Common egg envelope antigens are limited to the animal class

The antigens of the egg envelope (zona pellucida) in mammals are of special interest because of their possible involvement in immunoinfertility and as candidate targets for immunocontraception. Conserved zona epitopes from divergent species may present a suitable source and an animal model for investigation of the above factors. We compared egg envelope antigens from 6 species of vertebrates belonging to 3 different classes in order to demonstrate the existence of shared antigens. Egg envelopes from the trout, carp, turtle, hen, duck and quail were isolated and heat-solubilized. They were tested with rabbit polyclonal antisera against carp, trout and duck egg envelopes by the enzyme-linked immunosorbent assay. The results showed significant cross-reactions among egg envelopes of fish and birds. The examined solubilized preparations did not show cross-reactivity with egg envelopes from any other class, suggesting that divergent species did not share common egg envelope antigens, and that their use may not be appropriate in the investigation of immunoinfertility and immunocontraception in humans.     

Exogenous antigens and the stimulation of MHC class I restricted cell-mediated cytotoxicity: possible strategies for fish vaccines

An MHC class I restricted cytotoxic T lymphocyte (CTL) activity assay has recently been established for rainbow trout. MHC class I restricted cytotoxicity probably plays a critical role in immunity to most viral diseases in mammals and may play a similar role in fish. Therefore, it is very important to investigate what types of vaccines can stimulate this immune response. Although logical candidates for vaccine components that can stimulate an MHC class I restricted response are live attenuated viruses and DNA vaccines, these materials are generally not allowed in fish for commercial vaccine use due to potential safety issues. In mammals, however, a number of interesting vaccination strategies based on exogenous antigens that stimulate MHC class I restricted cytotoxicity have been described. Several of these strategies are discussed in this review in the context of fish vaccination.     

Induction of immunosuppression to bacterial antigens in carp, Cyprinus carpio

Typical primary antibody responses were found after injecting carp maintained at 25°C with formalin-killed (Fk) Vibrio anguillarum bacteria, but not in fish injected with the same antigen held at 12° C. There were no significant increases over the primary responses in fish receiving a second injection 60 days after the first injection.
The differences in the results between fish groups receiving three V. anguillarum antigens at two dosages were found to be insignificant in most of the experiments. A comparison of the antibody titres of fish groups after challenge with Fk bacteria showed no difference from control fish receiving a single injection similar to the challenge dose or fish receiving a previous injection with the antigens emulsified in complete Freund's adjuvant (CFA) at 25° C. However, significant immunosuppression was found in fish that were given the primary injection intracardially at 25° C or 12° C, or with CFA emulsion at 12° C.     

Serospecific antigens of Legionella pneumophila.

Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria.     

Multiple whole bacterial antigens in polyvalent vaccine may result in inhibition of specific responses in rainbow trout (Oncorhynchus mykiss)

The goal of fish vaccination today is to protect fish against multiple bacterial fish pathogens simultaneously using polyvalent vaccines. However, many immunological processes such as antigenic cross-reaction, antigenic competition, affinity maturation and antigen-induced suppression may affect the specificity, avidity and level of antibodies. Consequently, the biological function of antibodies may be markedly different from that predicted by conventional serologic tests. Here, we investigated the effects of vaccination and composition of vaccine on the plasma antibody levels, biological function of antibodies in opsonophagocytosis as well as the effects of vaccination on the blood leucocyte counts. Rainbow trout were vaccinated with saline or with two different polyvalent, mineral oil-adjuvanted vaccines. Vaccine 1 contained Aeromonas salmonicida, Listonella anguillarum and both Th and Fd serotypes of Flavobacterium psychrophilum antigens and vaccine 2 contained A. salmonicida, L. anguillarum and only Fd serotype of Fl. psychrophilum. The antibody-mediated opsonophagocytosis was determined as the respiratory burst (RB) activity of blood monocytes and granulocytes against the tested bacterial antigens. Three weeks after vaccination both vaccine groups and the control group showed increased RB activity against all bacterial strains. However, the increase in RB activities was non-specific and originated from the increased number of circulating granulocytes and monocytes. On the other hand, at 6 weeks post-vaccination both specific antibodies and antibody-dependent opsonophagocytosis appeared in both vaccine groups. However, the composition of the vaccine had a marked effect on the magnitude of specific responses. The Fd+Th vaccine enhanced the target specific opsonophagocytosis, to a lesser extent than the Fd vaccine. Both polyvalent vaccines appeared to mainly affect the numbers of circulating monocytes and our results suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis. Our results also suggest that the presented opsonophagocytic assay is an advantageous method to predict vaccine efficiency and that the number, and properties, of bacterial antigens in polyvalent vaccines should be carefully selected in order to avoid inhibitory effects of antigens on the specific response of fish.     

Immunization of Atlantic salmon against the salmon louse: identification of antigens and effects on louse fecundity

Injection of extracts derived from adult caligid copepods induced a partial immunity to Lepeophtheirus salmonis in Atlantic salmon. Antigens were derived from the supernatant of adult lice extracts and were partially purified by Con A affinity chromatography. The antigens were also present in the pellet derived from adult lice extracts, Immunohistochemistry showed that antibodies present in the serum of rats immunized with the Con A purified extract bound predominantly to the gut of L. salmonis. Components of apparent sizes > 205 000, 165 000, 133 000, 130 000, 125 000, 114 000, 110 000, 96 000, 82 000, 78 000, 65 000, 46 000, 35 000, 31 000 and < 29 000 were present in the extract. Although the numbers of attached lice were initially higher on those fish in the immunized group, throughout the course of the challenge experiment the total numbers of lice did not vary greatly between the immunized or control groups of fish. Initially, an overall average of about 17 attached stages were recorded from each fish sampled and by the end of the experiment this figure had fallen to about five adult lice per fish. However, compared with control fish fewer gravid female lice were present on immunized fish and furthermore these lice possessed fewer eggs (P<0.01). No major differences in egg hatching success were recorded. Further exploitation of this would require the isolation and purification of the antigens responsible for the observed effects. The possibilities for the development of a salmon louse vaccine are promising.     

Specificity of H-2 antigens expressed on mouse blastocysts

A highly sensitive cytotoxicity assay was used to detect H-2 antigens on mouse blastocyst stage embryos of the b, a, k, and d haplotypes. The assay was based on the principle that live embryos incorporate 3H-thymidine into DNA whereas embryos killed with antiserum and complement do not. The use of specific alloantisera showed that blastocysts of different haplotypes express different H-2 antigens. Thus, positive evidence was obtained for the expression of Kd and Dk molecules and negative evidence for the expression of Db, Kk, and Dd molecules. Evidence was also obtained that blastocysts express different H-2 antigens than those found on adult lymphocytes. Unexpected cross-reactions were found when some of the alloantisera were tested on blastocysts of different haplotypes. It is proposed that the aberrant expression of H-2 antigens on embryos might facilitate their escape from surveillance by the maternal immune system.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10(5) cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Characterization of the cell-mediated cytotoxic responses of isogeneic ginbuna crucian carp induced by oral immunisation with hapten-modified cellular antigens

A better understanding of the immune responses in fish elicited by oral immunisation is of importance for the development of new and effective oral vaccines for cultured fish. In the present study, we characterized specific cell-mediated cytotoxic responses in isogeneic ginbuna crucian carp (Carassius auratus langsdorfii) following oral immunisation with cellular antigens. Trinitrophenyl- (TNP) or dinitrophenyl- (DNP) modified syngeneic and allogeneic cells were used for studying the fine specificity and genetic restriction of orally-induced cytotoxic cells. Hapten-specific cytotoxic responses were detected in peripheral blood leukocytes (PBLs) of fish orally immunised with haptenated syngeneic cells. PBLs from orally immunised fish had cytolytic activity for haptenated syngeneic cells, but they showed little reactivity against both haptenated and unmodified allogeneic targets. Similarly, oral immunisation of fish with hapten-modified allogeneic cells did not induce hapten-specific cytotoxic cells which can lyse haptenated syngeneic targets. Although ginbuna crucian carp possess spontaneous cytotoxic cells that are capable of killing mammalian tumour cells, cold target inhibition studies suggested that such spontaneous cytotoxic cells were not involved in the killing of haptenated syngeneic targets. Oral immunisation of fish with haptenated syngeneic cells also induced hapten-specific cytotoxic memory responses. Oral administration of haptenated fixed cells also effectively induced hapten-specific cytotoxic cells in the treated fish. These findings suggest that oral immunisation with antigens can elicit antigen-specific cytotoxic cells that are capable of recognizing antigens in an MHC-restricted manner. In addition, our results provide indirect evidence that fish possess a mechanism for taking up exogenous non-replicating antigens from the alimentary tract and generating antigen-specific cytotoxic cells.     

Evidence for two newVibrio anguillarum K antigens

Surface polysaccharides from five strains ofVibrio anguillarum were studied by means of immunoelectrophoretic procedures. The study suggested existence of two new K antigens, displaying cross-reactivity, in strains derived from diseased feral fish. The importance of a detailed serologic characterization of isolates for ecologic and epidemiologic studies ofV. anguillarum is considered.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

Abstract The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10⁵ cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Immunoprecipitation analysis of radiolabelled protein antigens biosynthesized in vitro by S. mansoni. I. Identification of antigens uniquely recognized by protective antibodies

Protein antigens from 4-wk worms were metabolically radiolabelled with [3H]leucine or [35S]methionine. Three freeze-thaw cycles released a large proportion (50% to 60%) of the TCA-precipitable radioactivity from the worms. Immune serum from twice-infected Fischer rats (F-2x), which was shown to confer resistance in a passive immunization assay, and immune serum from twice-infected Wistar Furth rats (W-2x), which does not confer resistance, were used for analyzing antigens in this worm fraction. Antibodies in these antisera differed in their titers to the freeze-thaw released antigens (W-2x greater than F-2x) and in their relative affinities for these antigens (F-2x greater than W-2x). Gradient slab gel electrophoresis of immunoprecipitates of radiolabelled antigens under denaturing conditions revealed many components, which could be categorized into two main types: unique antigens, recognized only by F-2x antibodies, and nonunique antigens, recognized by both F-2x and W-2x antibodies. The potential relevance of these antigens in resistance was further examined by antibody absorption experiments in which 4-wk worms were used as an immunoabsorbent to remove 90% to 95% of the immunoprecipitating activity and 65% to 70% (p less than 0.005) of the capacity to confer resistance in a passive immunization assay. It was concluded that loss of both anti-schistosome activities was specific since antigen released by worms during absorption could account for only 16% of the reduction in antigen-binding capacity and the titer of antibodies directed against beta-galactosidase did not significantly change during absorption. Antigens recognized uniquely by F-2x antibodies are therefore candidates for immunization studies examining induction of resistance against Schistosoma mansoni.     

Detection of Vibrio anguillarum antigen by the dot blot assay

The dot blot assay, modified and adapted for detection of antigens from Vibrio anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii, Pseudomonas sp., or Yersinia ruckeri. The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cell-free extracts of V. anguillarum; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues.     

Synthesis of H-2 antigens by preimplantation mouse embryos

An enzyme-linked immunosorbent assay (ELISA), which utilized anti-H-2 monoclonal antibody, was used to detect H-2 antigens on preimplantation mouse embryos. All embryonic stages studied, including unfertilized eggs and 1-cell, 2-cell, 8-cell, and blastocyst-stage embryos, showed the presence of H-2 antigens. To prove that the H-2 antigens were not cytophilically adsorbed to the embryos, blastocysts were treated with papain to strip off the H-2 antigens, and then the embryos were further incubated to allow the H-2 antigens to regenerate. After a 3-h incubation time, 60% of the H-2 antigens on the embryos had reappeared, proving that the H-2 antigens were synthesized by the embryos themselves.     

Icthyophthirius multifiliis has membrane-associated immobilization antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

Antibodies to cell-surface antigens of plant protoplasts

Both mouse and rabbit polyclonal antibodies to plant cell-surface antigens were developed by immunization with cell membrane material from oat (Avena sativa L. cv. Garry) roots. We were able to quickly assess the activity of antisera by monitoring the degree of protoplast agglutination and by using an indirect immunofluorescence assay. Using polyclonal antibodies to cell-surface antigens, we have found that oat root protoplasts share common surface antigens with protoplasts from other plant tissues and species. From experiments with antisera treated with excess oat leaf or oat root protoplasts before our immunoassays, we have obtained evidence for the existence of organ-specific cell-surface antigens in higher plants.     

A rapid detection of circulating antigens of Dirofilaria immitis in dogs by a dot enzyme-linked immunosorbent assay

This report describes a dot enzyme-linked immunosorbent assay (Dot-ELISA) for detecting circulating antigens in the sera of dogs infected with Dirofilaria immitis (D. immitis). Circulating D. immitis antigens could be detected in 24 of 25 infected dogs. The remaining animal had two immature worms. However, non-infected dogs and dogs infected with other parasites were all negative. Few cross-reactions to different parasite antigens were observed. The advantages of the Dot-ELISA include; 1) there is no need for pretreatment and dilution of sera and samples could be immediately bound to nitrocellulose paper set into microfiltration apparatus, 2) this assay could be carried out within 2 h at room temperature, 3) the resulting enzyme-reaction could be measured by both visual observation and densitometric reading.     

Characterization of human K cells by surface antigens and morphology at the single cell level

Human lymphocytes killing bovine erythrocytes in vitro in antibody-mediated reactions were characterized at the effector cell level in the ADCC plaque assay. Five to 10% of highly purified peripheral blood lymphocytes are active K cells in this system. Forty to 50% of these were T gamma cells expressing the T cell-associated surface antigens T3 and Leu-1. These cells also expressed the T8/Leu-2a antigens (approximately 20%) or the T4/Leu-3a antigens. Although approximately 30% of the K cells were T4+ when examined after completion of the ADCC assay (18 hr), only less than 10% were T4+ (and Leu-3a+) when examined before the assay. The results indicated that exposure to antigen/antibody complexes during the assay induced increased T4 expression, probably linked to Fc gamma R modulation on some initially T4-/T3+ lymphocytes. The expression of the other antigens (including Leu-3a) was not affected by exposure of the lymphocytes to antigen/antibody complexes. Two-color fluorescence experiments further demonstrated that a minor fraction (10 to 20%) of the K cells carrying T cell-associated antigens also expressed the monocyte/null cell-associated antigen M1 as detected with the monoclonal antibody OKM1. A second major category of effector cells, composed of at least 25% of the K cells, were large granular lymphocytes (LGL) that lacked detectable T cell-associated antigens but expressed the HNK-1 (Leu-7) as well as the M1 antigen. As seen from the size of the plaques formed by different effector cells, K cells of the LGL type had a greater recycling capacity and/or cytolytic efficiency than those of T gamma type.     

A 125I-protein A-binding assay detecting antibodies to cell surface antigens

A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were then investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells.     

Age-related variability of serum protein antigens in sturgeons (Acipenseridae, Acipenseriformes)

We studied the variability of antigenic properties of proteins in two sturgeon species at different stages of postembryonic development. The deepest changes occurred in individual components of albumins and beta-globulins (transferrins) and were mostly related to an increased proportion of the protein accounting for these antigens. Transformation of the main component of albumins A1 into adult antigens was completed in 5-month old fry. The main component of beta-globulins A (component of transferrins) appeared in the blood flow much later than other proteins and could retain the fry features until the age of 3-4 years. Other antigens belonging to alpha1- and alpha2-globulins and the second component of transferrins were more stable and did not undergo substantial changes. The direction of ontogenetic variability of serum antigens in sturgeon fry did not depend on the habitat of adult fish in fresh or sea water.