Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro.

The loss of variable antigen from Trypansoma brucei during transformation from the bloodstream to the procyclic form in vitro has been monitored by agglutination and immunofluorescence reactions using antisera against both forms. Maximum agglutination of transforming trypanosomes with anti-culture form sera was obtained in 36–48 hr coinciding with loss of the surface coat as seen by electron microscopy. Agglutination with antisera against homologous bloodstream forms, however, reached a constant minimum but still positive level after 7–9 days: absorption of such antisera with culture or heterologous bloodstream forms reduced this period of persistent agglutinability to 72–84 hr, suggesting that the sera contained antibodies to “common” (surface membrane) antigens which became exposed when the surface coat was lost during transformation. The indirect immunofluorescence reaction provided a direct correlation of loss of antigen with loss of coat. The majority of trypanosomes lost detectable variable antigen by 36–48 hr, but a few flagellates, morphologically resembling bloodstream forms, retained the coat and capacity for labeling up to 84 hr; the numbers of such persistent bloodstream forms were shown to be sufficient to give a positive agglutination reaction for the population as a whole up to this time. Variable antigen appeared to be lost by dilution over the entire trypanosome surface rather than in patches or caps and the relevance of this observation to the process of antigenic variation is discussed.     

Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme-linked immunosorbent assay (Elisa)

Craig P.S. and Rickard M.D. 1981. Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme-linked immunosorbent assay (ELISA). International Journal for Parasitology11: 441–449. Crude somatic or cyst fluid extracts prepared from Taenia saginata, T. hydatigena or Echinococcus granulosus were partially purified by absorption against homologous and heterologous bovine or ovine antisera on immunoabsorbent affinity columns. Antigens in parasite extracts which were eluted after binding to the homologous anti-parasite antisera (bovine or ovine) coupled to CNBr-activated Sepharose were then passed sequentially through affinity columns containing heterologous anti-parasite Ig and the ‘run-through’ antigens collected. The level of cross reactions to these absorbed antigens, in an enzyme-linked immunosorbent assay (ELISA) using sera from cattle or sheep given heterologous parasite infections (including Fasciola hepatica), were significantly decreased. Absolute specificity was not achieved, and some loss in sensitivity occurred. The absorption of cross-reactive antigen(s) using affinity Chromatographie techniques may be a useful first step in the production of species-specific immunodiagnostic antigens for larval cestode infections.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

Production and characteristics of antisera to Spiroplasma citri and clover phyllody-associated antigens derived from plants

Antisera were produced to clover phyllody- and Spiroplasma citri-associated antigens partially purified from infected Vinca rosea plants. Separate antisera were made to ‘membrane fraction’ (MF) preparations comprising the resuspended pellet obtained by high speed centrifugation, and to ‘soluble fraction’ (SA) preparations, comprising the supernatant from high speed centrifugation concentrated by freeze-drying. All antisera showed considerable activity against normal plant antigens but after cross-absorption with extracts of healthy plants the MF antisera were used in F(ab')₂based ELISA tests to detect S. citri- or clover phyllody-associated antigens in infected plants. The ‘clover phyllody’ antiserum also reacted specifically with extracts of clover plants with phyllody, and with naturally-infected strawberry plants showing symptoms of green petal disease. Both the ‘clover phyllody’ and S. citri antisera were specific for their respective homologous antigens. No cross-reactions were observed in heterologous tests or between either antiserum and extracts of V. rosea infected with various MLOs obtained from different host plants.     

Exoantigens of Trypanosoma cruzi. I. Conditions for Their Detection and Immunogenic Properties in Experimental Infections1

ABSTRACT Exoantigens of Trypanosoma cruzi were produced in experimentally infected BALB/c mice. The exoantigens were detected by the counterimmunoelectrophoresis method (CIE), with antisera raised in rabbits by immunization with total homogenates of culture forms of ***T. cruzi in plasma from ***field animals obtained by centrifugation and filtration. Control experiments indicated that exoantigens are not somatic components of T. cruzi leaked during the preparative procedure. Exoantigens were detected in male and female mice, 11-90 days old, between 6 and 60 days of infection, and in all mice with patent parasitemia. After 13 days of infection, mice developed antibodies to exoantigens; by CIE up to three populations of antibodies were revealed in different groups of animals. In mice between 13 and 60 days of infection, the coexistence of exoantigens and homologous antibodies was also observed. The exoantigens are not strain specific since a cross reactivity between antigens from three strains of T. cruzi (Tulahuén, Higueras, and Alejandro) was seen. Finally, the presence of antibodies to exoantigens in humans with chronic Chagas’ disease was demonstrated.     

Observations on the antigenicity and serological relationships of stocks of Trypanosoma congolense from East and West Africa.

Antigenic relationships of 4 stocks of Trypanosoma congolense from different parts of Africa were examined by immunofluorescence (IFAT) and neutralization tests. Antisera to each stock were obtained from rabbits infected with trypanosomes transmitted by Glossina morsitans. Trypanosomes for use as antigens were obtained from local skin reactions developing on rabbits infected with 2 of the stocks. Using the IFAT and antisera at end-point dilutions approximately 40% of the trypanosomes fluoresced strongly and a further 30% less intensely with homologous antisera, indicating antigenic heterogeneity among the trypanosomes developing in the skin. Using antisera at low dilutions some samples gave cross-reactions with trypanosomes of heterologous stocks, but at higher dilutions there were no cross-reactions with either of the antigens. The lack of cross-reactions at high antiserum dilutions was interpreted as indicating antigenic differences between the 4 trypanosome stocks. Using neutralization tests only homologous antisera reduced the infectivity of trypanosome suspensions. Overall, these observations indicated that there were at least 3 different strains of T. congolense among the 4 stocks examined.     

Trypanosoma lewisi: effects of immunosuppression on the serological reactivities of exoantigens and cellular antigens of bloodstream parasites.

Groups of rats were immunosuppressed with antithymocyte serum (ATS) and infected with Trypanosoma lewisi. Immunodiffusion studies were performed which demonstrated that trypanosome exoantigens, present in the plasma of these animals, were precipitated by antibodies in the sera of rats undergoing a typical primary T. lewisi infection; extracts of trypanosomes which had been collected from ATS-treated rats contained antigens which also were precipitated by antibodies in these sera. These precipitating antibodies could not be detected using either the plasma of untreated infected rats or extracts of trypanosomes which had been collected from untreated rats. With the exoantigens, precipitating antibodies were detected in serum samples collected from rats 14 to 250 days after infection. With the extract, precipitating antibodies were found as early as 5 days after infection and could be detected as late as 90 days after infection. Antigens of trypanosome extracts partially blocked the precipitin reactions between antisera and exoantigens, suggesting the presence of common antigens in the two preparations. Intact trypanosomes were serologically more reactive when collected from immunosuppressed rats. Trypanosomes collected from ATS-treated rats were agglutinated by antisera at titers fourfold higher than trypanosomes collected from untreated hosts. Absorption with exoantigens from immunosuppressed infected rats blocked trypanosome agglutination, indicating that these antigens are of cell surface origin. The experiments suggest that a likely result of immunosuppressing the host is a trypanosome antigen preparation that is a more reactive serodiagnostic reagent.     

Leishmania mexicana and Leishmania tropica: Cross immunity in C57BL/6 mice

Leishmania mexicana and Leishmania tropica infection were comparatively studied in C57BL/6 mice. Infection with 10⁴ amastigotes of L. mexicana was followed by the appearance of a single lesion which ulcerated in 8 weeks and healed in 24 weeks. Mice infected with 10⁴ amastigotes of L. tropica developed less severe lesions which healed in 18 weeks. In both cases healing was accompanied by a delayed hypersensitivity response and an in vitro lymphocyte reactivity to leishmanial antigens. Mice recovered from a primary infection with L. mexicana or L. tropica were resistant to both homologous and heterologous challenge. In vitro and in vivo immunological tests indicated that L. mexicana and L. tropica share antigenic determinants which are involved in cell-mediated immune responses to these parasites.     

The passive cutaneous anaphylaxis (PCA) test in Cryptococcosis

Summary The PCA test appeared to be a useful indicator of past infection due toCryptococcus neoformans when the extracellular starch antigen was used. The whole cell antigen, crude cell wall antigen, ruptured cell antigen, crude carbohydrate antigen and the crude capsular polysaccharide antigen were not found to be effective.Serums from the nine rabbits injected withC. neoformans showed some degree of reactivity in the PCA test with the homologous extracellular starch antigen. Serums from rabbits injected withC. diffluens andC. laurentii gave no response.None of the rabbits exhibited evidence of previous sensitization and none were obviously infected. Six rabbits injected withC. neoformans were skin tested 4 weeks later with all of the antigens. Positive tests were obtained in four rabbits but only with the cell wall antigen or the ruptured cell antigen. PCA activity was not observed in the serums of mice injected with strains ofC. neoformans although some came from mice showing lesions and some came from fatally infected mice.Of the 500 human serums tested for PCA activity using the extracellular starch antigen, only one reacted strongly and with a sufficiently high titer to suggest previous infection. Serums from three cases of cryptococcosis failed to react.This investigation was supported by Communicable Disease Center grant CC 00151-01.The material in this paper is taken in part from a dissertation presented to the Graduate School of The University of Arizona in partial fullfilment of the requirements for the Ph.D. degree.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.     

Babesia bovis (Argentina): Studies on the composition and location of antigen associated with infected erythrocytes

Specific antisera against Babesia bovis (= argentina) antigens were prepared in rabbits by inoculation of precipitates from an extract of infected erythrocytes and absorption of the antisera with normal bovine components. Of three babesial antigens detected, one appeared to contain a modified stromal component. The antisera, when conjugated with fluoroscein isothiocyanate, stained aggregated infected erythrocytes in the microcirculation and located antigen in glomeruli and blood vessel endothelium. It was suggested that a babesial enzyme-fibrinogen complex contributes to the pathological changes of infection such as sludging and adherence of erythrocytes to blood vessel walls.     

Reaginic antibody response in rabbits infected with Taenia pisiformis

Homologous skin-sensitizing antibodies were detected in the sera of rabbits infected with Taenia pisiformis. They were demonstrated in the sera of some rabbits as early as 8 days after first exposure and peaked at 30 days post-infection before declining over a period of 1 month. A second challenge dose of eggs boosted the titers markedly. The physico-chemical characteristics of the antibody in gel-filtration and anion-exchange chromatography, and in response to heat treatment and reduction and alkylation, conform to those previously described for reagin or IgE. PCA reactions first became positive 4 h after sensitization, and were maximal at 48 h ,although positive reactions occurred at sensitized sites when challenge was delayed for 8 weeks. The significance of this reaginic antibody response is discussed in relation to its potential role in the mechanism of acquired resistance to experimental cysticercosis.     

Antigenic Relationships Among Strains of Vibrio parahaemolyticus

The antigenic relationships of 79 strains of Vibrio parahaemolyticus representing 46 assigned K-types were studied by tube agglutination. Homologous titers of 46 anti-K sera ranged from 80 to 2,560. All but three sera exhibited from one to six heterologous reactions, the majority of which gave titers of 相似文献   

Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.     

Echinococcus multilocularis: Cell-mediated immune response in early and chronic alveolar murine hydatidosis

The in vivo cell-mediated immune response of C57L/J mice infected with Echinococcus multilocularis cysts was investigated using a homologous antigen, a T cell-dependent heterologous antigen (oxazolone) and an alloantigen at 6 and 12 weeks postinfection. Mice infected for 12 and 14 weeks harbored a 9 to 18 times larger larval cyst mass than those infected for 6 weeks and showed splenomegaly and elevated antihydatid antibody titers. In mice infected for 6 weeks and challenged with hydatid antigen the 48-hr footpad swelling and histological picture of footpad were consistent with cell-mediated immunity. These parameters were significantly reduced in the mice infected for 12 weks. Their response to skin allograft and oxazolone, however, remained comparable to the mice 6 weeks postinfection and uninfected controls. The lymphoid tissues of the mice 6 weeks postinfection were histologically intact and showed moderate to intense lymphoproliferation. In the mice 14 weeks postinfection disorganization of lymphoid tissue, plasma-, and histiocytosis in the thymus-dependent areas coincided with a large larval cyst mass.