Ichthyophthirius 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 ceil surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-l14 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-dimensiona! 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.     

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.     

Can the surface immobilization antigens of Philasterides dicentrarchi (Ciliophora: Scuticociliatida) be used as target antigens to develop vaccines in cultured fish?

In order to test whether immobilization antigens (i-antigens) of Philasterides dicentrarchi could be suitable antigenic targets against scuticociliatosis, polyclonal olive flounder (Paralichthys olivaceus) sera were raised against P. dicentrarchi by immunization with lysates of ciliates grown using chinook salmon epithelial (CHSE) cells, and the ability of the immune sera to kill the ciliates via classical complement pathway was analyzed in relation to agglutination activity. The immune sera showed clear agglutination activity against the CHSE-cultured ciliates. However, the agglutinated ciliates were not killed but escaped from the agglutinated mass within a few hours. Ciliates isolated from fish artificially infected with the same population of CHSE-cultured ciliates were not agglutinated by the immune sera even at the lowest dilution. In antibody-dependent complement-mediated killing (ADCK), the immune sera completely killed the CHSE-cultured ciliates at relatively higher serum dilutions (showing low or no agglutination activity). However, CHSE-cultured ciliates were not killed completely at lower immune serum dilutions (showing high agglutination activity). In contrast to CHSE-cultured ciliates, the ciliates isolated from infected fish were killed at lower dilutions of the immune sera in spite of no agglutination response. Considering the presence of various i-antigen types, ability to change i-antigen type in response to corresponding antibody, and relatively low ADCK activity at high agglutination titer, i-antigens of P. dicentrarchi may not be good targets for subunit vaccine development. To develop subunit vaccines against scuticociliatosis, other surface antigens expressed constitutively or expressed specifically under the infection state for survival of the ciliates in the host fish might be more favorable to elicit protective antibodies than the surface i-antigens.     

In vitro oncospheral agglutination given by immune sera from mice infected, and rabbits injected, with eggs of Hymenolepis nana.

Oncospheral agglutination given by sera immunized with Hymenolepis nana eggs is described as a new way of assessing H. nana infection. All sera of mice which possessed acquired protective immunity against reinfection by H. nana eggs had the potency to induce oncospheral agglutination in vitro. Only oncospheres, which had been hatched, agglutinated, no agglutination occurred in sera from uninfected mice. Oncospheral agglutination was carried out by mixing 0-1 ml of serial two-fold dilutions of serum and 0-1 ml of Hanks' balanced salt solution containing about 600 hatched oncospheres. Titre of agglutinins was indicated as a reciprocal of the final dilution capable of giving agglutination clusters made of three or more oncospheres. Agglutinins developed within 14 days after a primary infection with 500 shell-free eggs. There was no rapid increase of agglutinins within 4 days following a secondary infection. The titre increase coincided with the increase in dosages of eggs. Agglutinins were thought to be immunoglobulins, because the potency of the serum to agglutinate oncospheres was extinguished after absorption of globulins with rabbit anti-mouse globulin serum. Agglutinins were produced in rabbits by intravenous injections of shell-free eggs. The titres of the rabbit sera were much higher than those of mouse sera.     

Tetrahymena pyriformis as a protective antigen against Ichthyophthirius multifiliis infection: comparisons between isolates and ciliary preparations

Channel catfish, Ictalurus punctatus , were immunized with cilia from three isolates of Tetrahymena pyriformis and challenged with Ichthyophthirius multifiliis using a reproducible quantitative procedure. Two different methods of deciliation were used in antigen preparation. Results indicate that T. pyriformis cilia elicit an immune response in channel catfish against I. multifiliis , and that the protective ability of the cilia varies between T. pyriformis strains.     

Antibodies against the predominant glycosaminoglycan of the mammalian cornea, keratan sulfate-I

Antibodies have been made in rabbits against bovine corneal keratan sulfate proteoglycan. Antisera were titered by their ability to agglutinate sheep red blood cells that had been coated with the proteoglycan. Immune antisera, but not preimmune sera, agglutinate coated cells. Uncoated cells are not agglutinated by either serum. Immune agglutination is inhibited by prior incubation of antiserum with the intact corneal proteoglycan fraction or with 2-mercaptoethanol. Immune agglutination is also sharply reduced by the glycosaminoglycans, keratan sulfate-I (corneal type), and keratan sulfate-II (cartilage type). Desulfated keratan sulfate-I is somewhat less effective as an inhibitor than keratan sulfate-I. In contrast, chondroitin 4- and 6-sulfates, heparin, and hyaluronic acid do not interfere with immune agglutination. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblot transfer of the proteins to nitrocellulose paper, incubation with antisera, and reaction with 125I-protein A suggest that the proteoglycan fraction contains high molecular weight antigenic components (Mr = approximately 300,000) whose mobility is sharply decreased by incubation with keratanase to that corresponding to molecular weights of approximately 55,000 and 40,000. No antigenic component appears sensitive to reduction by 2-mercaptoethanol. Chondroitinase ABC does not affect the mobility of proteins in the proteoglycan fraction. These results suggest that antibodies against corneal keratan sulfate proteoglycan may include some that react with the keratan sulfate chains, as well as those directed against the core protein. Keratan sulfate core proteins of two molecular weights may be present.     

The question of the validity of haptoglobin/G-streptococci relations in nonhuman sera]

The relationship between haptoglobin type and the agglutinability of some group G streptococci, which was observed in humans, could also be demonstrated in animal sera. In humans the haptoglobin type Hp 1-1 is correlated with a lacking or very low agglutinability. Sera of 10 rhesus monkeys showed Hp-type 1-1 and agglutinated the streptococci only to dilutions of the serum up to 1:4. The sera of 48 pigs contained an Hp 1-1 like type and a streptococcus agglutination titer of max. 1:10. Seven ahaptoglobinaemic sera gave the same values. Fifty examined sera of rabbits showed also an Hp 1-1 like pattern and did not agglutinate or in undiluted state only.     

Altered expression of Plasmodium knowlesi variant antigen on the erythrocyte membrane in splenectomized rhesus monkeys

Variant antigens are present on the surface of Plasmodium knowlesi malaria-infected erythrocytes as detected by the schizont-infected cell agglutination (SICA) assay. We found that parasitized erythrocytes passaged in splenectomized monkeys did not agglutinate with immune sera. On the first passage from intact to splenectomized monkeys, the SICA titers decreased 4- to 16-fold; after the second and subsequent passages in splenectomized monkeys, the infected cells became nonagglutinable to all sera tested, including sera from animals infected with the nonagglutinating parasites. This loss of agglutinability could have resulted from selection of a genetically distinct subpopulation of the original parasites or the ability of the original parasites to alter their phenotypic expression. We have designated the new nonagglutinable phenotype, SICA [-], and the agglutinable phenotype, SICA [+]. The loss of agglutinability indicates that the variant antigen normally expressed on the erythrocyte membrane of infected cells is altered or absent. Because SICA [-] parasites developed in the absence of the spleen, the major organ of host defense against malaria, then this organ may in some manner influence or modulate antigenic expression in P. knowlesi and possibly other malaria parasites.     

Stage-Dependent Protein Composition in the Life Cycle of Synchronous Ichthyophthirius multifiliis,a Ciliate Fish Parasite1

The sequential development of Ichthyophthirius multifiliis Fouquet, 1876 at 22°C was monitored from detachment from the fish through to infective tomites using one-dimensional polyacrylamide gel electrophoresis. Methods are described for synchronizing I. multifiliis cells to permit the collection of large numbers of cells at stages during mitotic division. The electrophoretic protein patterns of the encysted stage differ from those of the tomite stage as well as from those of the other measured intervals.     

Antiserum inhibition of rabbit spermatozoal adherence to ova

The in vitro effects of different normal and immune sera, rabbit oviductal fluid, and culture media from incubations of female reproductive tissues on the adherence of rabbit spermatozoa to rabbit and rat ova is described. There were no apparent differences in results due to species of ova. Adherence of spermatozoa to ova was not affected by treatment with normal sera. Antisera against materials that contained spermatozoa such as semen epididymal spermatozoa and testis completely inhibited the spermatozoa from attaching to ova and caused considerable agglutination of sperm cells in the incubation slides. When the agglutinating and immobilizing activities of rabbit and goat antisera were removed by papain treatment the antiadherent effect of the antisera was unaffected. Oviductal secretions and concentrated media from the culture of reproductive tissue from female rabbits isoimmunized with semen inhibited adherence of sperm.     

Effects of the antiserum against a fraction enriched in scrapie-associated fibrils on the scrapie incubation period in mice

An antiserum against a fraction enriched for scrapie-associated fibrils (SAF), was examined for its effects on scrapie incubation period by inoculating mice either intraperitoneally or intracerebrally with various dilutions of the serum mixed with scrapie-infected mouse brain homogenate. After intraperitoneal inoculation the mean time of the incubation period increased with increasing concentrations of the antiserum in a statistically significant fashion, when the serum dilutions were made with phosphate-buffered saline. After intracerebral inoculation, however, there were no statistically significant differences between the control group and any of the antiserum-groups. When the antiserum dilutions were made with pre-immune serum, the mice inoculated intraperitoneally also showed no significant differences between the two groups. These results indicate that the specific antibodies to SAF have no effect on the scrapie infectivity.     

Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.     

Antibodies to human immunodeficiency virus in human sera induce cell-mediated lysis of human immunodeficiency virus-infected cells

The capacity of human immunodeficiency virus (HIV) antibody-positive sera from homosexually active men without acquired immune deficiency syndrome to lyse the HIV-infected T cell lines MOLT-4f and CCRF-CEM (CEM) in cooperation with lymphocytes from normal donors was investigated. Twenty-seven HIV antibody-positive sera, most of which enhanced the killing of HIV-infected MOLT-4f and CEM target cells by normal mononuclear cells were studied in detail. HIV antibody-positive sera resulted in lysis at dilutions as high as 1/10,000. HIV antibody-negative sera did not augment lysis of infected target cells. In addition, lysis of uninfected targets was not enhanced in the presence of HIV antibody-positive sera. Because fractionation of the HIV antibody-positive sera on a protein A affinity column resulted in recovery of the activity from the IgG fraction, the extra cytotoxic activity mediated by nonimmune cells in the presence of immune sera appears to be antibody-dependent. Furthermore, the cytotoxic effector cells were in the nonrosetting fraction of lymphocytes and expressed Leu-11 (cluster designation (CD)15) antigens, which is characteristic of cells participating in antibody-dependent cellular cytotoxicity reactions. The antibody specificity of the sera, determined by radioimmunoprecipitation, provides evidence that antibody-dependent cellular cytotoxicity can occur even when there are no detectable antibodies directed against gag proteins. Sera which lacked detectable antibodies to the envelope protein gp120 by radioimmunoprecipitation did not mediate antibody-dependent cellular cytotoxicity.     

Trypanosoma gambiense: immunity with thymic cell transfer in mice.

This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopulation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 M effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were able to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of normal and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection.     

Rubella Hemagglutination Inhibition: Removal of Nonspecific Agglutination Due to Manganous Chloride

Nonspecific inhibitors of rubella hemagglutination can be removed by treatment of sera with heparin-manganous chloride for use in the hemagglutination-inhibition test. After removal of nonspecific inhibitors by this procedure, an excess of manganous chloride may remain. This may cause the cells to agglutinate, thus obscuring the reading at low serum dilutions. This disadvantage can be overcome by the addition of sodium carbonate, which forms an insoluble compound with manganous chloride and does not interfere with antibody determination. The procedure presents a further refinement of the hemagglutination inhibition test for rubella by increasing specificity and sensitivity; it permits detection of antibody levels as low as 1:4 in sera.     

A rabies agglutination test (RAT) for rabies antibody detection

An agglutination test has been developed for the detection of rabies antibodies after human vaccination. The rabies agglutination test (RAT) is based on the capability of specific antibody to agglutinate sensitized polystyrene (or latex) beads. In the RAT, latex beads were coated, in a first step, with inactivated and purified rabies virus (PV strain adapted and propagated on BHK-21 cells) and, in a second step, with bovine serum albumin. Negative control beads were coated with bovine serum albumin (BSA) only. To test for human antibody, several microliters of serum were mixed on a glass slide with an equal volume of virus-sensitized beads and the mixture was gently agitated. After a few minutes, agglutination was visible with sera which had been characterized as positive by the virus neutralization antibody (VNAb) technique. No agglutination was observed with negative sera tested with virus-coated beads or with positive sera tested with BSA-coated beads. Virus-sensitized beads were agglutinated when the virus neutralizing antibody titres were equal to or greater than 2.5 international units per ml (IU/ml) in human sera. The concordance between the RAT results and VNAb titres was about 97% when 2.5 IU/ml was taken as the cut off value for determining the positive sera with the VNAb technique. The possibility that clinicians might use the RAT as a simple means to determine sero-conversion at the end of the post-exposure treatment of patients is discussed.     

Preparation of Type 2 Herpes Simplex Virus Complement-Fixing Antigen

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.     

Mycoplasma-latex agglutination reaction

Morton, Harry E. (University of Pennsylvania, Philadelphia). Mycoplasma-latex agglutination reaction. J. Bacteriol. 92:1196-1205. 1966.-The building up of Mycoplasma cell mass through adsorption to carrier particles as a method for enhancing the agglutination reaction to identify Mycoplasma is described. Mycoplasma cells of human, avian, swine, goat, sewage, and tissue-culture origin were adsorbed to latex particles (0.81 mu) and then were agglutinated by immune sera. The adsorption was demonstrated by electron microscopy. Either the cells or their antibodies, depending on which came into contact with the latex particles first, were adsorbed. The test, completed in less than 2 hr, consisted of serially diluting immune sera with buffered saline, adding the antigen, incubating in a water bath, centrifuging, and reading the reaction under 50 x microscope magnification. The antigen in each reaction tube, representing the growth from about 1.6 ml of culture, was estimated to contain 23 mug of protein (approximately one-tenth the amount of Mycoplasma cells needed for a direct agglutination reaction). In the sera from rabbits undergoing immunization with Mycoplasma antigens, the presence of anti-Mycoplasma antibodies was detected much sooner in the Mycoplasma-latex agglutination reaction test than in the agar-gel diffusion reaction and the growth inhibition tests. Four different lots of latex particles showed excellent uniformity of behavior and stability during storage and testing.     

Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.     

THE STABILITY OF BACTERIAL SUSPENSIONS : III. AGGLUTINATION IN THE PRESENCE OF PROTEINS,NORMAL SERUM,AND IMMUNE SERUM.

1. The addition of proteins or serum to suspensions of bacteria, (Bacillus typhosus or rabbit septicemia) at different pH widens the acid agglutination zone and shifts the isoelectric point to that of the added substance. 2. The amount of serum required to agglutinate is much less near the acid agglutination point of the organisms. 3. The addition of immune serum prevents the salt from decreasing the cohesive force between the organisms, and agglutination therefore is determined solely by the potential, provided excess immune body is present. Whenever the potential is decreased below 15 millivolts the suspension agglutinates.