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Allergens present in the excretory/secretory (ES) products of adult Paragonimus ohirai were biochemically identified. Immunoblot analysis using sera from P. ohirai-infected rats revealed only two allergens to be major proteins in the ES products, with apparent molecular masses (Mr) of 27 and 29 kD. As the ES products contained a high proportion of acidic and neutral cysteine proteinases, we examined whether or not the allergens and the cysteine proteinases were identical. The acidic and neutral cysteine proteinases were biochemically purified from the ES products and showed Mr of 27 and 29 kD, respectively. The two cysteine proteinases had almost identical N-terminal amino acid sequences and were reactive with specific IgE in sera from the infected rats. The allergenicity of the cysteine proteinases was confirmed by 48-hour homologous passive cutaneous anaphylaxis. Immunoblot and immunocapture assays using anti-human IgE monoclonal antibody showed that the proteinase allergens were reactive with specific IgE of patients with paragonimiasis westermani. Also, the cysteine proteinases were reactive with specific IgG of both the infected rats and the patients. Therefore the acidic and neutral cysteine proteinases prepared from the ES products of P. ohirai will be useful allergens and antigens for the immunodiagnosis of paragonimiasis.

The tegumental glycocalyx of excysted juvenile (EJ) of Paragonimus ohirai was immunobiochemically characterized using a monoclonal antibody (MS-Mab). HPLC gel filtration showed that the antigens detected by two-site ELISA had a molecular weight of greater than or equal to 2 x 10(6) Da (dextran marker). On reduced SDS-PAGE, the glycocalyx antigen retained in the stacking gel was cleaved into several much smaller antigens after pronase treatment. The antigenic activity of the glycocalyx was stable in two-site ELISA to heat and acid treatments, but sensitive to alkali, periodate, base/borohydride, and pronase treatments. Precipitin formation in immunodouble diffusion between MS-Mab and EJ crude antigen was inhibited only by two monosaccharides: galactose and N-acetylgalactosamine. The purified glycocalyx bound strongly to PNA lectin, fairly well to RCA120 lectin, and slightly to SBA lectin, but not to Con A, WGA, UEA-1, DBA, or LFA lectins. Exo-beta-galactosidase treatment increased SBA binding, whereas it decreased PNA binding. PNA was observed to strongly bind to the body surface of living EJ. The antigenic activity of the glycocalyx was remarkably lost by incubation with exo-beta-galactosidase and O-glycanase. The glycocalyx was reactive with sera of P. ohirai-infected rats, and its reactivity was remarkably reduced by O-glycanase treatment. The ELISA level was higher in sera at an early stage of infection than in a late one. These studies show that the EJ tegumental glycocalyx is antigenic in infection, a marked, high molecular weight glycoprotein containing antigenic O-linked sugars, and that the sugar epitope is at the nonreducing terminal of the O-linked sugars and is composed of galactose and N-acetylgalactosamine.

Non-specific suppression of the immune response was investigated in Brugia pahangi-infected Lewis rats. The proliferative response of peripheral blood lymphocytes or splenic non-adherent cells to mitogens was significantly reduced by B. pahangi infection. The degree of hyporesponsiveness of splenic non-adherent cells to mitogens was comparable between microfilaremic and non-microfilaremic animals. The suppressed proliferative response of splenic non-adherent cells was restored by blocking with anti-CD8 monoclonal antibody. After separation of T cells into CD4+ and CD8+ subpopulations, only CD8+ T cells from B. pahangi-infected rats suppressed the proliferative response of normal spleen cells to concanavalin A. CD8+ T cells from normal rats had no suppressive effect. On the other hand, the proliferative response of CD4+ T cells to concanavalin A was comparable between normal and infected rats. These results suggest that CD8+ T cells participate in the non-specific suppression of immune response in experimental filariasis.

The role of mast-cell-derived eosinophil chemotactic factor A (ECF-A) on eosinophil-rich periovular granuloma formation was examined by using mast-cell-deficient WBB6F1W/Wv mice infected with Schistosoma japonicum. The average size of granulomas formed around newly deposited eggs in the liver of W/Wv mice was significantly smaller than that observed in control +/+ mice. The number of adult worms recovered or specific IgE titers were comparable between W/Wv and +/+ mice. In contrast, immediate-type hypersensitivity response to specific antigen and dialyzable low-molecular weight ECF in the serum was detectable only in infected +/+ mice. When naive bone marrow eosinophils were incubated with the dialyzable fraction of infected +/+ mice sera, chemotactic reactivity of eosinophils to ECF derived from S. japonicum eggs was significantly enhanced, although that to synthetic ECF-A was depressed. Similar effects were observed when naive eosinophils were treated with synthetic ECF-A. The dialyzable fraction of infected W/Wv mice sera had no such modulating effect on the chemotactic reactivity of eosinophils. These results suggest that an immediate-type hypersensitivity reaction is important in the formation of eosinophilic granulomas around S. japonicum eggs, mainly through the modulating effect of mast-cell-derived ECF-A on the chemotactic reactivity of eosinophils.

Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection.

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.

Allergen in crude extract of Dirofilaria immitis was purified and separated from IgG-inducing antigens by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 gel filtration and starch gel zone electrophoresis. The purified preparation was proved to be one protein band by sodium dodecyl sulfate polyacrylamide gel (SDS-gel) electrophoresis and one precipitin arc by immunodiffusion. The molecular weight of the purified allergen was estimated to be approximately 20,000 by gel filtration and 15,000 by SDS-gel electrophoresis. The carbohydrate content of the preparation was apparently low, about 2%. The allergen was positively charged, and its determinant group was protein in nature. It was resistant to tryptic, pepsic and chymotryptic digestion, periodate oxidation and DNase and RNase digestion but very sensitive to pronase digestion. Allergen was inclined to aggregate each other in the buffered solution. It was also very resistant to vibration, heat (80°C for 1 h) and acid (pH 2.5) and alkali (pH 11.0) treatments. Rats as well as mice immunized with allergen developed only a reaginic antibody and no hemagglutination antibody.

Rats reinfected with Paragonimus ohirai (P.o.) elicited little secondary IgE response to adult P.o. antigen. In contrast, rats immunized with X-irradiated (2–10 krad) metacercariae elicited not only a marked primary IgE response comparable to that in normally infected rats, but also a marked secondary IgE response after challenge infection. Rats immunized with 2 krad X-irradiated metacercariae yielded a higher secondary IgE response than with 5 or 10 krad irradiated ones, and elicited a secondary response lasting at least 1 year. An IgE response to antigen of newly excysted juvenile parasites (NEJ) was clearly different from the IgE response to adult P.o. antigen. The former IgE response was weak after infection or immunization, but after challenge infection a marked secondary response occurred at a level similar to the IgE response to adult P.o. antigen. The secondary IgE responses to adult P.o. and NEJ antigens were also elicited by reinoculation with X-irradiated metacercariae. Cross-absorption experiments confirmed that there were adult type and NEJ type allergens.

Ikeda T. , Oikawa Y. and Fujita K. 1982. Kinetics and localization of parasite-specific IgE in Paragonimus ohirai -infected rats. International Journal for Parasitology 12: 395–398. In Wistar rats infected with Paragonimus ohirai (P.O.), P.O. -specific IgE responses of the mesenteric and mediastinal lymph nodes and spleen were determined by homologous adoptive cutaneous anaphylaxis (ACA) assay since P.o. -specific IgGa was not detected by either 2 h or 4 h PCA. Intraperitoneal (i.p.) infection with metacereariae elicited similar patterns of ACA response in the three lymphoid tissues examined, with the mediastinal lymph node giving the highest response. ACA positive cells were detected 2 weeks after infection, peaked at 3 weeks and then declined. These kinetics of ACA responses nearly paralleled the kinetics of serum P.o. -specific IgE titre. In intrapleural infection with metacereariae, on the other hand, the mediastinal lymph node gave a high ACA response comparable to the lymph node in i.p. infection, but the mesenteric lymph node and spleen gave negligible ACA responses. In infection established by i.p. transplantation of 4–5-week-old worms, only the mediastinal lymph node of the three lymphoid tissues responded and its response was at a low ACA level. The level of serum P.O. -specific IgE was much lower in the above two infections than in i.p. infection with metacercariae.

In Guatemala, an indirect hemagglutination (IHA) test for onchocerciasis, performed with a crude saline extract of Onchocerca volvulus and blood samples taken on filter paper, showed a high level of sensitivity and specificity. IHA titers of blood samples from the ear lobe taken on filter paper and of sera obtained by venipuncture showed a high correlation.

In seven patients with paragonimiasis westermani, parasite-specific IgE and IgG levels in sera and pleural effusion were determined by an enzyme-linked immunosorbent assay (ELISA). The sensitivity to adult excretory-secretory (E-S) antigen was compared with the sensitivity to whole worm extract antigen, and the former was more sensitive in both an IgE-ELISA and IgG-ELISA. Both parasite-specific IgE and IgG could be detected by ELISA at levels much higher than those in control subjects using E-S antigen. When specific IgE and IgG levels in sera and pleural effusion of individual patients were compared, the latter had higher values. The difference between levels of specific IgE in pleural effusion and serum did not correlate with that of specific IgG. These results indicate that specific IgE and IgG antibodies form locally, i.e., in the lung, and that pleural effusions from patients with paragonimiasis are more suitable than serum for immunodiagnosis.

The amino acid analysis of the purified allergen from Dirofilaria immitis revealed that it had a total number of 98 amino acid residues. The allergen was remarkably rich on glutamic acid (15 residues), lysine (14 residues) and aspartic acid (12 residues). No half-cystine was obtained. The ratio of the acidic residues to the basic ones was 1.4. The weak positive charge of the allergen is contributed by the fewer number of basic residues relative to acidic residues.

Parasite-specific IgE response in rats infected with metacercariae of the lung fluke, Paragonimus ohirai, was enhanced about 8 times by Bordetella pertussis adjuvant. In rats infected by intraperitoneal transplantation of adult worms, the adjuvant markedly increased the usually low or absent parasite-specific IgE response. The most effective time of the adjuvant administration was different between the two modes of infections.

A sero-epidemiological study of onchocerciasis was carried out with the IHA test in Guate- mala. In the endemic area, 94.8% of 191 subjects with microfilariae, 82.1% of 172 cases with onchocercal nodules, and 22.5% of236 cases without either microfilariae or nodules gave positive reactions in the IHA test. On the other hand, the positive rate was 3.4% and 2.0% in each of 2 nonendemic populations. A follow-up study of reactors in the IHA with neither microfilariae nor nodules showed that 11 (42%) of 26 cases were microfilaria positive 6-7 mo after the first examination. A clear correlation was found between the results of the IHA test and skin biopsy in surveyed communities. The age distribution curve of the IHA test was closely associated with skin biopsy although the former was higher. IHA titers rose in proportion to microfilarial density. A sex-related difference was evident in the IHA positive rate in the subjects from medium and low endemic areas, but little difference was shown in individuals from a higher endemic area. Although onchocerciasis can be diagnosed by detecting microfilariae in the skin, skin biopsy is not sensitive, especially in light or early stages of infection. Therefore, a stan- dardized immunological test may offer a pref- erable method of diagnosis and for epidemi- ological studies. The immunodiagnosis of onchocerciasis has been attempted by various workers using the skin test and complement fixation test (as reviewed by Kagan, 1963), im- munofluorescent antibody test (Lucasse, 1962; Lucasse and Hoeppli, 1963; Woodruff and

In rats infected with Paragonimus ohirai (P.o.), IgE response against the parasite was influenced by both the site of metacercarial inoculation and the age of the inoculated worms, whereas the IHA response was not influenced by these variables. Intraperitoneal infection and oral infection with 10 metacercariae induced a high level of P.o.-specific IgE (maximum PCA titer of 1:128 and 1:111, respectively). In contrast, intrapleural infection induced little IgE (1:496, maximum titer). Thus migration of the parasite in the peritoneal cavity and the liver, or the former alone, was an important event in the production of IgE, because intrapleurally inoculated metacercariae normally develop into mature worms without migrating into the abdominal cavity. Intraperitoneal transplantation of five immature or adult worms produced significantly lower IgE levels and correlated with the age of the transplanted worms. Four-week-old worms were observed to induce negligible or no P.o.-specific, IgE antibody. The stage-specific, IgE resonse observed in infections established by transplantation appears to be partly dependent on migration of the parasite in the abdominal cavity. Worms in the pleural cavity may have suppressed IgE production.