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PubMed1976

Adhesive properties of Vibrio cholerae: adhesion to isolated rabbit brush border membranes and hemagglutinating activity.

Adhesion of vibrios to the small intestine may occur (i) by association of the bacteria with secreted mucus gel or (ii) by adherence of the bacteria to the surface of epithelial cells. In the present study, vibrios readily adhered to isolated brush border membranes obtained from rabbit intestinal epithelial cells. Adhesion was temperature dependent and required the presence of divalent cations such as calcium. The agglutination of human O erythrocytes by Vibrio cholerae was observed also, and the hemagglutination test appeared to detect the same mechanism that was involved in the adhesion of vibrios to brush borders. When the bacteria were grown in broth they were adhesive and hemagglutinating, but vibrios grown on agar plates or suspended in buffer for 15 min at 37 C lacked these abilities, even though they retained undiminished motility. These two model systems differed, however, in that strontium promoted only adhesion to brush borders. The significance of this difference remains to be determined. Vibrios were observed to penetrate intestinal mucus gel and occasionally to become entrapped in it. However, there was no evidence that vibrios attached to mucus gel.

ABO Blood-Group System↗
PubMed1977

Lipopolysaccharide serotyping of Neisseria meningitidis by hemagglutination inhibition.

The method of hemagglutination inhibition was used to investigate the antigenic diversity of lipopolysaccharide (LPS) from Neisseria meningitidis and to develop a serotyping systems based on this antigen. The system uses outer membrane complex prepared by a simple extraction procedure to inhibit homologous hemagglutination reactions involving sheep erythrocytes sensitized with purified LPS and rabbit antiserum raised to whole meningococci. Antisera with specificity for eight different LPS determinants were used as typing sera to serotype a cross section of 67 meningococcal strains. Only two strains (both group A) were not typable with the eight sera, and most strains had more than one type. Comparison of LPS type and bactericidal serotype suggests that the LPS and protein serotypes are independent serological markers.

Antibodies, Bacterial↗
PubMed1978

Quantitative measurements of cholera enterotoxin in cultures of toxinogenic wild-type and nontoxinogenic mutant strains of Vibrio cholerae by using a sensitive and specific reversed passive hemagglutination assay for cholera enerotoxin.

A sensitive and specific reversed passive hemagglutination (RPHA) assay for cholera enterotoxin has been developed. Equine anti-choleragenoid antibodies purified by immunoadsorption were covalently coupled to formalinized sheep erythrocytes, using bis-diazotized benzidine, and the antitoxin-sensitized erythrocytes were shown to agglutinate specifically in the presence of cholera enterotoxin. In a microtiter RPHA assay system, the smallest quantity of enterotoxin that caused hemagglutination was approximately 20 pg. A sensitive assay for antibodies to enterotoxin was also developed, based on inhibition of RPHA. Using such assays, we demonstrated that several nontoxinogenic (tox-) strains of Vibrio cholerae produced small but detectable yields of enterotoxin, 4 to 16 ng/ml, under conditions where the highly toxinogenic strain 569B Inaba produced approximately 16 microgram of enterotoxin per ml. The enterotoxin produced in small quantities by these tox- strains was found to be identical to the enterotoxin from V. cholerae 569B Inaba iv its immunological and biological activities. Strains of V. cholerae that produce intermediate yields of enterotoxin have been obtained by two techniques: (i) as less toxinogenic mutants derived from highly toxinogenic strains and (ii) as more toxinogenic mutants derived from tox- strains. Thus, the yield of enterotoxin in cultures of V. cholerae grown under standardized conditions is is a genetically controlled trait that can be altered by mutation.

Antibody Specificity↗
PubMed1979

Strain differentiation of Neisseria gonorrhoeae by reverse passive hemagglutination.

A reverse passive hemagglutination test that utilizes human erythrocytes coated with antibody to gonococci was developed to distinguish differences among 11 strains of Neisseria gonorrhoeae. Different rabbits were immunized with each strain of gonococcus. Antibody was purified by passing antiserum over an immunoadsorbent column containing homologous cell walls trapped in a cross-linked polyacrylamide gel. Antibody, after absorption with N. meningitidis, was used for coating 11 individual suspensions of erythrocytes, each with antibody to one gonococcal strain. The panel of coated erythrocytes was added to microtiter trays containing dilutions of homologous bacterial lysate and lysates from 10 heterologous strains. Agglutination titers were highest with homologous lysates, although cross-reactions occurred among some heterologous lysates. Lysates of nongonococcal Neisseria species and of other genera did not agglutinate coated erythrocytes. The reverse passive hemagglutination test can be a useful procedure to distinguish differences among strains of N. gonorrhoeae.

Antibodies, Bacterial↗
PubMed1979

Comparison of enzyme-linked immunosorbent assay and passive hemagglutination method for quantification of antibodies to lipopolysaccharide and tetanus toxoid in rats.

In a comparative study, the enzyme-linked immunosorbent assay, using peroxidase labeled anti-rat immunoglobulin M and immunoglobulin G, and the passive hemagglutination test were applied to determine the primary and secondary antibody response to lipopolysaccharide and tetanus toxoid in rats. In the enzyme-linked immunosorbent assay, the antigens were bound to the wells of polystyrene microplates, tetanus toxoid directly, and lipopolysaccharide after complexing it with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled anti-immunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with their respective antigens. The enzyme-linked immunosorbent assay proved to be more sensitive than the hemagglutination reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, the enzyme-linked immunosorbent assay is a convenient method for measuring both immunoglobulin M and immunoglobulin G antibodies. At low serum dilutions of lipopolysaccharide antisera, inhibition of the reaction in the enzyme-linked immunosorbent assay occurred. This phenomenon could be prevented by heating the sera at 56 degrees C for 30 min. Lipopolysaccharide was immunogenic in rats over an extremely wide dose range (from 10 pg to 1 mg); the optimal immunogenic dose of lipopolysaccharide for young adult rats was 0.1 to 1,000 mug when administered intravenously, and that of tetanus toxoid was 5 to 10 lines of flocculation, as determined by the Ramon flocculation test.

Animals↗
PubMed1981

Nonimmunoglobulin fraction of human milk inhibits bacterial adhesion (hemagglutination) and enterotoxin binding of Escherichia coli and Vibrio cholerae.

Human milk and colostrum samples were divided into an immunoglobulin and a nonimmunoglobulin fraction by immunosorbent chromatography. The ability of these fractions to inhibit bacterial cell adhesion and enterotoxin receptor binding of Vibrio cholerae and various Escherichia coli isolates was then tested by in vitro assays. The strongest effect was generally seen with the nonimmunoglobulin fractions, which were shown to significantly inhibit E. coli cell adhesion (hemagglutination) mediated by CFA/I, CFA/II, or K88 fimbriae (but not type 1 pili) and V. cholerae hemagglutination, as well as the binding of cholera toxin and E. coli heat-labile enterotoxin to GM1 ganglioside. Also, the immunoglobulin fractions had significant inhibitory activity in some of these systems. The results are interpreted to suggest that human milk and colostrum may contain secreted structure analogs of the cell receptors for some bacterial adhesions and enterotoxins; this might contribute to the protective effect of milk against enteric infections.

Bacterial Physiological Phenomena↗
PubMed1981

Experimental retrograde pyelonephritis and cystitis induced in rabbits by a group D Streptococcus sp.: serum antibody assay by a hemagglutination test.

Experimental models of urinary tract infection caused by a group D Streptococcus sp. in rabbits are described: retrograde pyelonephritis was induced by injecting 10(9) group D streptococci into the renal pelvis and obstructing the ureter by ligature for 24 h; cystitis was induced by injecting 5.10(9) group D streptococci through a transurethral catheter. The animals were observed for 3 months. Microbiological data from the urine and from the renal parenchyma were well correlated in 12 of 14 animals. A hemagglutination test for titration of serum antibodies in these infected rabbits is described. Before the beginning of experimentation, hemagglutination titers for all animals were below 160. During experimentation, titers for rabbits with cystitis were always below 640; in those with pyelonephritis, the highest titer was much greater than 640. These results show a positive correlation between serum antibody levels and the localization of urinary tract infection.

Animals↗
PubMed1981

Serum resistance and hemagglutination ability of marine vibrios pathogenic for fish.

Representative strains of marine vibrios pathogenic for fish were shown to be resistant to the bactericidal activity of normal (nonimmmune) rainbow trout (Salmo gairdneri) serum, and loss of this resistance coincided with a marked reduction in virulence. Thermal lability and a requirement for Mg2+, but not for Ca2+, suggested that a mechanism for serum killing was the alternative complement pathway. In the case of Vibrio anguillarum, serum resistance was not coded for by the virulence plasmid pJM1. Additional testing showed that these pathogenic vibrios were able to agglutinate a variety of eucaryotic cells and that selected strains agglutinated trout erythrocytes; however, a correlation between strain virulence and the ability to agglutinate fish erythrocytes was not apparent. Moreover, whereas mannose was found to inhibit the agglutinating activity of several strains, two of the high-virulence strains displayed a transient, mannose-resistant hemagglutinating activity. No relationship between the carriage of pJM1 by the V. anguillarum strains and hemagglutinating activity was demonstrable.

Animals↗
PubMed1982

Hemagglutination activity and colonization factor antigens I and II in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli isolated from humans.

We examined 205 enterotoxigenic strains of Escherichia coli for colonization factor antigens (CFA) I and II, using an immunodiffusion technique with specific antisera. A total of 36 strains of serogroups O63, O78, O114, O128, and O153 and 1 rough strain possessed CFA/I and gave a single precipitin line; 47 strains of serogroups O6, O8, O80, and O115 possessed CFA/II. The latter strains gave a major precipitin line (component 3) when tested with specific antisera prepared against strain E1392 or PB-176 (both E. coli O6.H16; biotype A). However, all 16 strains of E. coli O6.H16 belonging to biotype A gave a second precipitin line (component 1) when tested with both antisera. When CFA/II-positive strains were tested with a specific antiserum prepared against E. coli O6.H16 strains of biotype B or C, all strains gave component 3, but 16 of 17 strains of E. coli O6.H16 belonging to biotype B, C, or F gave a second precipitin line (component 2) not given by strains of biotype A. CFA/II-positive strains of serogroups other than O6 gave only component 3 in tests with all specific antisera. Nine enterotoxigenic strains of serotypes O7, O15, O25, O115, and O128 gave mannose-resistant hemagglutination of human or calf erythrocytes but lacked CFA/I or CFA/II. Although mannose-resistant hemagglutination was common in non-enterotoxigenic strains of E. coli, none of the non-enterotoxigenic strains possessed CFA/I or CFA/II; these strains included fecal strains of serogroups O6, O8, O63, and O78, fecal strains of enteropathogenic serogroups, and strains from extraintestinal sources.

Animals↗
PubMed1972

Indirect hemagglutination test for varicella-zoster infection.

An indirect hemagglutination test has been adapted for use with varicella-zoster serology. Antigen was made in infected WI-38 human diploid cell line. The indirect hemagglutination antigen seemed to be a subunit of the virus particle. The antibodies measured in the test are well correlated with neutralizing and complement-fixing antibodies in cases of zoster but not in cases of varicella.

Antibodies, Viral↗
PubMed1989

Type 3 fimbriae among enterobacteria and the ability of spermidine to inhibit MR/K hemagglutination.

The distribution of the gene cluster encoding type 3 fimbriae among various isolates of the family Enterobacteriaceae was investigated by using 112 clinical and nonclinical isolates. Closely related DNA sequences were detected in all Klebsiella strains, in most Enterobacter isolates, in a smaller number of Escherichia coli and Salmonella spp., and in a single isolate each of Yersinia enterocolitica and Serratia liquefaciens but not in isolates of Morganella or Providencia species or Serratia marcescens. Except for E. coli and Salmonella strains, the presence of gene sequences was correlated with the phenotypic expression of either the MR/K hemagglutinin or fimbriae that reacted with specific antibodies. In one isolate of Y. enterocolitica the expression of type 3 fimbriae was plasmid determined. The polyamine spermidine was identified as an inhibitor of MR/K hemagglutinating activity, exhibiting an MIC of 1.2 mM. Spermidine inhibited the hemagglutination of 37 MR/K-positive clinical isolates from various genera. However, one clinical isolate of Enterobacter cloacae and most (four of five) nonclinical Klebsiella isolates were not completely inhibited.

Antibodies, Monoclonal↗
PubMed1989

Immunochemical identification and preliminary characterization of a nonfimbrial hemagglutinating adhesin of Bacteroides gingivalis.

A cell-bound hemagglutinating adhesin (HA-Ag2) of Bacteroides gingivalis was identified by crossed immunoaffinity electrophoresis as one of the common antigens of the species. A polyclonal antiserum with a restricted specificity for HA-Ag2 was produced by immunizing with the relevant immunoprecipitate excised from crossed-immunoelectrophoresis gels. The immunoglobulin G fraction of this monospecific antiserum inhibited hemagglutination. The antiserum was used against a cell surface extract of B. gingivalis in immunoblotting experiments, and we detected two antigens with apparent molecular masses of 33 and 38 kilodaltons in B. gingivalis ATCC 33277 and W83. Monoclonal antibody, C1.17, produced in another laboratory against B. gingivalis 381 and characterized as showing reactivity with a hemagglutinin of this strain (Y. Naito, K. Okuda, T. Kato, and I. Takazoe, Infect. Immun. 50:231-235, 1985), was also used to produce immunoblots of extracts of strains ATCC 33277 and W83. The apparent molecular masses of the major polypeptides recognized by monoclonal C1.17 in the immunoblots were the same as those detected by the polyclonal monospecific antiserum, i.e., 33 and 38 kilodaltons. Significantly, none of the polypeptides identified in this study corresponded to the polypeptide appearing in the 41- to 43-kilodalton region and identified by Yoshimura and co-workers (F. Yoshimura, K. Takahashi, N. Yoshinobu, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) as the fimbrial protein characteristic of the species. Enzyme-linked immunosorbent assay inhibition experiments with the monospecific antiserum indicated that the cell surface extracts from strains ATCC 33277 and W83 were strong inhibitors, whereas the fimbria-enriched preparations from both strains failed to inhibit binding of antibodies to the cell surface antigens. As a whole, our study indicates that a nonfimbrial surface protein complex demonstrating erythrocyte-binding capacity, HA-Ag2, is common to three strains of B. gingivalis and is composed of at least two associated polypeptides with apparent molecular masses of 33 and 38 kilodaltons which share at least one antigenic determinant.

Antibodies, Bacterial↗
PubMed1999

Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis.

Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis. P. gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity. P. gingivalis grew well at all three temperatures. An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01). A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0. 01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased, P. gingivalis adopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.

Adhesins, Bacterial↗
PubMed1966

Adenovirus antibody measured by the passive hemagglutination test.

Lefkowitz, Stanley S. (Variety Children's Research Foundation, Miami, Fla.), Julia A. Williams, Bernard E. Howard, and M. Michael Sigel. Adenovirus antibody measured by the passive hemagglutination test. J. Bacteriol. 91:205-212. 1966.-Rabbits immunized intravenously with adenovirus type 5 antigen were tested for antibody titers by use of the passive hemagglutination test (PHA). Primary and secondary responses were studied, and the class of antibody was determined by means of density gradient centrifugation and reduction with 2-mercaptoethanol (ME). It was found that the PHA was 10 to 100 times more sensitive than complement-fixation and neutralization tests for the detection of antibodies to adenovirus. The immunological response to primary immunization was dependent on the dose of antigen, with antibody appearing in as early as 3 days. After secondary stimulation with the same antigen, there was a rapid response which appeared to be less dose-dependent. It was found that a heavy 19S antibody sensitive to ME was produced early and was followed by a lighter, presumably 7S, ME-resistant antibody. Upon secondary stimulation, both 7S and 19S antibody increased to levels greater than those of the primary injection.

Adenoviridae↗
PubMed1968

[Detection by hemagglutination of antibodies to group A and group E streptococci by the use of O-stearoyl derivatives of their cell wall carbohydrate-grouping antigens].

The streptococcal group A and group E cell wall polysaccharide antigens were extracted with trichloroacetic acid from the cell or cell wall and esterified with stearic acid. The stearoyl derivatives contained 5 to 8% (by weight) of the ester. Sheep or human red blood cells were sensitized with the esterified antigens and were shown to agglutinate in the presence of specific rabbit antisera. Sera from (i) children hospitalized with group A streptococcal respiratory disease and (ii) swine possessing group E streptococcal lymphadenitis were shown to possess antibody titers significantly higher than the controls. The use of the two esterified antigens as controls for each other established the specificity of the reaction in each case. The general shape of the antigen-antibody precipitin curves was not changed when the stearoyl antigens were used; however, the quantitative aspects differed markedly. Oligosaccharides which inhibit the normal antigen-antibody precipitin reaction did not inhibit the hemagglutination reaction. The adsorption of antisera with whole streptococcal cells reduced the hemagglutination titer in relation to the quantity of cells employed. Data are given on the (i) optimal concentration of stearoyl antigen for sensitization, (ii) time of adsorption of antigen to red cells, (iii) use of albumin as diluting fluid, and (iv) condition of red cells. Properties of the esterified antigens and the mechanism of the agglutination reaction are discussed. The results indicate that polysaccharide antigens of other bacteria may be esterified and employed in a similar manner.

Animals↗
PubMed1975

Evaluation of turkey erythrocyte hemagglutination assay for the detection of hepatitis B antigen.

A recently described hemagglutination test for hepatitis B antigen (HBsAg) using turkey erythrocytes coated with horse antibody to HBsAg purified by affinity column chromatography was evaluated on a comparative basis with 100 HBsAg-positive and -negative serum samples. The turkey erythrocyte hemagglutination test (TEHA) was found to be less sensitive than radioimmunoassay (RIA) but gave far better results than counterimmunoelectrophoresis. Quantitative titration of HBsAg in serial dilutions of the samples appeared to be more reliably performed by TEHA than by RIA. TEHA is a simple and sensitive technique for the detection of HBsAg and may offer several practical advantages over RIA.

Animals↗
PubMed1979

Direct hemagglutination technique for differentiating Bacteroides asaccharolyticus oral strains from nonoral strains.

A simple and economical method for differentiating Bacteroides asaccharolyticus of oral sources from nonoral sources is described. The present data indicate that oral strains of B. asaccharolyticus strongly agglutinate sheep erythrocytes, whereas isolates from various nonoral sites typically are devoid of hemagglutination activity. The direct hemagglutination test may aid in determining the source of B. asaccharolyticus present in an infection, and thus the procedure has potential value as a means of biotyping.

Animals↗
PubMed1979

Neutralization test in influenza: use in individuals without hemagglutination inhibition antibody.

In influenza immunization trials, patients who were seronegative by the hemagglutination inhibition test could be divided into two groups according to preimmunization levels of neutralizing (Neut) and neuraminidase inhibition (NI) antibodies. The group with low levels of Neut and NI antibodies usually developed adequate levels of hemagglutination inhibition antibody after one dose of vaccine, and the group with undetectable Neut and NI antibodies did not.

Antibodies, Viral↗