Inhibition of the development of spontaneous autoimmune thyroiditis in the obese strain (OS) chickens by in vivo treatment with anti-CD4 or anti-CD8 antibodies.

The involvement of CD4+ and CD8+ T cells in pathogenesis of spontaneous autoimmune thyroiditis (SAT) in obese strain (OS) chickens has not been studied in depth until now. We depleted CD4+ or CD8+ T cells in OS chickens by treatment with murine monoclonal anti-CD4 or anti-CD8 antibodies at 3 day intervals beginning at hatching. The birds were killed at 19-25 days of age. Treatment with anti-CD4 antibody completely prevented SAT development, while treatment with anti-CD8 antibody partially inhibited SAT. These results show the critical role of CD4+ T cells in the development of SAT in OS chickens, and indicate that CD8+ T cells are also involved in SAT pathogenesis.

Helper activity of chicken V beta 1+ and V beta 2+ alpha beta T cells for in vitro IgA antibody synthesis.

Chickens have only two T cell receptor variable beta gene families: V beta 1 and V beta 2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of V beta 1+ alpha beta T cells by treatment with a TCR V beta 1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both V beta 1+ and V beta 2+ chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of V beta 1+ T cell-depleted chickens to produce IgA is not caused by the inability of V beta 2+ T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).

Coordinate induction of tetrahydrobiopterin synthesis and nitric oxide synthase activity in chicken macrophages: upregulation of GTP-cyclohydrolase I activity.

Biosynthesis of nitric oxide (NO) and tetrahydrobiopterin (BH4) was investigated during cytokine-mediated activation of chicken macrophages. Monocyte derived macrophages and HD11 cells, a chicken macrophage cell line, constitutively synthesize BH4. Treatment of these cells with chicken macrophage activation factor (ChMAF) causes up to 10-fold increases of intracellular BH4 and of nitrite concentrations in the cell culture supernatant. Elevated BH4 levels correlate with an increase in GTP-cyclohydrolase I (GTP-CH) activity. Kinetic studies show a joint upregulation of GTP-CH activity and NO synthase activity first detectable 4 hr after stimulation. A corresponding increase in the mRNA for GTP-CH was detected by Northern blot analysis with a chicken GTP-CH specific cDNA probe. These results demonstrate that cytokine-induced BH4 synthesis by chicken macrophages is at least partially regulated through increased GTP-CH gene expression. The functional relevance of BH4 formation for NO production is shown by experiments using 2,4-diamino-6-hydroxypyrimidine (DAHP) as a specific inhibitor of GTP-CH. Monocyte derived macrophages stimulated in the presence of DAHP show a significant decrease in NO synthesis. The effect of DAHP was reversed by adding sepiapterin, which allows synthesis of BH4 through a salvage pathway.

[Systemic availability of bovine immunoglobulin G and chicken immunoglobulin Y after feeding colostrum and whole egg powder to newborn calves]. / Zur systemischen Verfügbarkeit von bovinem Immunglobulin G und Hühner-Immunglobulin Y aus gefüttertem Kolostrum bzw. Volleipulver bei neugeborenen Kälbern.

In connection with a study on the prophylaxis of infectious diarrhea with specific egg yolk antibodies, the systemic availability of colostral bovine immunoglobulin G (bIgG) and chicken immunoglobulin Y (IgY) after feeding egg powder was investigated on 26 newborn calves from 23 different farms. Blood was sampled daily and at the same day time from these calves in the first 14 days of life. During the feeding of colostrum, the mean bIgG concentration was highest at day 1 post natum with a value of 9.3 mg/ml serum. Thereafter, the mean bIgG level was reduced continuously to a significant lower concentration of 4.9 mg/ml serum at day 12 post natum and remained nearly constant at 5.2 mg/ml till to the end of the observation period. Total protein concentrations in the serum did not change and plateaued at a mean value of 56.2 mg/ml (SD 11.2). The number of colostrum meals had no significant effect on the mean bIgG concentrations during that period. The individual variation of bIgG concentrations was very high on every day of the sampling period. The mean coefficient of variation was at 52.1 % (SD 5.7). After having described the individual bIgG concentration curves mathematically with a regression curve, two groups with significantly different bIgG elimination constants (k) could be obtained. Thus in one group (n = 10) with k-values of < -0.02 a mean half time of serum bIgG of 24.3 days (SD 4.6) was calculated. In the other group of calves (n = 16) with elimination constants of k > -0.02, a mean half time of 68.5 days (SD 36.7) could be calculated, possibly because these calves started earlier with their endogenous bIgG production. Additionally, to 18 of these calves 20 g egg powder with an IgY concentration of 15 mg/g was fed up to day 14. Calves had a maximal mean IgY concentration of 1.9 micrograms/ml serum if egg powder feeding started already during the first 12 hours of life. Starting at a later time resulted in a significant reduction of IgY levels. For example, the mean initial IgY concentration dropped to 0.035 micrograms/ml serum after having had the first egg powder application between 25 and 48 hours post natum. Using the individual IgY elimination constant derived from a regression analysis (r2 = 0.84) of the IgY concentration curve, a mean IgY half time of 5.0 days (SD 2.5) could be calculated. To prevent the absorption of heterologous antibodies and consecutively, also to prevent a possible systemic effect, egg powder for prophylactic purposes in newborn calves should be fed after the first 24, better 48 hour, post natum. Most important for the prophylactic effect of specific antibodies on infectious diarrhea is not their systemic but their high local intestinal availability.

Preferential expression of V beta gene families in CD8 memory cells of apparently healthy donors.

Antigen stimulation may lead to expansion or deletion of T-cells expressing T-cell receptors that belong to specific V beta gene families. Since such stimulation at the same time will lead to conversion from naive to memory T-cells, we have asked whether a bias in V beta families can be observed when comparing these two populations. We have studied the expression of V beta 3, 8, 13.3, 19, and 22 in peripheral blood T-cells for 12 apparently healthy male donors. For flow cytometry 100,000 CD4+ or CD8+ cells each were analysed in three-colour immunofluorescence for percentage of V beta families among CD45R0- naive and CD45R0+ memory cell. Greater than twofold excess was found in the CD8+CD45R0+ cells in four cases (1 x V beta 13.3, 2 x V beta 19, 1 x V beta 22) and a greater than twofold decrease in CD8+CD45R0+ cells in two cases (1 x V beta 8, 1 x V beta 22). In contrast, among CD4+ cells no such bias was detected. The excess in CD8+CD45R0+ memory cells showed no substantial fluctuation over time in that it was found to be stable for 19 to 70 days. Finally, in vitro conversion of purified CD8+CD45R0- cells to CD45R0+ cells by polyclonal stimulation with PHA did not result in the excess of V beta usage observed in vivo. These data suggest that specific antigen stimulation during past infection or allergy may be responsible for the excess of certain V beta gene families. Clinical studies looking for disease associations will have to test CD4 and CD8 naive and memory subsets in order to precisely identify a bias in V beta usage and these studies will have to consider the pronounced changes observed in healthy controls.

Differential binding of two chicken beta-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells.

Plant lectins can be potent modulators of vertebrate immune functions. Biochemical characterization of lectins from animal tissues enables the determination of whether these endogenous activities display a comparable immunological potency. Focusing on chicken beta-galactoside-binding lectins, the monomeric intestinal (CL-14) and the dimeric liver lectin (CL-16) were purified and the lack of cross-contamination was ascertained. In very close agreement with the molecular masses of 14,974 and 14,976 calculated on the basis of the available sequence data (Y. Sakakura et al., J. Biol. Chem. 265, 21573-21579, 1990), electrospray mass spectrometric analysis yielded values of 14,969 (CL-14) and 14,972 (CL-16), the reasons for the deviation in gel electrophoretic behavior being unclear. Solid-phase assays with immobilized lactosylated poly-L-lysine demonstrated a comparatively lower affinity and higher extent of binding at saturation for the monomeric lectin than for the dimeric protein, whose properties were similar to those of an immunomodulatory plant lectin. Flow cytometry revealed homogeneous and strong binding of the dimeric lectin within the chicken peripheral blood lymphocyte population, whereas the monomeric lectin stained two subpopulations at different intensities. Two-color flow cytometry disclosed preferential binding of this lectin to B cells. When a B cell line was employed for determination of affinity constants and extents of binding at saturation, qualitatively comparable parameters to those for the solid-phase assays were obtained. The similar profile of lectin-binding glycoproteins in blots of cellular extracts underscored that accessibility to ligands, not qualitatively different ligand display, may explain the differences for the cell line. At up to a concentration of 10 micrograms/ml of the lectins no stimulation of [3H]thymidine incorporation was seen for blood and spleen cell populations. However, the dimeric lectin reduced stimulation of cells that were responsive to an anti-TcR2 antibody. Thus, this lectin can apparently exhibit inhibitory activity to this kind of T cell activation in vitro.

Preferential TCR V beta 1 gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis of the obese strain of chickens.

We studied T cell receptor variable beta (TCR V beta) gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis (SAT) of obese strain (OS) chickens. Chicken alpha beta T cells may express either V beta 1 or V beta 2 genes, the products of which can be recognized by TCR2 and TCR3 monoclonal antibodies, respectively. Selective depletion of V beta 1+ or V beta 2+ T cells in OS chickens was accomplished by repeated injections of TCR2 or TCR3 antibodies into embryonic and 1-3-week-old chickens. The birds were killed at 20 days of age and their spleens and thyroid glands evaluated by immunohistochemistry. We found that V beta 1+ T cells preferentially infiltrated OS chicken thyroid glands. Antibody treatments resulted in a 41% reduction in frequency of V beta 1+, and a 87% reduction of the frequency of V beta 2+ cells in the circulation, and in a profound decrease of the respective T cells in spleens and thyroid glands. Selective suppression of V beta 1+ T cells partially inhibited SAT development in that thyroid-infiltrating cells and destruction of thyroid follicles were reduced by more than 50%. Thyroglobulin autoantibody serum levels were also reduced in V beta 1+ T cell-depleted OS chickens, whereas selective depletion of V beta 2+ T cells did not inhibit SAT development. These findings indicate preferential TCR V beta 1 gene usage by autoreactive T cells in SAT of OS chickens.

[Intestinal absorption of homologous and heterologous immunoglobulin G in newborn calves]. / Untersuchungen zur intestinalen Absorption von homologem und heterologem Immunglobulin G bei neugeborenen Kälbern.

Studying the prophylactic effects of specific yolk antibodies against diarrhea in newborn calves, also the intestinal absorption of unspecific heterogeneous avian antibodies as well as their effects on the uptake of maternal bovine colostral antibodies (bIgG) was investigated. Two groups of newborn calves received egg powder (16 g or 8 g per day) for the first 10 days of their life beginning with the first meal. A third group was kept as a control without any egg powder in their diet. Blood samples (5 to 10 calves per sampling time) were taken from 123 calves at 6, 12, 24, 48, or 96 h postnatally. With both doses the highest chicken IgG (cIgG) levels (3.1 micrograms resp. 1.2 micrograms per ml serum) have been measured 12 h after birth. These concentrations decreased continuously to the levels of 1.1 micrograms resp. 0.2 micrograms cIgG per ml serum at 96 h postnatally. The uptake into blood at 6 h postnatally has roughly been estimated as approximately 23% (bIgG) and 7% resp. 6% (cIgG) of the IgG dosages given with the first meal. The time-course (6 to 96 h) of the bIgG level in blood was quite stable, plateauing already after 6 h at a mean of 5.9 mg per ml serum. Significant differences between the bIgG levels of calves with yolk antibodies in their diet (6.2 resp. 6.1 mg bIgG per ml serum) and those of the control group (5.4 mg per ml serum) could not be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The specificity and affinity of polyclonal antibodies raised in rabbits against a hapten conjugated to rabbit serum albumin.

In this study, rabbit antisera to hapten-rabbit serum albumin conjugates were investigated regarding antibody titer, affinity, specificity, and affinity distribution. Methyl phosphonic acid p-amino-phenyl 1,2,2-trimethylpropyldiester (MATP) served as model hapten. Four MATP-rabbit serum albumin conjugates with various hapten densities (with and without spacer) were synthesized and used for immunization of rabbits. Antisera were collected over a 130 day-period and characterized with different ELISA methods. We found that immunogens with rabbit serum albumin gave antisera with lower titers, but similar affinity as compared to polyclonal or monoclonal antibodies obtained with non-rabbit protein as carrier protein. Immunogens with a low hapten density led to higher final titers without affecting antibody affinity or specificity. Immunogens containing a bridging group resulted in higher antibody affinity with a changed specificity. The pattern of antibody affinity distribution differed considerably among individual rabbits and showed a non-Gaussian subpopulation distribution.

[Significance and course of a cold agglutinin in Eperythrozoon suis infection of swine]. / Bedeutung und Verlauf eines Kälteagglutinins bei der Eperythrozoon suis-Infektion des Schweines.

After infection with Eperythrozoon suis, pigs began to produce cold agglutinins of immunoglobulin type IgM, that because of the similarity between the pathogenic antigen and antigen on the erythrocyte membrane caused agglomeration. The progression of this cold agglutinin was measured with an enzyme immunoassay especially invented for this purpose, and compared with serum-IgM, agglutination strength, pathogenic effect as well as number of erythrocytes in the blood. The cold agglutinin extracted from the erythrocyte membrane at 40 degrees C showed, in comparison to the initial figures, a higher level (1259 micrograms/ml) at 6 days after the illness peak, reaching its maximum (2435 micrograms/ml) at 12 days. Similar results were achieved at lower extraction temperatures (22 degrees C, 0 degree C). A high correlation could be shown between the levels of IgM and of cold agglutinin, as well as the parallel increase in the agglutination strength of the blood. At the time of maximal pathogenic effect, no agglutination of blood was observed. The number of erythrocytes decreased in acute phases of an attack to a constant mean of 2.32 Mill./microliters of blood and then increased, at the same rate as the cold agglutinin level decreased, almost reaching normal values. These experiments confirm the fact that an organism following an infection with Eperythrozoon suis, begins to produce cold agglutinins. Due to structural similarities between the pathogen and the erythrocyte antigen the cold agglutinin causes temperature dependent agglutination.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of antigenic differences of recombinant and pituitary bovine growth hormone using monoclonal antibodies.

For characterization and determination of recombinant bovine GH (rbGH) eight monoclonal antibodies (MAb) were produced against rbGH from Monsanto. The various MAb showed different affinities to rbGH, pituitary bovine GH (pbGH), and pituitary ovine GH (poGH). With epitope analysis several MAb were shown to recognize different epitopes of rbGH. The MAb MUC-rbGH-3A11 and MUC-rbGH-1E5 were used to develop a Sandwich ELISA. By checking the specificity of the assay no cross reactivity was found with pituitary porcine GH, pituitary human GH, bovine or ovine prolactin and little cross reactivity with poGH could be found. The Sandwich ELISA detected various rbGH (Monsanto, Elanco, Cyanamid) with different N-terminal amino acids and discriminated between rbGH and pituitary bovine GH by an affinity factor of 2.0. The detection level was 2 ng rbGH per ml PBS buffer. The recovery was about 86% in bovine serum. It might therefore be possible to detect rbGH-treated cows using a Sandwich ELISA, but this would need a field study.

[New possibilities in oral immunoprophylaxis of newborn diarrhea in calves--a field study using specific egg antibodies]. / Neue Möglichkeiten in der oralen Immunprophylaxe der Neugeborenendiarrhoe des Kalbes--ein Feldversuch mit spezifischen Eiantikörpern.

To investigate the effect of oral immunoprophylaxis in diarrhea of newborn calves egg powder with antibodies specific to E. coli K99 (ETEC) and rotavirus were used in a field trial in south west Germany. Fourteen farms with a total of 105 calves were selected. Egg powder (21 g per day) was fed as a supplement to the regular diet for the first 14 days after birth. Animals of the control group received egg powder derived from hens not immunized with the antigens. Frequency, duration and severity of diarrhea, fecal dry matter and weight gain were examined in order to evaluate the influence of the specific egg powder. Using a Lactovac test kit pathogens were detected in the feces of 76.2% of the calves, with 24.7% infected with E. coli K99, 39.1% with rotavirus, 19.0% with coronavirus and 32.4% with cryptosporidia. An overall reduction in diarrhea frequency from 68.5% to 52.9% was observed in calves fed with specific antibodies. Animals with an E. coli K99 infection showed a reduction from 92.3% to 30.7% and those infected exclusively with E. coli K99 from 83.3% to 0%. The duration of diarrhea was significantly reduced (42 h) in animals fed with specific antibodies in comparison to the control animals (60 h). With the exception of animals infected with coronavirus a marked reduction in the severity of diarrhea was observed in antibody treated calves. During the first 14 days after birth antibody treated calves showed a weight gain of 5.6 kg on average in comparison with 3.5 kg in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Chicken egg antibodies for immunohistochemical labeling of growth hormone and prolactin in bovine pituitary gland.

We describe the production of polyclonal chicken antibodies specific for bovine growth hormone (bGH) and prolactin (PRL). Antibodies were generated by immunization of laying hens with recombinant bGH (rbGH), pituitary derived bGH (pbGH), and ovine PRL (oPRL). After the lipoprotein fraction was removed by dextran sulfate precipitation the antibodies were isolated from the egg yolks by ammonium sulfate precipitation. Immunization with rbGH and oPRL generated large amounts of specific antibodies, as revealed by ELISA and Western blot analysis. Antibodies against pbGH showed pronounced crossreactions with oPRL. The antibodies against rbGH and oPRL were well suited for sensitive and specific labeling of the GH- and PRL-synthesizing cells in bovine pituitary glands by immunohistochemistry. In addition, a quick and sensitive procedure for demonstration of both bGH- and PRL-synthesizing cells in a single paraffin section by double immunohistochemistry is presented. The chicken anti-bGH antibodies showed excellent results in combination with rabbit anti-PRL antibodies. The main advantage of avian antibodies in double immunostaining methods is the lack of crossreactions between avian antibodies and mammalian immunoglobulins and receptors which bind to the crystalline fragment of mammalian immunoglobulins (Fc receptors).

Characterization of new monoclonal antibodies identifying avian T lymphocyte antigens.

Six monoclonal antibodies (mAb) were produced to identify and characterize surface antigens of chicken T cells. Determination of their reactivity with different lymphatic cells using immunofluorescence analysis demonstrates that mAb KH8, NA6, PD4 and TH8 stained 32-43% blood lymphocytes, 72-77% thymocytes and 19-27% spleen cells, mAb OC5 approximately 99% thymocytes and 55% blood and spleen lymphocytes each, and mAb OC2 36% blood lymphocytes, 79% thymocytes and 62% spleen cells. The KH8, NA6, PD4 and TH8 antibodies immunoprecipitated from lysates of surface-labeled chicken thymocytes a polypeptide of M(r) 60,000 under non-reducing conditions and the OC5 antibody a glycoprotein of M(r) 68,000 under reducing conditions. MAb OC2 precipitated a single polypeptide of M(r) 40,000 under both conditions. The mAb KH8, NA6, PD4, TH8 and OC2 inhibited ConA-induced proliferative responses of blood T cells in vitro. However, sepharose-bound or soluble OC5 antibody was able to increase DNA synthesis significantly. These results indicate that (a) the mAb KH8, NA6, PD4 and TH8 identify the avian homologue of the mammalian CD4 molecule, (b) the mAb OC2 detects the avian CD2 antigen, and (c) the mAb OC5 recognizes the putative avian CD5 homologue.

In vivo depletion of chicken T-cell subsets.

In the chicken three types of T-cell receptors can be defined by monoclonal antibodies TCR1, TCR2 and TCR3, which recognize gamma delta T cells, and V beta 1- and V beta 2-expressing alpha beta T cells, respectively. In the present report we have analysed means of selectively depleting the gamma delta T cells and the V beta 1+ alpha beta T cells. gamma delta T cells, which represent up to 66% of all T cells in blood of a 6-month-old chicken, can be effectively depleted by neonatal thymectomy (Tx) to levels as low as 1%. Immunohistology demonstrates a similar depletion in lymphoid organs while intestinal epithelium-associated gamma delta T cells are affected by Tx to a lesser extent. V beta 1-bearing alpha beta T cells, which comprise about 80% of the alpha beta T cells, were depleted by embryonic and neonatal injection of the TCR2 antibody. In the thymus such treatment depleted only the V beta 1+ alpha beta T cells with high density expression of T-cell receptor. Therefore, we thymectomized TCR2-treated animals in order to prevent development of mature V beta 1+ alpha beta T cells from the low density immature thymocytes. Treatment of chickens with a total of 22 mg of TCR2 antibody plus Tx reduced V beta 1+ alpha beta T cells from an average of 65% to 10% of all T cells. In these TCR2 antibody-treated animals the V beta 2-expressing alpha beta T cells become the predominant type of T cell (average 85%).

A homogeneous immunological detection system for soman using the in vitro protection of acetylcholinesterase by a monoclonal antibody.

In this study a monoclonal antibody (MAb) based soman detection system was investigated. Since the MAb F71D7 recognizes the pinacolyl group of soman, non-toxic soman analogues are also detected when using an indirect competitive ELISA. This can lead to falsely positive results. The toxic effect of soman is, however, independent of the pinacolyl group. In the described homogeneous enzyme immunoassay (EIA), the inhibitory effect of soman on acetylcholinesterase (AChE) was combined with its specific binding to the MAb F71D7 in order to minimize false positive results and enhance the specificity of the detection system. In this rapid EIA no incubation or washing steps are necessary, so only time for pipetting and reaction have to be considered. Soman could be detected in concentrations of 1.6-25 nM using the EIA. This corresponds to 8 pg soman per 25 microliters sample and means that compared to other ELISA systems, besides enhanced specificity, the limit of detection could be improved by 3 orders of magnitude.

The cytotoxic T lymphocyte response in reticuloendotheliosis virus-infected chickens is mediated by alpha beta and not by gamma delta T cells.

We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h 51Cr-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alpha beta and TCR gamma delta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gamma delta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, > 98% pure alpha beta and gamma delta T cells, it was found that alpha beta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific 51Cr-release, while gamma delta T cells showed only 2-4% 51Cr-release. These data indicate that REV-specific CTL response is mediated by alpha beta T cells and that gamma delta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.

Quantification of the immunoglobulin classes IgG and IgA in the young and adult pigeon (Columba livia).

Pigeon immunoglobulin classes IgG and IgA were purified and specific isotype antisera were produced in rabbits. The antisera were used to develop a quantitative assay for both immunoglobulins. Serum IgG concentrations in relation to age showed a similar pattern in pigeons to that in chickens. The same applies to the transfer of maternal immunoglobulins via egg. Besides this transfer mechanism, an additional transfer of immunoglobulins exists in the pigeon via feeding of crop milk. Crop milk contains considerable amounts of IgA (1.45 mg/ml) and significantly less IgG (0.34 mg/ml). During day 1 intestinal absorption of IgA is possible to a very low extent. Most of the IgA, as well as IgG, remains in the intestine to provide local immunity.

Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B.

The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography. The alpha-toxin has a mol. wt of 190,000 and does not contain any disulfide cross-linkages. It consists of a single polypeptide chain. The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC. The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice. Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5). One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol. wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test. The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain. The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine. By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.