[Bordetella pertussis agglutinogens in cultivation dynamics].

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.

[Pertussis toxin and cross-reactive antigens in dynamics of Bordetella pertussis cultivation].

Cultures of Bordetella pertussis from phases of exponential growth, retarded growth and from stationary phase were obtained during periodic dynamic cultivation. Preparations for intravenous immunization of rabbits were made from these cultures. Levels of IgG to pertussis toxin, cell walls preparations from 12 bacterial species, 4 organo-specific antigens, and 7 organospecific human antigens were measured in obtained sera. It was shown that higher levels of IgG to pertussis toxin were found in sera of rabbits immunized with cultures from exponential growth phase whereas decrease of this level in 8 times was observed in sera of rabbits immunized with cultures from retarded growth phase or end of stationary phase. After immunization with culture from exponential growth phase increase of IgG levels to cross-reactive antigens was not observed compared to levels of these antibodies in control sera obtained before immunization. After immunization with cultures from retarded growth phase or end of stationary phase increase of IgG levels to preparations of cell walls of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, to denaturated DNA, elastin, and renal and liver microsomal fractions was detected compared to control sera. Described data can substantiate usefulness of obtaining the most specific diagnostic sera and test-systems using cultures of B. pertussis from the phase of exponential growth.

[Perspectives for development of polyvalent meningococcal vaccine].

Experimental whole-culture oral polyvalent meningococcal vaccines against serogroups A, B and C consisting of three monovalent components in different proportions have been developed and evaluated. Kinetics of IgG response to meningococcal antigens (outer membrane proteins, polysaccharide, lypooligosaccharide of these serogroups) in sera of rabbits immunized orally with these preparations was measured. Sharp rise of IgG levels (on 2 - 3 orders) compared to baseline has been detected as well as persistence of high titers during the observation period (322 days).

[Influence of the growth phase of Neisseria meningitidis on the accumulation of antigens cross-reacting with human antigens in the culture].

The content of N. meningitidis antigens with epitopes cross-reacting with human antigens was studied in the dynamics of the meningococci cultivation. The absence of these antigens in the culture in the middle of the exponential phase of growth and their presence in the culture at the end of the stationary growth phase of growth were shown. Thus the expediency of using the culture in the middle of the exponential phase of growth for obtaining meningococcal (serogroups A, B and C) vaccines was demonstrated. The trend, found to be promising for the development of immunologically safe vaccines which contained no antigens having common epitopes with human antigens, was determined.

[Level of IgG antibodies to bacteria of 12 species in the blood sera of rabbits, immunized with preparations obtained from Neisseria meningitidis, grown under the stress conditions].

The blood sera of rabbits, immunized with preparations obtained from N. meningitidis of serogroups A, B or C, cultivated under the stress conditions, were studied. These sera were found to contain IgG antibodies not only to N. meningitidis antigens, but also to the bacterial antigens of 12 species. The sera of rabbits, immunized with meningococcal preparation of serogroup A, were found to have the elevated levels of IgG antibodies, in comparison with the control, to the antigens of 3 other bacterial species; the blood sera of rabbits, immunized with meningococcal preparation of serogroup B, were found the elevated levels of IgG antibodies to the antigens of 11 other bacterial species; and the blood sera of rabbits, immunized with meningococcal preparation of serogroup C, to the antigens of 9 other bacterial species. The study of serogroup B meningococci, used as an example, revealed the influence of the growth phase of the culture on the content of cross-reacting antigens. Their greatest amount was determined at the stationary phase when the stressor effect on the culture reached its maximum and their least amount, at the exponential phase when the stressor effect on the culture was minimal. It was, therefore, found to be expedient to obtain immunodiagnostic and test systems from N. meningitidis cultures, grown to middle of the exponential phase of growth.

[Cold shock in bacteria]. / Kholodovoi shok u bakterii.

Various species and genera of bacterial show the same responses to low temperature. Cold induces cold shock genes, by causing significant changes in the regulation of protein synthesis. The synthesis of major proteins in the microbial cell is suppressed. However, there is synthesis of a great deal of new proteins, the so-called cold shock ones. The chief protein in this family is E. coli CspA that activates the translation of other cold shock genes and negatively regulates the expression of its own gene. E. coli CspA homologies were identified in many bacteria. They can be also identified in other microorganisms, including the pathogens of infectious diseases. This can be attributable to the presence of common antigens in different bacteria. The data presented should be borne in mind in identifying bacteria and in designing immunodiagnostic agents if there is a culture cooling stage in their technology.

[Impact of stressors on serogroup B neisseria meningitidis outer membrane proteins during cultivation in bioreactor]. / Vliianie stressorov na belki naruzhnoi membrany neisseria meningitidis serogruppy B v protsesse (periodicheskogo) kul'tivirovaniia (v bioreaktore).

The authors examined the antigenic complexes obtained from serogroup B Neisseria meningitidis strain 125 cells grown in the bioreactor to different phases of growth in the reaction of gel precipitation and counter immunophoresis in a periodical fashion. A set of serotypic antigens was shown to increase from 3 to 9 with cultural growth. Their largest quantities were seen in the cells grown till the end of the stationary growth phase, the concentration of high-molecular weight peptides rising from 19% (in the exponential growth phase cells) to 54% (in the stationary growth phase ones). The revealed regularities were evidenced by gel precipitation examinations of the sera from rabbits immunized with meningococci grown till different growth phases. At the same time, there were the largest quantities of serotypic antigenic precipitation bands when the sera obtained in the culture grown till the end of the stationary growth phase were used. It cannot be excluded that the results depend on the combined effects of several stressors on Neisseria meningitidis in the transition from the exponential to stationary growth phase, as resulted from the increased synthesis of high-molecular weight proteins that seem to act as protectors.

[Antigen properties of oral intake whole-culture preparations from Neisseria meningitides of serogroups A, B and C]. / Antigennye svoistva peroral'nykh tsel'nokul'tural'nykh preparatov iz Neisseria meningitidis serogrupp A, B i C.

A new technology of deriving the whole-culture peroral meningococcal mono-vaccines of serogroups A, B and C from the acetone-inactivated cultures was experimentally substantiated. The latter cultures were obtained through computer-aided continuous-flow cultivation of Neisseria meningitides of the above serogroups in the bioreactor with a synthetic nutrient medium. The whole-culture meningococcal mono-preparation of 3 serogroups comprised a complete set of antibodies, i.e. polysaccharides (PS), external membrane proteins (EMP) and lipopolysaccharides (LPP), in their native condition. High levels of IgG to PS, EMP and LPP were detected in blood sera of rabbits, which had been perorally immunized by the above preparations; such high levels persisted for a long time (303 days--observation period). It is noteworthy, that in accumulating of IgG to all investigated antigens of meningococcus of serogroups A and C, the optimal doses of whole-culture peroral monovaccines did not exceed the culture doses used to derive the hyper-immune sera at intravenous immunization.

[Common stressor proteins in bacteria]. / Obshchie stressornye belki u bakterii.

Research data on common stressor proteins of bacteria obtained during recent 10 years are updated and analyzed. Bacteria of one and the same species were shown to give similar response to the action of different stressors; the main stressor proteins of different bacteria appeared to be homologous; bacteria have cross protection from different stressors. In addition, some common stressor proteins of bacteria were found to be homologous with human antigens that is of great importance for immunobiotechnology.

[Acidic stress in bacteria]. / Kislotnyi stress u bakterii.

Information on acidic stress in bacteria, studied not only on the phenomenological, but also molecular and genetic levels, are systematized. Acidic stress in bacteria, appearing as the result of the acidification of the medium, is characterized by many events on the level of gene regulation. An increased expression of some genes and a decreased expression of others result in growth deceleration, quantitative and qualitative changes in the synthesis of proteins. Some of the newly synthesized proteins ensure the survival of bacteria in a medium with higher acidity and their protection from other stresses. The applied importance of acidic stress is relevant to some aspects of biotechnology, immunobiology and medicine.

[Improved culture of Corynebacterium diphtheriae for obtaining toxin-anatoxin]. / Sovershenstvovanie kul'tivirovaniia difteriinykh korinebakterii dlia polucheniia toksina-anatoksina.

The work deals with the optimization of the cultivation of C. diphtheriae with a view to obtaining diphtheria toxin--toxoid on the basis of the study of the kinetics of microbial growth and toxin formation. The combined cultivation process consisting of 3 cycles has been experimentally developed and realized under industrial conditions. The use of this cultivation method has made it possible to obtain more balanced cultures and to standardize the biological properties of the biomass and toxin thus obtained. The characteristic features of the toxoid preparations obtained on the basis of this method are a high degree of purification, good immunogenic properties and constant fractional composition; their degree of homogeneity approximates the molecular homogeneous state.

[Starvation of bacteria as a stress caused by substrate limitation]. / Golodanie bakterii--stress, obuslovlennyi limitom substrata.

The analysis of literature has made it possible to establish the priority of Russian research works made in the 1970-80s on the subject of starvation of bacteria caused by substrate limitation as well as research made in the 1990s concerning starvation of bacteria. This state is characterized by synthesis of additional proteins, so-called stress proteins, which not only ensure the survival of bacteria under the conditions of substrate limitation, but also protect them from a number of other stressors. In spite of the fact that genetic mechanisms regulating the synthesis of some stressor proteins have been revealed their significance for microbiological technology is not yet clear.

[Methodological approach to the analysis of the scientific effectiveness of studies with regard to the development and improvement of medical biological preparations]. / Metodicheskii podkhod k analizu nauchnoi éffektivnosti issledovanii po sozdaniiu i usovershenstvovaniiu meditsinskikh biologicheskikh preparatov.

A new methodologic approach to analysis of the scientific effectiveness of investigations aimed at the development and improvement of medical biological preparations has been worked out. This approach comprises the classification of research work and choice of parameters characterizing scientific effectiveness. The use of relative values (percentage) as characteristics of scientific effectiveness has made it possible to compare the results of research on 260 themes, carried out in one of medical research institutes.

[Improved technology of culturing Bordetella pertussis based on a study of the kinetics of the process]. / Sovershenstvovanie tekhnologii kul'tivirovaniia kokliushnykh bakterii na osnove izucheniia kinetiki protsessov.

The article deals with the study of the kinetics of the processes occurring in the population of B. pertussis grown in a semisynthetic culture medium. On the basis of the mathematical description of the specific growth rate the factors permitting the regulation of the processes of the cultivation of B. pertussis have been revealed. New prolonged multicyclic combined exponential and continuous processes of the cultivation of B. pertussis have been worked out and introduced into practice. These processes are important for the improvement of the modern technology of obtaining B. pertussis biomass, suitable for the production of prophylactic preparations.

[Cultivation of Neisseria meningitidis serogroups A, B and C in a synthetic nutrient broth]. / Kul'tivirovanie Neisseria meningitidis serogrupp A, B i C v zhidkoi sinteticheskoi pitatel'noi srede.

The processes of the cultivation of N. meningitidis, serogroups A, B and C, in a liquid synthetic culture medium have been studied. Strictly group-specific biomass has been obtained. The maximum productivity at all stages of the batch cultivation of N. meningitidis strains 125 and 133 in this medium does not differ from that at similar stages of cultivation in modified Cohen-Wheller semisynthetic medium. In the serotype antigen preparations obtained from N. meningitidis strain 125 grown in the above-mentioned liquid synthetic culture medium basic polypeptides with a molecular weight of 33000, 36000 and 41000 D have been detected. Their presence in N. meningitidis cells is linked with the growth phase of the population.

[Various aspects concerning the intensification of bacterial preparation production]. / Nekotorye aspekty intensifikatsii proizvodstva bakteriinykh preparatov

The authors present a classification of the process of cultivation of the microorganisms, which are divided into periodic, quasi-continuous (semi-continuous and multicyclic) and continuous. The advantages of the quasi-continuous processes in comparison with the periodic ones (obtaining the required amount of biomasses at shorter periods) and the continuous ones (no special expensive apparatus is required) are demonstrated. The question on the expediency of organizing the structural subdivision for the cultivation of different microorganism species at the bacterial preparation plants is raised.

[The criterion of cell division synchronization for assessing the stressor exposure of a Salmonella typhi population]. / Kriterii sinkhronizatsii deleniia kletok dlia otsenki stressornogo vozdeistviia na populiatsiiu Salmonella typhi.

Criterion of the synchronization (CS) of cells division for S. typhi population is proposed. The criterion is based on the assumption of the normal distribution of cells with different generation time in the population after stressor (shock) action. CS is equal to the ratio of the dispersion of the generation time of cells in the population to the average generation time of the whole population and determined from the parameters of the mathematical model. The quantitative values of the parameters of the mathematical model were obtained by the minimization of error between the calculation and experimental data. CS was used for the evaluation and choice of the optimum stressor action in the synchronization of the division of S. typhi.

[Pathology and physiology of microbes. IV. Economic and metabolic coefficients in assessing the functional states of Salmonella typhi]. / Patologiia i fiziologiia mikrobov. Soobshchenie IV. Ekonomicheskii i metabolicheskii koéffitsienty v otsenke funktsional'nykh sostoianii Salmonella typhi.

The study of economic and metabolic coefficients under different conditions of batch and continuous cultivation with the use of S. typhi, a facultative anaerobe, as a model has demonstrated that the functional state of the microbial population is determined by the phase of the development of the culture, the composition of the culture medium, the conditions of aeration and stirring, the concentration of the energy-providing substrate and the dilution rate in continuous cultivation. The economic coefficient with respect to the energy-providing substrate has been found to be the main characteristic of the functional state of the population: the high values of this characteristic correspond to the physiological state of the population. The decrease of the economic coefficient below a certain critical value is indicative of the pathological state of the population.

[The optimization of the batch cultivation process for Pseudomonas aeruginosa to produce exotoxin A]. / Optimizatsiia periodicheskogo protsessa kul'tivirovaniia Pseudomonas aeruginosa s tsel'iu polucheniia ékzotoksina A.

The experimental study of conditions for the optimization of the batch cultivation of P. aeruginosa has been made. As revealed in this study the aim of this cultivation can be achieved by using exponentially growing culture in a dose of 1.10(9) cells/ml as seed material and by ensuring the conditions of rational air supply during the main cultivation process.

[The effect of the conditions of the controlled cultivation of Neisseria meningitidis on the lipopolysaccharide yield]. / Vliianie uslovii upravliaemogo kul'tivirovaniia Neisseria meningitidis na vykhod lipopolisakharida.

The preparations of lipopolysaccharide (LPS) were obtained from N.meningitidis cells grown in synthetic nutrient medium in a fermenter. Different N.meningitidis strains, groups A, B, C, were found to give different yield of the preparation of LPS: the maximum yield was obtained from N.meningitidis cells C 133 and the minimum yield, from N.meningitidis cells B 125. The comparison of group B strains 125 and 150 showed that the former gave a higher yield of LPS. The influence of the phase of growth on the yield of the preparation of LPS obtained from N.meningitidis cells 125 was shown to be manifested under the conditions of oxygen excess; in the exponential phase the highest yield of the preparation was observed. Oxygen dissolved in the medium was found to influence the yield of the preparation of LPS: with the same content of glucose in the medium the yield of LPS was higher under the conditions of the limited concentration of oxygen; with the same concentration of oxygen in the medium the yield of LPS increased with the increase of the concentration of glucose.