Schistosoma japonicum: immunological characterization and detection of circulating polysaccharide antigens from adult worms

The antigenic constituents of a trichloroacetic acid (TCA)-soluble fraction of adult Schistosoma japonicum were studied with immunoelectrophoresis, and compared with those of Schistosoma mansoni. Eight TCA-soluble antigens of S. japonicum were demonstrated, five of which showed immunological identity with S. mansoni antigens. Of the eight antigens, five antigens with anodic motility were found as circulating antigens in S. japonicum-infected hamster and rabbit sera; the major circulating antigen was the circulating anodic antigen (CAA). Two other antigens, with cathodic motility, including the circulating cathodic antigen (CCA), were demonstrable as circulating antigens in S. mansoni infections, but not in S. japonicum infections. Most of the circulating antigens were shown to be gut-associated. Only one antigen, line 2, which was not demonstrable as circulating antigen and which was present in the parenchyma of the worms, was found to be specific for S. japonicum. Using an ELISA for the detection of CAA in the sera of S. japonicum-infected rabbits, a lower detection level of 100 ng CAA/ml serum was achieved. Moreover, at 7-8 weeks after infection, a direct relationship between worm burden and CAA level was demonstrated.     

Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse,hamster, and human infections

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.     

Schistosoma mansoni: Use of antigens from excretions and secretions in immunodiagnosis

The use of antigens from excretions and secretions (ESA) of Schistosoma mansoni in two immunodiagnostic tests, the enzyme-linked immunosorbent assay (ELISA), and the defined antigen substrate spheres (DASS) system, has been extensively investigated. In comparison with total adult worm antigens (AWA), the sensitivity of the DASS tests remained the same, while that of the ELISA increased slightly when ESA was used. For further analysis, the ESA preparation was fractionated according to molecular weight, by gel filtration. The humoral immune response of immunized rabbits, infected mice, and humans to each of these molecular-weight fractions was determined by incubating an equal, nonsaturating amount of each ESA fraction in a double-antibody sandwich system, using Sepharose beads as a carrier. The humoral immune response of rabbits immunized with ESA was primarily directed against antigens with molecular weight between 50,000 and 70,000. In contrast, immunoglobulins from sera of infected mice or humans, reacted well with antigens from a large molecular-weight range. Screening of a large number of sera for the presence of specific antibodies is most conveniently executed with tests in which antigens, instead of antibodies, are bound to a matrix. However, binding of antigens to Sepharose beads or polystyrene microtiter plates was shown to decrease considerably with decreasing molecular weight of the antigen. Therefore, of all ESA fractions, those containing the high-molecular-weight antigens (MW > 200,000) gave the most sensitive DASS and ELISA tests. These high-molecular-weight excretory and secretory antigens, in contrast to a total-worm homogenate, and excretory and secretory antigens with a molecular weight lower than 200,000, possessed a high specificity for S. mansoni. The specificity of the high-molecular-weight preparation was shown to be mainly due to the presence of the circulating anodic polysaccharide antigen, since removal of this antigen by immunoadsorption led to a considerable decrease in specificity.     

Comparison of Schistosoma mansoni Soluble Cercarial Antigens and Soluble Egg Antigens for Serodiagnosing Schistosome Infections

A Schistosoma mansoni cercarial antigen preparation (cercarial transformation fluid – SmCTF) was evaluated for detection of anti-schistosome antibodies in human sera in 4 collaborating laboratories. The performance of SmCTF was compared with that of S. mansoni egg antigens (SmSEA) in an indirect enzyme-immunoassay (ELISA) antigen assay, the latter being used routinely in 3 of the 4 participating laboratories to diagnose S. mansoni and S. haematobium infections. In the fourth laboratory the performance of SmCTF was compared with that of S. japonicum egg antigens (SjSEA) in ELISA for detection of anti-S. japonicum antibodies. In all 4 laboratories the results given by SmCTF in ELISA were very similar to those given by the antigen preparation routinely used in the respective laboratory to detect anti-schistosome antibodies in human infection sera. In so far as the ELISA results from SmCTF are thus so little different from those given by schistosome egg antigens and also cheaper to produce, the former is a potentially useful new diagnostic aid for schistosomiasis.     

Production and properties of reaginic antibodies in rabbits infected with Clonorchis sinensis or Schistosoma japonicum

The production of reaginic antibodies detected by homologous passive cutaneous anaphylaxis (PCA) was demonstrated in all rabbits experimentally infected with either Clonorchis sinensis or Schistosoma japonicum. The antibodies appeared in the sera as early as 3 weeks after exposure and persisted with relatively high titers for at least 7 weeks in some animals. The antisera of rabbits infected with C. sinensis were found to be cross reactive against heterologous trematode antigens, although PCA titers were less than 3% of the titer by the homologous antigen; no cross reaction was observed between S. japonicum antiserum and the heterologous antigens. PCA activity of the antisera was completely destroyed in some samples by heat treatment at 56 C for 2 hr, but partially in the others even after heating for 6 hr. However, the physicochemical properties of these antibodies were analogous to human IgE; the PCA activity was eluted with 0.035 M phosphate buffer from a DEAE-cellulose column and recovered in the ascending portion of the IgG peak by Sephadex G-200 gel filtration. PCA activity was found in a β region in preparative agar electrophoresis.     

Screening Diagnostic Candidates for Schistosomiasis from Tegument Proteins of Adult Schistosoma japonicum Using an Immunoproteomic Approach

Background

Schistosomiasis is one of the world’s most prevalent zoonotic diseases and a serious worldwide public health problem. Since the tegument (TG) of Schistosoma japonicum is in direct contact with the host and induces a host immune response against infection, the identification of immune response target molecules in the schistosome TG is crucial for screening diagnostic antigens for this disease.

Methodology/Principal Findings

In this study, an immunoproteomics approach used TG proteins as screening antigens to identify potential diagnostic molecules of S. japonicum. Ten spots corresponding to six proteins were identified that immunoreacted with sera from S. japonicum-infected rabbits but not sera from uninfected rabbits and their specific IgG antibody levels declined quickly after praziquantel treatment. Recombinant phosphoglycerate mutase (PGM) and UV excision repair protein RAD23 homolog B (RAD23) proteins were expressed and their diagnostic potential for schistosomiasis was evaluated and compared with schistosome soluble egg antigen (SEA) using ELISA. The results showed high sensitivity and specificity and low crossreactivity when rSjPGM-ELISA and rSjRAD23-ELISA were used to detect water buffalo schistosomiasis. Moreover, antibodies to rSjPGM and rSjRAD23 might be short-lived since they declined quickly after chemotherapy.

Conclusion/Significance

Therefore, the two schistosome TG proteins SjPGM and SjRAD23 were identified as potential diagnostic markers for the disease. The two recombinant proteins might have the potential to evaluate the effectiveness of drug treatments and for distinguishing between current and past infection.     

KCl-Extractable surface antigens of Schistosoma mansoni: immunological characterization and applicability in immunodiagnosis

A Schistosoma mansoni antigen preparation was obtained by extraction of adult worms with a 3 M KCl solution. An indirect immunofluorescence reaction on cryostat sections of adult worms showed that the extracted antigens mainly originated from the tegument. The complex antigenic composition of the tegument extract was shown by immunoelectrophoresis against serum from infected mice and immunized rabbits, which gave up to 9 and 17 precipitation lines, respectively. When we compared the use of adult worm antigens and the tegument antigen preparation in the DASS and ELISA tests for immunodiagnosis of human schistosomiasis, the average sensitivity of the tests with the two preparations was about equal, although considerable differences between individual sera occurred. Analysis of tegument antigens, fractionated by gel filtration, showed that the main serological activity of the tegument antigen preparation was due to high molecular weight antigens.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Immunological aspects of murine infection with the rat nematode Strongyloides ratti Sandground, 1925

In a study of the immune response of the rat to infection with the nematode Strongyloidis ratti, the antigens of the infective larval stage (L3) and of the parasitic, parthenogenetic female (Fp) were investigated. From both the larvae and the adult females, one metabolic (exoantigen) and two somatic antigens were extracted. Of the two somatic antigens, one was soluble and obtainable by physical means while the other was separated by chemical means from the tegument of the parasite. Humoral responses to the various antigens were evaluated by immunodiffusion and ELISA techniques, while the overall immune response was assayed by the worm burden in the immunized and subsequently infected rats. Agar-gel double diffusion yielded precipitin bands only with larval somatic antigens. ELISA proved positive at a titer of 20,000 with larval metabolic antigen and sera of rats immunized against either larval metabolic or somatic antigens. By 20 days post challenge infection, however, this titer diminished to 4000. In vivo studies of worm burden in rats immunized with the various antigens and then exposed to the live L3 of the nematode showed that there were significantly fewer adult worms in the rats immunized with larval somatic antigen and adult metabolic antigen than in those immunized with adult somatic antigen or larval metabolic antigen.     

Presence of Schistosoma mansoni antigens in liver, spleen and kidney of infected mice: a sequential study

In the present study the kinetics of the uptake and deposition of Schistosoma mansoni antigens in liver, spleen and kidney of S. mansoni infected Swiss mice have been investigated in relation to duration of infection and infection dose (50, 100, 200 cercariae). The presence of antigen was studied with a direct immunofluorescence reaction on frozen sections of the organs, using a number of fluorescein isothiocyanate (FITC)-labeled antisera produced against various antigen preparations isolated from different life-cycle stages of the parasite. The presence of antigen was demonstrable with two of the antisera, directed against the circulating anodic antigen (CAA) and against total soluble egg antigen (SEA). CAA was demonstrable from 1 week post infection (p.i.) onwards in Kupffer cells in the liver, from 2-3 weeks p.i. onwards in macrophages in the marginal zones in the spleen and from 3 weeks onwards in kidney glomeruli. Immunofluorescence reactions on CAA in kidney glomeruli, however, were only weak positive until 12 weeks p.i., whereafter strong positive reactions were found. SEA was demonstrable from 5 weeks p.i. onwards in Kupffer cells in the liver and from 4 weeks p.i. onwards in macrophages of the spleen. In contrast to CAA, SEA was not detectable in kidney glomeruli.     

Hymenolepis diminuta: The origin of protective antigens

Mice were infected orally with 1,6, or 30 cysticercoids of Hymenolepis diminuta. These were allowed to develop for different periods of time before elimination with anthelminthic, thus exposing the hosts to antigens from the prestrobilate, early strobilate, or fully strobilate worms. Other groups of mice were immunized by intraperitoneal (ip) implantation of a live strobilate worm or by ip implantation of live worms from cysticercoids excysted in vitro. Strong protection against challenge with a surgically transplanted strobilate worm was achieved by prior infection with 6 or 30 worms eliminated as early as Day 3 of infection. By this time these worms would not have strobilated. Conversely, a single worm, strobilating extensively over 16 days, stimulated only weak protection. Parenteral implantation of excysted worms protected mice but parenteral implantation of a strobilate worm had no effect. It is suggested that (i) the tapeworm protective antigens are primarily related to the scolex and/or the germinative region; (ii) the number of worms and the duration of antigenic stimulation in an immunizing infection determine the magnitude of a protective secondary response.     

Fasciola hepatica: host responders and nonresponders to parasite glutathione S-transferase.

Fasciola hepatica glutathione S-transferase (FhGST) was isolated from adult worms by glutathione agarose affinity chromatography. SDS-PAGE shows three proteins of M(r) ranging from 29-27.8 kDa. Western immunoblot analyses using SDS-PAGE separated adult worm extracts and probed with a rabbit anti-FhGST antiserum reveal two bands in the same M(r) range. Mice and rabbits immunized with purified FhGST develop copious amounts of anti-FhGST antibodies. Moreover, antisera to F. hepatica adult worms and excretion-secretion products also react with FhGST. Cross-reactivity with schistosomes is evidenced in the reactivity with FhGST of anti-Schistosoma mansoni adult worm antisera and, to a lesser extent, antisera to S. mansoni-soluble egg antigens. The time of appearance of anti-FhGST antibodies in different species of animals infected with F. hepatica was determined. Sheep and a New Zealand white rabbit developed anti-FhGST antibodies detectable by ELISA as early as 2 weeks postexposure with F. hepatica. However, neither mice nor calves infected with F. hepatica developed antibodies to FhGST through the 5-10 weeks of infection tested. But mice infected with S. mansoni developed anti-FhGST cross-reacting antibodies by 6 weeks of infection. Calves immunized with a Fasciola/Schistosoma cross-reactive, cross-protective antigen complex in which a 12,000-kDa protein (Fh12) has been shown to contain immunoprophylactic activity, also developed antibodies to FhGST. Since FhGST is a novel potential vaccine, its protection-inducing capability in a multivalent vaccine combined with Fh12 clearly warrants study. In summary, it appears that hosts with fascioliasis are either responders to FhGST (rabbits, sheep) or nonresponders (mice, cattle), offering interesting models for studying the immune response.     

Model multiple antigenic and homopolymeric peptides from non-repetitive sequences of malaria merozoite proteins elicit biologically irrelevant antibodies

Three model peptides containing B-epitopes from conserved, non-repetitive regions of the merozoite surface antigens, MSA2 and MSA1, and the erythrocyte binding protein EBP of Plasmodium falciparum were synthesised. The peptides incorporated GPG spacers and C residues at the N and C termini, and were polymerised by oxidation to form cystine bridges. Multiple copies of essentially the same peptide sequences were also synthesised on a branching lysyl matrix to form a tetrameric multiple antigen peptide. Rabbits were immunised with the polymerised and multiple antigen peptides, in alum followed by Freund’s adjuvant, and the antibody responses examined by IFA and ELISA. Reproducible antibody responses were obtained against the MSA1 and EBP but not MSA2 peptides. IgG antibody levels detected by ELISA after three injections of antigen in alum, increased significantly after further immunisation in Freund’s adjuvant. IgG levels were largely maintained for at least 23 weeks after the final immunisation. IgM antibodies, generally detectable only after immunisation in Freund’s adjuvant, were absent 23 weeks later. Antibody titres against the native protein on fixed parasites, assayed by IFA, were three to five orders of magnitude lower than the corresponding ELISA titres against the peptides. Antibody-dependent inhibition of P. falciparum growth in vitro could not be demonstrated with the immune rabbit sera. The MSA1 and EBP peptides elicited cross-reactive antibodies. The results suggest that the selected non-repetitive sequences are conformationally constrained in the native proteins and only a small proportion of the anti-peptide antibodies bind to the native proteins. The significance of the findings for the development of peptide vaccines and the use of peptides in immunoassays is discussed.     

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.     

Schistosoma japonicum: Immediate hypersensitivity reactions to egg antigen in humans and rodents

Immediate hypersensitivity reactions in schistosoma japonicum infections were examined in both man and experimental animals. In man higher reaction to soluble egg antigen than to adult worm antigen was detected by the use of the radioallergosorbent test (RAST). Blood-collecting filter paper can be used in RAST for seroepidemiological study in place of a skin test. Reaginic antibody formation against egg antigen was detected at the approximate time of egg deposition in strains of mice, Mongolian gerbils, cotton rats, and laboratory rats by the use of passive cutaneous anaphylaxis or Prausnitz-Küstner-type skin tests. At the same time circumoval precipitin tests were positive. Results with athymic nude mice suggest that these reactions are T-cell dependent. No detectable reagin synthesis against adult worm antigen was found in the animals so far examined, confirming stronger allergenic reaction to egg antigen than to that of adult worms in S. japonicum infections in man and animals.     

Kinetics of antibodies and antigens in serum of mice experimentally infected with Echinostoma caproni (Trematoda: Echinostomatidae)

The present study reports on the kinetics of antibodies and antigens in serum of mice experimentally infected with 75 metacercariae of Echinostoma caproni during the first 12 wk postinfection (wpi). Antibody titers in the serum of mice were determined by an indirect enzyme-linked immunosorbent assay (ELISA) using excretory/secretory (ES) antigens of E. caproni. The early detection of antibodies against ES antigens of E. caproni is feasible using indirect ELISA. Mice developed significant antibody responses at 2 wpi, and the values progressively increased until the end of the experiment. This may be related to the intestinal absorption of adult worm antigens that induces humoral responses. The presence of E. caproni circulating antigens was determined by a capture ELISA based on polyclonal rabbit antibodies against ES antigens of E. caproni. High levels of seroantigens in mice were detected by 1-2 wpi, probably because of the local inflammatory responses in mice induced by the adult worms. A drop in circulating antigen levels was observed at 9 wpi, which could reflect changes in the intestinal tissues over the course of the infection.     

Uncinaria stenocephala: assessment of antigens for the immunodiagnosis of canine uncinariosis

Although Uncinaria stenocephala is the most frequent hookworm in the intestine of dogs from Northern, Central and Southern Europe, little is known about its host-parasite relationship. Three groups of sera from dogs (Group 1: dogs naturally infected only by U. stenocephala; Group 2: helminth-free dogs at necropsy, and Group 3: dogs parasitized by other helminths) were analyzed by ELISA using U. stenocephala antigens from adult worms (somatic and excretory-secretory antigens) and from L3 larvae (somatic antigens). All three sources of antigens were found to be suitable for immunodiagnosis of canine uncinariosis with up to 90% efficacy. However, an analysis to assess the diagnostic value of the different antigens demonstrated that the adult excretory-secretory antigens had a higher diagnostic efficacy (96.7%), indicating that this is the best antigen source for the diagnosis of Uncinaria infection.     

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.