Detection of Brucella antigens by dot immunoassay using specific antibodies labelled with colloid gold particles]

Dot immunoassay was developed to improve the quality of laboratory diagnosis of brucellosis. Particles of colloid gold were used as a marker of specific antibodies. The method was used for detecting Brucella antigens in artificially contaminated environmental objects (soil and water) and in biological material (milk, blood serum, and visceral homogenates of animals). The sensitivity of the test system was 19.5.10(3)-62.5.10(4) CFU/ml. Specificity of the assay was tested with 10 heterologous antigenically closely related bacterial species. The proposed test system is simple, economic, highly sensitive and specific, and requires no expensive equipment and reagents.     

Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis

Hybridomas producing monoclonal antibodies (MAb) to Yersinia pseudotuberculosis, serovars I-IV, responsible for serovar appurtenance, were obtained. Virtually all MAbs reacted with protein antigens in immunoblotting. The only exclusion was MAb 3A2 presumably reacting with a glycoprotein epitope of complex structure. Variability of Y. pseudotuberculosis antigenic structure, depending on culturing temperature, was confirmed. Polypeptides with mono- or polydetermined antigenic specificity were determined using MAbs.     

Study of Yersinia pseudotuberculosis surface antigen epitopes using monoclonal antibodies

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O-side chains. The concentration of these epitopes increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.     

Detection of brucella antigens with colloid metal particles as markers for specific antigens

Gold and silver sols were comparatively approved as markers of specific IgG isolated from hyperimmune Brucella antisera for the detection of brucellar antigens. The sensitivity of the test system using gold immunosol proved to be some higher (3.1-9.8 ng/ml of soluble and 2.0 x 10(4)-5.3 x 10(6) CFU/ml of corpuscular brucellar antigens) than that achieved with the use of silver immunosol (5.7-18.4 ng/ml of soluble and 6.1 x 10(4)-8.0 x 10(6) CFU/ml of corpuscular brucellar antigens). At the same time silver sol was a cheaper and more available marker. Both test systems were found to be highly specific. False positive results were observed only with Yersinia enterocolitica O:9 at high concentrations due to the fact that they had common polysaccharide haptens incorporated into lipopolysaccharides of these microorganisms. The proposed test systems with colloid metal particles used as markers of specific antibodies for the detection of Brucella antigens are technologically simple, economic, rapid, highly sensitive and specific. Their use in combination with other serological methods will make the results of analyses more informative, thus improving the quality of laboratory diagnostics of brucellosis.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia Yop proteins by Western-blot

The results obtained with the use of the western-blotting showed that antibodies for released proteins YopD (33-36 kDa) were the most frequently detected antibodies in serum samples from patients suspected for yersiniosis. Reactions between serum samples studied and the YopD protein were very intense, suggesting that protein is the strongest immunogen among the utilised, released proteins Yop of Yersinia. Antibodies IgM were more often diagnosed in patients with abdominal pain in the contrary to antibodies IgA which were characteristic to patients with reactive arthritis. Detailed analysis of the results of western-blotting on serum samples obtained several times from individuals with yersinosis during the course of infection in this investigation have showed also that antibodies of the IgA class hold longer in serum of individuals with arthritis compared with individuals with yersinosis not complicated by arthritis. In joint-fluid samples obtained from patients with arthritis antibodies for particular released proteins Yop were detected in the same class of immunoglobulins like in serum samples obtained from those individuals.     

Heterogenetic antigens of Yersinia pseudotuberculosis in human erythrocytes and organs

The study of the antigenic relationship between Y. pseudotuberculosis in red blood cells and different highly vulnerable tissues of the human body (in the liver, the spleen, the kidneys, the lymph nodes, the intestine) has been carried out; as a result, the presence of heterogeneous substances has been shown. Y. pseudotuberculosis heterogeneous antigens play an important role in the formation of severe forms of the disease and determine the frequency of relapses; they also determine the specific action of diagnostic and therapeutic preparations.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia lipopolisacharydes and Yop proteins by ELISA

The antibodies against the somatic antigens of Y. enterocolitica O3, O8, O9, O5,27,Y. pseudotuberculosis I, and released proteins Yop were detected using the ELISA in 1634 serum samples and 84 synovial fluids collected from 1290 persons suspected for yersiniosis, as well as 200 serum samples from healthy individuals (blood donors). The presence of antibody in diagnostically significant titres for somatic antigens of Yersinia were detected by ELISA in 20.5% and 50.6%, antibodies for released proteins Yop in 11.5% and 28.4% respectively of blood donors and patients suspected for yersiniosis. The antibody against the O3 antigen of Y. enterocolitica was the most frequently detected antibody while the most infrequent was the antibody for the antigen from the 08 serologic group. The results of the study showed that the humoral response picture to Yersinia antigens in the course of yersiniosis in humans is dependent on the age and sex of the patient, duration of the infection, and clinical manifestations. Most frequently the elevated antibody levels were detected among patients with erythema nodosum and patients with gastrointestinal symptoms. The frequency of occurrence of antibodies for most antigens of Yersinia, together with age increased reaching its peak, on the average, among individuals aged 21 - 40 years. Analysis of individual cases showed that by the end of the first week of infection, elevated levels of antibodies for somatic antigens of Yersinia are evident. On the other hand, antibodies for released proteins Yop as a matter of rule appear in the second week from the onset of clinical symptoms. Within this early phase of infection immunoglobulins of the A and M classes dominate reaching their highest level in the second to third week of the infection. In majority of the individuals studied antibodies of the IgG class reached their highest level much later in relation to those of the IgA and IgM classes. Significant differences were found in IgA antibody detection among individuals with clinical manifestations of stomachaches and arthritis. Nevertheless, among individuals with clinical symptoms of stomachaches, these immunoglobulins as a matter of principle disappear with a period of 2-3 months from the onset of clinical symptoms. In individuals with arthritis however the aforementioned immunoglobulins maintained at considerable levels even after a year. In joint-fluid samples obtained from patients with arthritis antibodies for Yersinia antigens were detected in similar levels just as obtained simultaneously serum from those individuals.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. IV. Specificity of antigens

The specificity of the lipopolisacharydes and released proteins (Yop) of Yersinia was tested using the sera of rabbits immunised with pathogenic and non-pathogenic strain of Y. enterocolitica and Y. pseudotuberculosis as well as selected sera of patients. The results of this study showed a cross-reactions between the different serotypes of Y. enterocolitica with the strongest reactions between the pathogenic serotypes O:3 and O:9 and pathogenic serotype O:5,27 and non-pathogenic serotype O:5. Sera positive for B. burgdorferi and from patients with Graves' disease showed a slight cross-reactivity with Yop proteins of Yersinia. However, the higher cross-reactivity was observed between the LPS of Yersinia and Salmonella spp. Due to the evidence of cross-reactivity the results of serological investigations should be interpreted with caution.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to enterobacterial common antigen (ECA)

The antibodies against the Enterobacterial Common Antigen (ECA) were detected using the ELISA in 293 serum samples collected from 185 persons suspected for yersiniosis, as well as 115 serum samples from healthy individuals (blood donors). The presence of IgA antibody in diagnostically significant titres for ECA were detected by ELISA in 3.5%, IgG in 13.0%, and IgM in 5.2% of blood donors. Statistical analysis showed that the frequency of detecting antibodies for ECA among the patients with yersiniosis was significantly higher (p < 0.05) in relation to the blood donors. Most frequently the elevated antibody levels were detected among patients with reactive arthritis (IgA 29.2%, IgG 35.4%, IgM 16.7%) while the most infrequent among patients with abdominal pain in acute phase of yersiniosis (IgA 14.9%, IgG 25.3%, IgM 19.5%). The level of antibodies for ECA, together with age increased reaching its peak, on the average, among individuals aged 41 - 60 years. In majority of the individuals studied antibodies of the IgG class reached the level much higher in relation to those of the IgA and IgM classes. The obtained results showed that the detection of antibodies to ECA may be useful in serodiagnosis of Yersinia infections.     

Molecular genetic monitoring of Yersinia pseudotuberculosis using PCR O-genotyping

Irkutsk PCR O-genotyping of 117 Yersinia pseudotuberculosis serotype O:1-O:4 strains isolated in Siberia and Far East was performed. These methods allowed both O-genotype and its variants (a, b, c) to be detected. It was demonstrated that three Y. pseudotuberculosis O-genotypes were circulated in Siberia (O:1a, O:1b and 0:3) and six genotypes (O:1a, O:1b, O:1c, O:2a, O:3 and O:4b) were circulated at Far East. Genotype O:1b dominates at both regions (87.8%). PCR-algorithm for identification of Y. pseudotuberculosis O-genotypes having epidemic significance was developed. The identification of the etiological agent O-genotype without bacteriological isolation of the stimulus is possible by PCR analysis of the clinical material.     

Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis

Aims:  Gas chromatography (GC) was utilized to investigate the cellular fatty acids (CFAs) composition of 141 Yersinia pestis isolates from different plague foci of China, and 20 Yersinia pseudotuberculosis strains as well.
Methods and Results:  The whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS). Y. pestis and Y. pseudotuberculosis strains are quite similar in major CFA profiles, which include 16:0, 17:0 cyclo, 3-OH-14:0, 16:1ω7c and 18:1ω7c, accounting for more than 80% of the total CFAs.
Conclusions:  Yersinia pestis could be easily differentiated from Y. pseudotuberculosis by plotting the ratios of some CFA pairs, i.e.,14:0/18:0 vs 18:1ω7c/18:0, 3-OH-14:0/18:0 vs 18:1ω7c/18:0, 16:1ω7c/18:0 vs 18:1ω7c/18:0, 12:0/18:0 vs 18:1ω7c/18:0 and 12:0 ALDE/18:0 vs 16:1ω7c/18:0 fatty acids.
Significance and Impact of the Study:  In the present study, the normalized Sherlock MIS and Sherlock standard libraries were used to analyse the fatty acid composition of different strains of Y. pestis and Y. pseudotuberculosis . Meanwhile, ratios of certain CFA components were found to serve as chemical markers for differentiating the two closely related bacteria that are difficult to be differentiated by simply comparing CFA profiles based on other researches.     

Characterization of Yersinia pseudotuberculosis heat stable serovar specific polypeptides with the use of monoclonal antibodies

The capacity of Y. pseudotuberculosis to express serovar specific polypeptides with different specificity of antigenic determinants was proved with the use of monoclonal antibodies (McAb). For the first time Y. pseudotuberculosis O antigens were found to have heat stable protein components carrying linear epitopes complementary to serovar specific MaAb and ensuring the serological specificity of the infective agent. The possibility of improving intraspecific classification of Y. pseudotuberculosis and their differentiation from other pathogenic Yersinia on the basis of the capacity of these bacteria for synthesizing species and serovar specific proteins is substantiated.     

Characterization of an amorphous and soluble hemagglutinin from Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis which were screened out depending on auto-agglutination and Ca2+ dependency, were examined for their production of hemagglutinin (HA), and its purification and characterization were performed. The HA with a broad reactivity with various mammalian erythrocytes was recovered from the culture supernatant of these strains grown at 37 C but not 25 C. HAs from two strains, R148R and T1040, were purified by salt precipitation, gel filtration and anion-exchange chromatography by HPLC. Both purified HAs were cysteine-deficient acidic protein with an apparent molecular weight in the range of 15,000 to 16,000. N-terminal amino acid sequences of the first 25 residues were found to share 12% identity with that of afimbrial adhesin from enterotoxigenic Escherichia coli 2230. Immunoelectron microscopy and immunodiffusion test with polyclonal antiserum raised against the purified R148RHA demonstrated that the HA was associated with the amorphous aggregates which were detached from bacteria. These results suggest that the HA of Y. pseudotuberculosis belongs to a third type of HA produced by the yersinial species.     

Pathogenetic significance of the psychrophilia of Yersinia pseudotuberculosis

The work presents the data indicating that the temperature of Y. pseudotuberculosis cultivation is very important in regulating the activity of pathogenicity factors, necessary for the initiation of the pathogenic process in the cells of the macroorganism. Low temperature (8-10 degrees C), necessary for the growth of Y. pseudotuberculosis, facilitates the activation of invasive and toxic pathogenicity factors. At a growth temperature of 37 degrees C the inhibition of such necessary attributes of virulence as adhesion and invasion into epithelial cells occurs in Y. pseudotuberculosis, which decreases the capacity of these bacteria for inducing the infectious process. The virulence of Y. pseudotuberculosis population, lost as the result of its cultivation in synthetic culture media at a temperature of 37 degrees C, has been found to be restored at low temperature.