalho, extratos antigênicos foram testados pelas técnicas de ELISA, Immunoblotting e IFI, utilizando larvas filarióides de Strongyloides venezuelensis, parasita de roedores, que mostram reação cruzada com epítopos de Strongyloides stercoralis. Sensibilidade de 89, 85, 57 por cento para a reação de ELISA e de 100, 100 e 96 por cento, para o Immunoblotting com os antígenos SAL, ZWIP e ZW, e especificidade de 90, 60 e 81 por cento para o ELISA e 96, 92 e 91 por cento para o Immunoblotting para os mesmos antígenos, foram encontradas nestes ensaios.

Strongyloidiasis affects 30 million people in 70 countries. This enteral parasitosis is usually diagnosed using parasitological tests based on hydrotropism or thermotropism of larvae eliminated in feces, but these tests have been shown to have low sensitivity. In this study, antigenic extracts were tested by means of ELISA, immunoblotting and IFI, using filariform larvae of Strongyloides venezuelensis, a parasite of rodents that shows cross-reactions with Strongyloides stercoralis epitopes. Sensitivity of 89, 85 and 57 percent for the ELISA reaction and 100, 100 and 96 percent for immunoblotting with the SAL, ZWIP and ZW antigens, and specificity of 90, 60 and 81 percent for ELISA and 96, 92 and 91 percent for immunoblotting with the same antigens, were found in these assays.

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794)

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

Protective immunity elicited by ultraviolet-irradiated third-stage infective hookworm (Necator americanus and Ancylostoma caninum) larvae in mice and hamsters.

The protective immunity elicited by ultraviolet-irradiated third-stage infective larvae of Necator americanus (UV-NaL3) and Ancylostoma caninum (UV-AcL3) was evaluated in laboratory mice (a non-permissive model) and hamsters (a permissive model). After optimizing the time of exposure to UV-irradiation, both oral and subcutaneous vaccination routes with UV-AcL3 in mice were explored. Oral vaccination was more effective at reducing the number of challenge AcL3 entering the lungs, whereas subcutaneous vaccination was more effective at blocking muscle entry. When UV-irradiated NaL3 and non-irradiated AcL3 were used as vaccines in hamsters, both of them were effective at reducing adult hookworm burdens. However, the length of protection afforded by UV-irradiated L3 was substantially greater than that resulting from immunization with non-irradiated L3. A single dose was less effective than multiple doses. The protective immunity elicited by UV-irradiated NaL3 given once every other week for a total of three immunizations was similar to that elicited by non-irradiated AcL3 given during the same schedule. Protection was not significantly affected by administering the L3 on a weekly basis for a total of three immunizations, even though the antibody titers were reduced using this schedule. These studies will facilitate the elucidation of the mechanisms underlying larval protection.

Estudo da resposta imune humoral (IgG específica) para antígenos de larvas infectantes (L3) de Wuchereria bancrofti, entre portadores e não portadores de filariose bancroftiana no Município de Olinda-PE / Study of the immune humoral response (IgG specific) for antigens of infective larvae (L3) of Wuchereria bancrofti, between bearers and not bearers of elephantiasis filarial in the city of Olinda-PE

A filariose linfática bancroftiana é uma doença parasitária humana de grande complexidade em sua dinâmica de infecção, necessitando ainda de maiores esclarecimentos, principalmente, em aspectos relacionados à tolerância e imunopatologia. A existência daimunidade protetora em comunidades endêmicas de filariose permanece como objeto de intenso debate e o grupo denominado “endêmicos normais”, tem sido alvo de interesse para elucidar muitas questões referentes à imunologia da doença. O presente trabalho tem como objetivo verificar através de um estudo do tipo caso-controle, as diferenças entre portadorese não portadores de filariose linfática bancroftiana pelo reconhecimento humoral de bandas protéicas de extrato secretório-excretório de larvas infectantes de Wuchereria bancrofti. Osquatro setores censitários de Olinda-PE, com maior prevalência de filariose, foram escolhidos como área de estudo. Consideramos “grupo controle” portadores da filariose bancroftiana e “grupo de casos” os de endêmicos normais. O extrato antigênico foi obtidopor incubação de larvas infectantes em meio Hanks por 72h em 5% de CO2. As bandas protéicas foram separadas por eletroforese (SDS-PAGE) e por western-blot, transferidas e incubadas com os soros humanos. Foram considerados portadores da doença os positivospela técnica da gota espessa ou filtração e endêmicos normais os residentes de área endêmica negativos pelo teste de detecção parasitária e captura de antígenos (Og4C3). Participaram do estudo 325 indivíduos, 130 considerados endêmicos normais e 195portadores de filariose bancroftiana. As bandas protéicas da composição do extrato de L3 foram as de 200, 175, 138, 105, 100, 76, 55, 49, 42, 39, 38, 32, 28, 14 kDa. Na comparaçãodo reconhecimento das proteínas entre os grupos estudados, apenas as proteínas 175 e 105 kDa não diferiram significativamente, os portadores de filariose bancroftiana não reconheceram a proteína 175 e no grupo...

Immune reactions and allergy in experimental anisakiasis

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.

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti / Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae

A caracterização protéica dos extratos de larvas infectantes (L3) de Wuchereria bancrofti foi realizada por eletroforese em gel de poliacrilamida, em presença de dodecil sulfato de sódio (SDS-PAGE) e o reconhecimento antigênico das proteínas por Western-blot. O maior número de bandas protéicas reconhecidas foi evidenciado nos extratos AgSE (105, 100, 76, 55, 49, 39 e 32 kDa) e AgS (100, 76, 55, e 49 kDa) na presença de soros de indivíduos endêmicos normais. As bandas de 49 e 55 kDa foram reconhecidas pelos soros dos microfilarêmicos, endêmicos normais (residentes de área endêmica livres de infecção filarial) e portadores da forma crônica da doença. As larvas infectantes foram obtidas pela dissecção de mosquitos Culex quinquefasciatus infectados com sangue microfilarêmico de voluntários portadores de microfilaremia, residentes do Município de Olinda-PE. Nos 792 indivíduos investigados pela técnica da gota espessa mensurada (60æl de sangue) 87 foram positivos (11 por cento). A diferenca da positividade entre homens e mulheres não foi significativa e a faixa etária de 11 a 19 anos foi a de maior prevalência.

Pineapple juice for digestion of swamp eel viscera for harvesting infective-stage larva of Gnathostoma spp.

Third-stage larvae were used as antigen in the diagnosis of gnathostomiasis in Western blot analysis. Normally, the larvae were obtained from digestion of eel's liver (Fluta alba) by the enzyme pepsin. We used pineapple juice (Ananus comosus) instead of enzyme pepsin in harvesting Gnathostoma spinigerum third-stage larvae. The difference in recovered larvae numbers, between pineapple juice and pepsin, were not statistically significantly different (p>0.05). The larvae from pepsin and pineapple juice digestion were cultivated on BME for 7 days; the survival rates were not significantly different (p>0.05). Thus, pineapple juice is another enzyme of choice for recovering Gnathostoma spinigerum third-stage larvae.

Defense reactions of Dermatobia hominis (Diptera: Cuterebridae) larval hemocytes

The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis. The cellular defense performed by larval hemocytes was observed under electron microscopy. China ink particles were phagocytosed by granular cells 5 h after injection. E. coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering. H. capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation. Nylon thread was encapsulated 24 h after the introduction into the hemocoel. Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation. In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material.

Specific monoclonal antibodies to Strongyloides stercoralis: a potential diagnostic reagent for strongyloidiasis.

In this study, specific hybridomas secreting monoclonal antibodies (MAb) to antigen of Strongyloides stercoralis filariform larvae were produced. Specific epitopes targeted by the MAb were protein in nature and located in situ in the internal content of the filariform larvae of the parasite but not in the esophagus. The MAb reacted to the homologous antigen in an indirect ELISA but did not reveal any reaction to the SDS-PAGE separated-homologous antigen in a Western blot analysis (WB) suggesting a conformational epitope specificity. The MAb were of IgG1 isotype which is the isotype known to have high affinity to this epitope so they were used in a dot-ELISA to detect the antigen of the parasite. The assay could detect the epitopes in 78 ng or more of the crude filariform larval extract but did not reveal any positive result when applied to detect antigen in stool samples of parasitologically confirmed strongyloidiasis patients. The negative antigen test results can be explained as follows. Either the MAb were filariform stage-specific and thus did not recognize the rhabditiform larval antigen mainly contained in the patient's stool or the amounts of antigen in the stool samples were too small and/or unevenly dispersed. In the latter instance, the MAb developed in this study would have a diagnostic potential if used in an immunological test design where more volume of fresh stool sample could be accommodated in the test, e.g. a sandwich plate ELISA.

Comparative immunopotentiating properties of saponin and incomplete Freund's adjuvant coupled to affinity purified larval antigen of Hyalomma anatolicum anatolicum.

Comparative immunostimulatory properties of saponin and incomplete Freund's adjuvant (IFA) were studied using affinity purified 39 kDa larval antigen of Hyalomma anatolicum anatolicum. Significant antibody response to 39 kDa antigen was detected in the sera of rabbits immunized with both 39 kDa plus saponin and 39 kDa plus IFA in comparison to their corresponding control animals. Insignificant differences were noted in the antibody response between the animals of two immunized groups. Upon challenge, the animals immunized with 39 kDa plus IFA rejected 76.2 +/- 9.7% of larvae and 45.8 +/- 4.1% of adults while in group of animals injected with 39 kDa plus saponin rejected 80.9 +/- 11.2% of larvae and 47.2 +/- 5.7% of adults.

Sandwich ELISA detection of excretory-secretory antigens of Toxocara canis larvae using a specific monoclonal antibody.

We produced a new monoclonal antibody (mAb) to the excretory-secretory (ES) antigens of Toxocara canis larvae. The mAb (IgG1) reacts specifically with the 120 kDa protein of many ES molecules and does not have any cross-reactivity with adult T. canis antigens. Sandwich ELISA to detect the ES antigens was performed using the mAb and rabbit polyclonal antiserum. The lower limit for the detection of ES antigen was 4 ng/ml; assay was proportional within a concentration range of 4 ng/ml to 1 microg/ml of ES antigen. This assay system may prove valuable when seeking to quantify parasite burden early in infection and when determining the efficacy of anthelmintic treatment.

Interleukin-4 production in BALB/c mice immunized with Anisakis simplex

We investigated the interleukin (IL-4) levels in BALB/c mice immunized with Anisakis extract in single or multiple doses and in mice orally infected with a larva. From animals immunized maximum responses were obtained with the multiple doses with an only IL-4 peak. Conversely, in the mice inoculated with a larva per os, the IL-4 levels showed two peaks of different rates

Production of monoclonal antibodies to the cuticle of advanced third-stage larva of Gnathostoma spinigerum.

This study has demonstrated that sera from Balb/c mice infected with live advanced third-stage larvae (aL3), but not those immunized with crude larval extract, immunoprecipitated the 25-kDa protein from surface-iodinated extract of aL3. Hybridoma cell lines derived from spleen cells of an infected mouse secreted antibodies that reacted with several tissue of aL3 including the esophagus, intestine, muscle and cuticle by immunofluorescence assay. However, none of the cuticle-positive hybridoma cell lines produced antibodies that recognized surface-iodinated protein of aL3 by immunoprecipitation. Western blot analysis showed that monoclonal antibodies (MAbs) secreted by clones derived from one of the cuticle-positive hybridoma lines recognized proteins of molecular weights ranging from 55-96 kDa. The MAbs most likely reacted with the collagenous component of the cuticle.

Specific IgE antibody responses to somatic and excretory-secretory antigens of third stage G. spinigerum larvae in human gnathostomiasis.

Specific IgE antibody levels in the serum of patients with proven gnathostomiasis and in those with intermittent cutaneous migratory swelling (CMS) were determined by the enzyme-linked immunosorbent assay (ELISA) using somatic extract and excretory-secretory (ES) products of Gnathostoma spinigerum infective larvae as antigens. The third stage larval used were obtained from naturally infected eels. There was an increase in specific IgE antibody to both antigens in these patients. The mean levels of these specific IgE antibodies were significantly higher than that of the healthy control (P<0.01). Comparison between using somatic extract and ES products in the test showed, a positive result in the group of suspected patients with gnathostomiasis or CMS was significantly higher when using ES products (81.81%) than somatic extract (59.09%) as the antigens (P<0.05). However, both somatic and ES antigens cross-reacted with other parasitic sera. The overall sensitivity of the ELISA for these IgE antibodies detection were 71.87 per cent and 87.50 per cent with somatic and ES antigens, respectively. The specificity was 57.53 per cent when somatic antigen was used and increased to 69.86 per cent when ES antigen was used. The positive and negative predictive values of the test were 42.59 per cent and 82.35 per cent by using somatic antigen. Both of these values, were also increased to 56.00 per cent and 92.72 per cent by using the ES antigen. It is obvious that more potential components may be present in ES products than those in the somatic extract. The ES antigen may have to be further purified and may be suitable for evaluation of the effectiveness of chemotherapy. As such, the antibody responses to secreted products are more closely related to active infection than the anti-whole worm antibody that may persist following the death of the parasites. However, in this disease, the effect of the IgE antibody on its pathophysiology it is still not known.