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To study the role of active programmed cell death induced by Vibrio cholerae antigens in alteration of peritoneal macrophages of experimental animals.Apoptosis was assessed by cytofluorometric analysis with propidium iodide using cytofluorometer "Coulter" as well as on characteristic morphological changes of cells in stained histological preparations.Performed experiments carried out by both methods provide evidence that V. cholerae and its antigens (cholera toxin, neuraminidase, chitinase, and lypopolysaccharide) cause apoptosis of mice peritoneal macrophages, which leads to their alteration.Our results demonstrate that programmed cell death of phagocytes is one of the causes of cytotoxic effect of V.cholerae and its antigens. Performed experiments show the role of apoptosis of macrophages in formation of postimmunization immunosuppression after vaccination against cholera.

The comparative study of the genomes of V. cholerae O139 isolated from humans and from water of surface reservoirs was carried out with the use of single- and double-primer polymerase chain reaction (PCR). The profiles of polymorphic DNA fragments obtained in this study made it possible to find out differences between groups of strains, as well as the individual features of some of them. The comparison of strains isolated from humans and from water in single-primer PCR revealed that they, in spite of the general similarity of their genomes, essentially differed, which was probably due to changes in the genome of this infective agent. Strains of aqueous origin lacked genes ctx and tcpA, which made them epidemiologically unimportant.

Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells.

Vibrio cholerae 01 and non 01 neuraminidase may be rapidly detected with the use of a synthetic 4-methylumbelliferyl-neuraminic acid substrate. Other vibrios and Enterobacteriaceae show no neuraminidase activity against this substrate. The method may be used with microorganism cultures grown in solid and liquid nutrient media. The time of analysis is 15-20 min.

The quantitative method for determination of penicillinacylase activity is described. The method is based on detection of phenylacetic acid (PAA) formed during hydrolysis of benzylpenicillin. PAA is extracted with toluol and nitrated with potassium nitrate solution in concentrated sulphuric acid followed by reduction of nitrophenylacetic acid into aminophenylacetic acid with zinc powder. Aminophenylacetic acid interacts with p-dimethylaminobenzaldehyde in acid medium forming a coloured compound (Schiff base). The optic density of the latter was determined with spectrophotometer SF-16 at 430 nm. The method was used for determination of acylase in Yersinia species and some other bacteria.