Influence of endotoxin-protein in immunoglobulin G isotype responses of mice to Brucella abortus lipopolysaccharide.

Brucella abortus endotoxin preparations, containing approximately 5 to 6% protein, induce strong immune and adjuvant immunoglobulin G (IgG) responses as compared with Escherichia coli endotoxin preparations, with equivalent amounts of protein, which induce responses in which IgM antibody predominates. Using an enzyme-linked immunoassay with isotype-specific conjugates, we found that antibody of all four subclasses of IgG were evoked during the course of the immune responses of C3H/HeAu mice to B. abortus endotoxin. Secondary responses of endotoxin-hyporesponsive C3H/HeJ mice were similar to those seen in C3H/HeAu mice, although lower levels of antibody were produced during their primary responses. The primary responses of BALB/c athymic mice consisted almost entirely of IgG3, and IgG1 appeared following a second injection. The effects of lipopolysaccharide (LPS)-associated protein on the immunogenic properties of B. abortus endotoxin were examined by comparing responses to endotoxin with those to a purified B. abortus LPS containing less than 1% protein. The endotoxin evoked strong primary and secondary responses in which antibody directed to LPS determinants consisted mainly of IgG3 and those to the protein determinants were largely IgG1 antibody. Primary and secondary responses to purified LPS consisted mainly of IgG3 antibody. The potential mechanism of the contribution of protein to the immunogenic properties of the endotoxin as well as possible immune mechanisms involved in these responses are discussed.

Effect of stage of lactation on transport of colloidal carbon or Staphylococcus aureus from the mammary gland lumen to lymph nodes in guinea pigs.

Guinea pig mammary glands which were either lactating, involuting or dry were infused with colloidal carbon or killed staphylococci. At different time intervals following infusion, animals were killed and the superficial inguinal lymph nodes examined for the presence of carbon. Sides which had nodes with visible carbon were designated 'positive'. The time intervals from infusion to positive for the three groups were compared using logistic regression. The times required for 50% of the sides to be positive were estimated to be approximately 4 h for lactating glands, 32 h for those in involution, and 13 min for dry glands. Histological differences in distribution of carbon in the mammary tissue suggest that differences in transit time may have been due to different mechanisms of transport through the glands in the three different physiological states. The distribution of bacteria was similar to that of the carbon in the corresponding tissues.

Use of polymer casts or metal particle infusion of ducts to study antigen uptake in the guinea pig mammary gland.

Microcorrosion casts were made of the duct system of guinea pig mammary glands by intramammary infusion of Mercox polyester resin following involution of the glands after the first lactation. The acinar configuration of the involuted gland was apparent on examination of the casts by scanning electron microscopy (SEM). Surface features, which were readily identified as those of imprints of ductal epithelium, were visible at higher magnifications. The morphology of these casts corresponded to the patterns observed by SEM of ethanol cryofractured specimens of mammary tissue. Cryofractured specimens of guinea pig mammary glands were also examined by SEM following intramammary infusion of tantalum. Tantalum particles were observed within the lumina of many ducts. Large phagocytic cells within the lumina were shown to contain tantalum by using back scatter imaging in conjunction with secondary imaging.

Induction of immune and adjuvant immunoglobulin G responses in mice by Brucella lipopolysaccharide.

The immunogenic and adjuvant properties of Brucella abortus and Escherichia coli lipopolysaccharides (LPSs) were studied in endotoxin-responsive, athymic, and euthymic BALB/c mice and in responsive C3H/HeAu mice and congenic nonresponsive C3H/HeJ mice. Consistent with previous reports, E. coli LPS did not stimulate significant primary or secondary antibody responses in C3H/HeJ mice and induced the production of immunoglobulin M (IgM) and low levels of IgG in C3H/HeAu mice. In contrast, B. abortus smooth and rough LPS stimulated primary and secondary antibody responses and induced the production of IgM and high levels of IgG in both responsive and nonresponsive strains of C3H/He mice and in nude mice. When used as adjuvant, B. abortus LPS augmented the IgG plaque-forming-cell response of C3H/HeAu and BALB/c euthymic mice to the T-dependent antigen sheep erythrocytes. E. coli LPS augmented only the IgM plaque-forming-cell response in the same mouse strains. Neither B. abortus nor E. coli LPS was adjuvant for C3H/HeJ or nude mice. The dichotomy between the antibody and adjuvant responses of both C3H/HeJ mice and athymic mice to B. abortus LPS may be a function of the true thymus independence and dependence of these responses. In addition, the refractiveness of C3H/HeJ and nude mice to B. abortus LPS as adjuvant, but not as mitogen or polyclonal B cell activator, clearly dissociates these phenomena.

Immunochemical characterization of rough Brucella lipopolysaccharides.

Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its reaction of partial identity in immunodiffusion. Monospecific mouse sera against B. ovis R-LPS agglutinated only the homologous bacteria but not R cells of other species of Brucella. B. ovis R-LPS contained more 2-keto, 3-deoxyoctonate, and glucosamine as a percentage of dry weight than any other R-LPS tested. B. abortus R-LPS was identified by the absence of an unidentified sugar present in the other R-LPS molecules, and B. melitensis R-LPS could be differentiated from B. canis R-LPS by its higher content of fatty acids. In contrast to S-LPS, all of the R-LPS studied lacked quinovosamine. In electron micrographs, Brucella R-LPS had a granular appearance, in contrast to typical lamellar structures formed by Brucella S-LPS and Escherichia coli R-LPS.

Interaction of Escherichia coli matrix protein with Brucella abortus peptidoglycan and chitin.

Although 10-20 mM MgCl2 in SDS at 37 degrees C extracted significant amounts of Brucella abortus 45/20 peptidoglycan associated proteins (PGAP) from 50 degrees C SDS-extracted cell envelopes, extraction of PGAP of Escherichia coli K-12 required at least 50 mM MgCl2. To study the basis for this difference, PGAP were bound to peptidoglycans and chitin in the presence and absence of lipopolysaccharide and the conditions for their reextraction examined. E. coli PGAP bound to both E. coli and B. abortus peptidoglycans. In either case, 50 mM MgCl2 was necessary for their reextraction regardless of the presence or absence of lipopolysaccharide. E. coli PGAP also bound to chitin and this binding was identical to the binding to peptidoglycans with respect to the temperature of extraction in SDS and, in presence of lipopolysaccharide, the MgCl2 concentration required for reextraction. However, when lipopolysaccharide was absent, 10-20 mM MgCl2 was enough for partial reextraction of PGAP. These results, and our previous finding that B. abortus lipopolysaccharides are not stabilized by interactions with divalent cations, suggested that E. coli lipopolysaccharide remaining in the 50 degrees C SDS-extracted envelopes interfered with the extraction of matrix proteins by MgCl2.

Isolation, purification, and partial characterization of Brucella abortus matrix protein.

Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along with lipopolysaccharide. At temperatures below 60 degrees C, the protein did not bind SDS strongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly, and the apparent molecular weight was 38,000. Urea at 5 M did not alter the electrophoretic mobility of this 38,000-molecular-weight form. Immunoelectrophoresis in detergents with antisera to cell envelopes, carbohydrate staining of SDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodies by mice immunized with the purified protein indicated that lipopolysaccharide was present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purification by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chromatographic analysis showed residual lipid tightly associated with the protein. The results suggested that the interactions between matrix proteins and other outer membrane components are stronger in B. abortus than in Escherichia coli, which was used as a control throughout.

Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope.

Cell envelopes prepared from smooth and rough strains of Brucella were characterized on the basis of lipopolysaccharide and protein content. The action of three kinds of detergents on Brucella cell envelopes and Escherichia coli control cell envelopes was examined on the basis of the proteins and lipopolysaccharides that were extracted. As compared with those of E. coli, Brucella cell envelopes were resistant to nonionic detergents. Zwittergents 312 and 316 were most effective in extracting E. coli cell envelopes, and Zwittergent 316 was most effective in extracting Brucella cell envelopes. Sarkosyl extracted proteins but extracted only trace amounts of lipopolysaccharides from cell envelopes of both bacteria. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the Sarkosyl-resistant proteins revealed a composition similar to that of the proteins exposed on the surfaces of viable cells, as determined by the lactoperoxidase-125I radioiodination method. EDTA, with either Tris-HCl or Tris-HCl-Triton X-100, did not have detectable effects on Brucella cell envelopes. Ultracentrifugation of purified lipopolysaccharides in detergents and EDTA demonstrate that, in contrast to that of E. coli, Brucella lipopolysaccharide was not stabilized by divalent cations. Sarkosyl was ineffective in dispersing lipopolysaccharides, whereas the action of Zwittergents was related to the length of their alkyl chains.

Biological activities of Brucella abortus lipopolysaccharides.

Purified lipopolysaccharide (LPS) from smooth (s) and rough (R) strains of Brucella abortus and lipid A isolated from S-LPS by mild acid hydrolysis were examined in several assays of biological activity. Brucella S- and R-LPSs and Brucella lipid A activated the complement cascade. Previously reported mitogenic activation by Brucella LPSs of spleen cells from endotoxin-resistant C3H/HeJ mice was confirmed and also produced by isolated Brucella lipid A. Mitogenicity was not inhibited by polymyxin B, and amino acid analysis showed no binding of polymyxin B to Brucella LPS under conditions in which mitogenicity of phenol-water-extracted Escherichia coli LPS was inhibited. S and R Brucella LPSs and lipid A all produced equivalent polyclonal stimulation of C3H/HeJ and C3H/HeAU spleen cells. Crude and purified LPS from S but not from R B. abortus was toxic for outbred mice, with 50% lethal doses approximately six times greater than that for E. coli LPS. S- and R-LPSs were abortifacient in pregnant outbred mice. S Brucella LPS was lethal for carrageenen-pretreated C3H/HeJ and C3H/HeAU mice, whereas only C3H/HeAU mice were killed by E. coli LPS. The data are consistent with the hypothesis that the unique fatty acid composition of Brucella lipid A is responsible for its biological activity in endotoxin-resistant C3H/HeJ mice. The participation of the protein strongly bound to the lipid A cannot be excluded, but its mode of action, if any, is different from that of the lipid A-associated protein of enterobacterial LPS.

Immunochemical characterization of Brucella lipopolysaccharides and polysaccharides.

Purified lipopolysaccharide (LPS) extracted with phenol-water from smooth Brucella abortus was hydrolyzed with 1% acetic acid at 100 degrees C. The degraded polysaccharide (AH) released gave reactions of identity with the native polysaccharide hapten (NH) in phenol-water- or trichloroacetic acid-extracted endotoxin preparations of B. abortus and with the polysaccharide (poly B) extracted by trichloroacetic acid from rough B. melitensis strain B115. Poly B was present in the soluble cytoplasmic fraction but not in the membrane fraction, of disrupted B115 cells. It could not be extracted from three rough mutants of B. abortus or from B canis or B. ovis cells. Both AH and NH shared determinants present on smooth LPS and missing from poly B. Sugars found in purified LPS, NH, and AH included mannose, glucose, quinovosamine, glucosamine, and 2-keto-3-deoxyoctonate. Poly B contained only a trace amount of quinovosamine and no 2-keto-3-deoxyoctonate detectable by the thiobarbiturate assay. Sera from some rabbits immunized with pure smooth LPS and some, but not all, cows infected with field strains of B. abortus recognized the determinants missing from poly B. A subclass-specific enzyme-linked immunoassay showed that most of the antibody in sera from infected cows which binds to smooth LPS and to NH is of the immunoglobulin G1 subclass.

Evaluation of a radial immunodiffusion test with polysaccharide B antigen for diagnosis of bovine brucellosis.

A radial immunodiffusion (RID) test employing a polysaccharide antigen (poly B) was compared with tests currently used in the diagnosis of bovine brucellosis. Over 1,000 sera from vaccinated and infected cattle, all of which had been examined bacteriologically, were used to determine the sensitivity and specificity of the RID, card, Rivanol, and complement fixation tests. The RID test identified 90% of the cattle that were shedding Brucella in their milk. Although the complement fixation test was more sensitive, it was less specific than the RID test in cattle vaccinated as adults with Brucella abortus strain 19. A sensitive screening test, such as the card test, in combination with the RID test could be used in diagnostic laboratories, or even in the field, with little additional expense or technical expertise. An additional advantage is that the RID could be applied to sera from adult cattle as early as 2 months after vaccination, when postvaccinal agglutinins and complement-fixing antibodies may still be present. The indirect hemolytic test was used with some of the sera and was found to be a very sensitive test which could be useful in areas of low incidence but would not be practical for large-scale testing in adult-vaccinated herds.

Secretory immune response in intestinal mucosa and salivary gland after experimental infection of pigs with transmissible gastroenteritis virus.

Pigs 8 to 10 weeks of age were orally infected with transmissible gastroenteritis (TGE) virus or infected by inoculation of the virus into Thirty-Vella loops of jejunum. Concentrations of immunoglobulin (Ig) A, IgM, and IgG in serum, saliva, jejunal secretions, loop secretions, and bile were determined by solid-phase radioimmunoassay for TGE virus-infected and control pigs. A multiple-staining fluorescent antibody technique was used to determine the relative numbers of IgA-, IgM-, and IgG-producing plasma cells in intestinal mucosa, mesenteric lymph node, spleen, iliac lymph node, and submandibular salivary gland. The numbers of IgA- and IgM-producing plasma cells were greater in the jejunal mucosa of pigs infected and reinfected orally with TGE virus than in that of the control pigs. There was also an increase of IgA- and probably of IgM-cells in the submandibular salivary glands. Similar numerical increases of IgA- and IgM-cells were observed in jejunal mucosa and salivary glands of all pigs with intestinal loops whether exposed to TGE virus or not. Increases in plasma cells in mucosa or salivary gland were not associated with increases in concentrations of IgA or IgM in the respective secretions or serum. The data support the hypothesis that after stimulation, IgA- and IgM-producing cells leave the intestinal mucosa and are trapped by distant secretory epithelial. The absence of a simultaneous increased concentration of IgA and IgM in saliva and intestinal secretions indicates that in an intact epithelium, the transport of IgA and IgM mediated by secretory component is probably saturable.

Characterization of Brucella abortus soluble antigen employed in immunoassay.

A soluble antigen extract of Brucella abortus (BASA) has been prepared by the National Veterinary Services Laboratories and furnished to a number of workers who are examining antibody-mediated and cell-mediated immune responses of cattle infected with B. abortus. Three lots of BASA were examined. There were quantitative but not qualitative differences among lots by content of protein, total carbohydrate, hexose, fatty acid, and 2-keto-3-deoxyoctonic acid. The presence of smooth lipopolysaccharide was demonstrated by the presence of 2-keto-3-deoxyoctonic acid and lipid, by Limulus lysate gelation activity, and by formation of characteristic lipopolysaccharide precipitates in immunoelectrophoresis. A polysaccharide antigen as well as two nonsurface antigens, A2 and C, were also identified. BASA is a satisfactory antigen for use in the enzyme-linked immunosorbent assay since the smooth lipopolysaccharide component bound to polystyrene and functioned in the test. Normal murine spleen cells showed a mitogenic response to BASA similar to that produced by purified smooth lipopolysaccharide. BASA has been used in other laboratories to stimulate peripheral blood leukocytes from cattle infected with B. abortus. Because BASA is a mixture of antigenic components shown to have mitogenic effects in the mouse system, questions on the nature of its stimulatory effect on bovine cells are raised.

Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice.

Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.

Enzyme-linked immunosorbent assay for bovine immunoglobulin subclass-specific response to Brucella abortus lipopolysaccharides.

An enzyme-linked immunosorbent assay was developed to follow the bovine response, by immunoglobulin class and subclass, to defined smooth and rough lipopolysaccharides (LPS) of Brucella abortus. Binding to smooth LPS of immunoglobulin G1 (IgG1) and IgG2 in sera from Brucella-infected animals was significantly greater than binding in sera from normal uninfected animals. Competition or steric blocking among IgM, IgG1, and IgG2 for binding sites on smooth LPS was shown to occur. Binding of IgM to Brucella smooth LPS with sera from uninfected animals was elevated above the assay control levels, and attempts to eliminate this nonspecific IgM binding were not successful. The same levels of nonspecific IgM binding were also seen with Brucella rough LPS, Escherichia coli LPS, and Pseudomonas solanacearum LPS. Sera from some, but not all, Brucella-infected animals showed elevated binding of IgG1 and IgM to both E. coli LPS and Brucella rough LPS as well as to Brucella smooth LPS. This was interpreted as specific antibody. Cross-reactions between B. abortus smooth or rough LPS and E. coli LPS could not be shown by immunodiffusion.

Purification and characterization of smooth and rough lipopolysaccharides from Brucella abortus.

In an attempt to obtain pure and well characterized smooth lipopolysaccharide (S-LPS) and rough lipopolysaccharide (R-LPS), smooth and rough strains of Brucella abortus were extracted by two different modifications of the phenol-water method. S-LPS was obtained in the phenol phase, and R-LPS was obtained in the aqueous phase. Further purification was accomplished by treatment with enzymes, detergents, NaI as a chaotropic agent to separate non-covalently bound contaminants, and by gel filtration. The degree of purity of the molecules was determined by chemical and immunological analysis and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Lipid identification by gas-liquid chromatography showed seven major fatty acids. Palmitic acid accounts for about 50%, stearic acid accounts for about 10%, and hydroxylated fatty acids account for less than 5% of total fatty acids. 2-Keto-3-deoxyoctonate but not heptose was detected in the sugar analysis. Protein was found to be firmly bound to S-LPS but not to R-LPS.

Antibody response to antigens distinct from smooth lipopolysaccharide complex in Brucella infection.

The smooth lipopolysaccharide complex of the outer surface of smooth Brucella abortus cells is believed to be the antigenic component involved in serological tests routinely used for the diagnosis of brucellosis. Sera from cattle vaccinated or infected with B. abortus generally contain antibody directed toward the smooth lipopolysaccharide complex. The brucella organism contains a large number of other antigenically distinct components. The biological significance of some of these antigens has been demonstrated by showing that sera from infected cattle have precipitins to these components. These sera revealed up to seven distinct lines in immunoelectrophoresis with a protein-rich antigen mixture prepared from rough strain B. abortus 45/20, whereas sera from strain 19-vaccinated cattle did not reveal these lines at 4 or more months after vaccination. Monospecific antisera were prepared against six antigens in this mixture, and the purification of two of them by antibody affinity chromatography is described.