Erythrocyte diagnostic kits for detection of Pseudomonas aeruginosa exotoxin A

The use of formulated chick red blood cells loaded with IgG preparations and affinity-purified antibodies, in comparison with initial immune serum to P. aeruginosa exotoxin A (ETA), has been shown to increase the sensitivity of antibody erythrocyte diagnosticum (AbED) 17-fold and to ensure the detection of ETA at a concentration of 1.2 mg of protein per ml. The passive hemagglutination (PHA) test with AbED has proved to be a more sensitive method for the detection of ETA than the antibody neutralization test with the use of antigenic erythrocyte diagnosticum, the latex agglutination test, the coagglutination test and the enzyme immunoassay. The PHA test has permitted the detection of ETA in the culture fluid of 80% of P. aeruginosa cultures under study.     

Use of a polyvalent erythrocyte diagnostic agent for determining antibodies to Pseudomonas aeruginosa in clinical practice

The use of polyvalent erythrocyte diagnosticum prepared on the basis of 5 polysaccharide antigens of P. aeruginosa slime, isolated from strains belonging to the most widespread serovars, makes it possible to check up the humoral response of donors after their immunization with P. aeruginosa polyvalent corpuscular vaccine with the aim of obtaining anti-P. aeruginosa donor plasma. Antibody titers, determined in the passive hemagglutination test with the use of the proposed diagnosticum and corresponding to a serum dilution of 1:320 and greater, age tentatively diagnostic, which may be indicative of P. aeruginosa in the development of purulent septic complications in patients. The use of the passive hemagglutination test with the newly developed polyvalent erythrocyte diagnosticum makes it possible to check up the specific response of patients having P. aeruginosa infection in the process of their treatment with anti-P. aeruginosa hyperimmune plasma used as a part of complex therapy.     

Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.     

Detection of pneumococcal antibodies in children with acute pneumonia and pleurisy by an immunoenzyme method

The samples of sera and pleural fluid from sick children have been analyzed by means of EIA techniques. To detect the time course of antibody production, the antigenic preparations of pneumococci (monovalent capsular polysaccharides, polyvalent polysaccharide vaccine and complex pneumococcal antigen) have been used. Antibody response observed in the forms of pneumococcal infection, studied in this investigation, has proved to be highly variable. It is expedient to determine antibodies to polysaccharide antigens not earlier than on days 10-12 from the beginning of the disease. But, besides the positive dynamics of antibodies, their unchanged level is sometimes observed in the patients at the beginning of the disease. As a rule, there is a coincidence between the dynamics of antibody formation in response to polysaccharide antigens and to complex pneumococcal antigen.     

The immunobiological properties of the antigenic preparations obtained from a vaccinal strain of Klebsiella pneumoniae 204 grown in media of differing compositions

In the technology of the cultivation of K. pneumoniae vaccine strain 204 with a view to obtaining biomass for the production of antigenic preparations the traditionally used culture medium with full nutritional value has been replaced by the alternative variant of synthetic medium. The specific physiological and morphological features of this strain grown in synthetic culture medium have been studied and described. Irrespective of the composition of the culture media used for cultivation, the antigenic preparations have been shown to have no difference in their chemical composition, immunogenic and toxic properties.     

Immunochemical study of the lipopolysaccharides of Yersinia enterocolitica serovars O5 and O5.27

Y. enterocolitica lipopolysaccharides (LPS), serovars O5 and O5.27, have similar antigenic specificity. The LPS of serovar O5.27 has been shown to contain no factor O27. Residual alpha-L-rhamnose, glycosylated by D-xylulose, has been found to play an important role in the formation of factor O5, common for both serovars.     

Trial of an erythrocyte antigenic diagnostic agent for detecting antibodies to Coxiella burnetii

A new method for the preparation of Q-fever erythrocyte antigenic diagnosticum, adaptable to large-scale production, has been developed, and the diagnosticum thus obtained has been found to be highly specific and sensitive. The combined use of the complement fixation and passive hemagglutination tests enhances the effectiveness of the serological study, as it not only ensures a more complete detection of antibodies to C. burnetii, phase I, in humans and cattle, but also gives more precise indications on the time of infection.     

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.     

Continuous synchronously dividing cultures of the B meningococcus serogroup

Cultures of Neisseria meningitidis Bc5S No. 125 with continuous and synchronous cell fission have been obtained. The synchronization index is 0.59-0.63. The synthesis of protein at different periods of the synchronized cycle occurs at constant speed. The synthesis of RNA and DNA is periodical, the maximum accumulation of DNA occurring at the period prior to cell fission. The amount of polysaccharide in the whole culture and the cells is considerably greater than in the supernatant fluid. At the period of fission the content of polysaccharide per 1 billion cells decreases. The indices characterizing the increase of biomass, the synthesis of macromolecular compositions and polysaccharide, the titers of the reaction of agglutination remain unchanged during the whole period of cultivation, which is indicative of the stability of the continuous and synchronous process. The use of this process holds considerable promise for obtaining N. meningitidis synchronized cultures in large amounts, for the study of the biological properties of meningococci and for obtaining immunoprophylactic preparations.     

Choleragen-anatoxin chemical cholera vaccine enriched with Ogawa O-antigen

The cultural fluid of Vibrio cholerae strains of serovar Ogawa, grown under the conditions of submerged cultivation, has been shown to contain a large amount of soluble O-antigen which sharply differs from all other concomitant components in its molecular weight. By enriching the commercial chemical cholera vaccine known as Choleragen Toxoid with purified Ogawa O-antigen a new preparation, consisting mainly of cholera toxoid and Ogawa and Inaba O-antigens and capable of producing pronounced immunity to V. cholerae of both serovars, has been obtained.     

Development of antilipase immunoglobulin polymer diagnosticum for the detection of Vibrio cholerae eltor, possessing hemolytic and lipase activity

The possibility of using a heterogeneous, but structurally similar antigen--the commercial preparation of Pseudomonas sp. lipase (Sigma, USA)--for the development of polymer diagnosticum aimed at determination of lipase production in cholera vibrios was shown. The new diagnosticum (antilipase antibodies) on a polymer carrier was used in the serological volume agglomeration test for the detection of hemolytic atoxigenic V. eltor, obtained from environmental, objects, which produced lipase in 80% of cases. The differentiating capacity of the diagnosticum was confirmed on 120 V. eltor cultures isolated from environmental objects. The newly developed diagnosticum makes it possible to determine the lipase activity in cholera vibrios of different biovars and serovars.     

Purification, characterization and serological properties of a glycolipid antigen reactive with a serovar-specific monoclonal antibody against Leptospira interrogans serovar canicola

A glycolipid antigen possessing a serovar-specific antigenic determinant of Leptospira interrogans serovar canicola was purified from a chloroform/methanol extract of the organism. The purification procedures included silicic acid column chromatography and preparative thin-layer chromatography (TLC). Antigenic activity was detected by a TLC-enzyme immunostaining technique using monoclonal antibody CT3, which specifically agglutinates serovar canicola and only weakly serovar sumneri but no other serovars of Leptospira. The purified glycolipid reacted with CT3 antibody, indicating that the glycolipid possessed a serovar-specific antigenic determinant. Infrared spectrum and proton nuclear magnetic resonance analyses showed that the glycolipid contained sugar and lipid moieties, which possessed amide linkages and an acetyl group. Gas-liquid chromatography-mass spectrometry analysis showed that the glycolipid contained two unknown sugars, one of which (unknown sugar II) appeared to be associated with the antigenic determinant specific for canicola. The serovar-specific antigenic determinant was destroyed by mild alkali treatment of the glycolipid. These findings suggested that the antigenic determinant was an alkali-labile moiety which may be related to the unknown sugar II.     

Immunologic properties of purified and complex preparations of group-B meningococcal polysaccharide

The complex preparations of group B meningococcal polysaccharide have been found to be capable of inducing primary immune response in mice, while purified group B polysaccharide has proved to be immunologically inert. As revealed in this investigation, the intravenous injection to mice of the optimum doses of the complex preparation of group B polysaccharide leads to the increased number of specific B-antibody-forming cells in their spleens and to a rise in B-antibody titers in their sera; besides, the time course of the process has been studied. Both preparations have been found capable of forming the immunological memory in mice if booster immunization is made with the complex preparation of group B polysaccharide. The immunological inertness of purified group B polysaccharide is attributed, supposedly, to the action of some specific suppressor mechanism. Considering the pronounced antigenic activity of the complex preparation of group B polysaccharide and the insignificant admixture of endotoxin in this preparation, the suitability of its future use as vaccine for the prophylaxis of meningitis caused by group B meningococcus is indicated and the tentative immunization schedules are discussed.     

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.     

The development of a process for culturing Bordetella pertussis immobilized on a polyurethane carrier

The processes of the cultivation of Bordetella pertussis, immobilized on polyurethane carrier in a fermenter, were carried out and studied. Acellular pertussis preparations were produced from the culture fluid obtained in the batch and multi-cycle cultivation processes with immobilized cells, as well as in the process with interrupted fermentation (for confirming the possibility of the preservation of cell viability). The content of protein and B. pertussis toxin in these preparations, as well as their leukocytes-stimulating and hemagglutinating activity, did not differ from similar characteristics of preparations obtained from culture fluid in homogeneous cultivation.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.     

Characterization of Salmonella enterica subspecies I genovars by use of microarrays

Subspecies 1 of Salmonella enterica is responsible for almost all Salmonella infections of warm-blooded animals. Within subspecies 1 there are over 2,300 known serovars that differ in their prevalence and the diseases that they cause in different hosts. Only a few of these serovars are responsible for most Salmonella infections in humans and domestic animals. The gene contents of 79 strains from the most prevalent serovars were profiled by microarray analysis. Strains within the same serovar often differed by the presence and absence of hundreds of genes. Gene contents sometimes differed more within a serovar than between serovars. Groups of strains that share a distinct profile of gene content can be referred to as "genovars" to distinguish them from serovars. Several misassignments within the Salmonella reference B collection were detected by genovar typing and were subsequently confirmed serologically. Just as serology has proved useful for understanding the host range and pathogenic manifestations of Salmonella, genovars are likely to further define previously unrecognized specific features of Salmonella infections.     

High-molecular antigenic complex of the pneumococcus: its properties

The study of an antigenic preparation isolated from pneumococci, serotype 3, by the method of alkaline hydrolysis has revealed that the preparation contains a high-molecular complex protein-polysaccharide composition of the cell wall of this microorganism. The preparation contains at least two immunologically active components: the type-specific polysaccharide of serotype 3 pneumococcus and a nonspecific protein component. This antigenic preparation is capable of the formation of immunological memory in mice and protecting them from experimental challenge with pneumococci of homologous and heterologous serotypes.     

Monoclonal antibodies to antigens of cholera vibrios of the Ogava serovar

A set of hybrids is obtained synthesizing monoclonal antibodies to the surface antigenic determinants of the choleric vibrio of the Ogava serovar. The antigenic structure of vibrios of the typical and atypical strains isolated from a man and from environmental objects is studied using a collection of highly specific immunoglobulins. The high-specific diagnostic preparations for identification of the 01-group cholera agent serovar may be created on their base.     

Restriction endonuclease DNA analysis of antigenic variants of leptospires selected by monoclonal antibodies

The genome of antigenic variant CV (CT3)-1 derived from Leptospira interrogans serovar canicola was compared by cleavage with restriction endonucleases with the parent and serovar bafani, to which the variant was serologically most closely related. No differences were observed between the parent and variant in DNA restriction endonuclease patterns using eight restriction endonucleases. Serovar bafani was different in the patterns from the parent and antigenic variant CV (CT3)-1. The two antigenic variants derived from serovar hebdomadis, HV (H16)-1 and HV (H19)-1 which belonged serologically to serovars jules and hebdomadis, respectively, were compared by restriction endonuclease DNA analysis with the parent and serovar jules. No differences were observed between the parent and variants in DNA restriction endonuclease patterns using the same enzymes. But some differences were observed in DNA restriction endonuclease patterns between HV (H16)-1 and serovar jules. Thus, the antigenic variant selected from the parent by the anti-parent monoclonal antibody and serologically different from the parent, being identified either as a new serovar or as a known one, was found to be similar to the parent by the restriction endonuclease DNA analysis.