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The integration of achievements in population ecology and the molecular genetics of pathogenic microorganisms into nowadays epidemiology of infectious diseases and the formation of its corresponding trends are discussed. The decisive role of highly complicated key problems of general epidemiology, such as the regularities of the autochthonous existence of pathogenic bacteria in the environment, the mechanisms of the pathogenicity regulation of infective agents and the formation of their epidemic variants, the mechanisms of the prolonged reservation of pathogenic bacteria at interepidemic (interepizootic) periods, the mechanisms of transformation of active (vegetative) and dormant forms in bacterial populations at different stages of epidemic and epizootic cycles, is demonstrated. The detection and analysis of such processes, and especially their latent mechanisms, become possible with the use of new concepts and methodological approaches, highly productive for the progress of epidemiology.

The ultrastructural organization of Y. pseudotuberculosis in the process of the transition of vegetative cells into the dormant (noncultivable) state in interaction with blue-green algae of the species Anabaena variabilis was studied by the method of transmission electron microscopy. The use of type specific Y. pseudotuberculosis serum made it possible to identify Y. pseudotuberculosis cells in the bacterial association and to find out whether their antigenic properties remained intact in time. The dormant forms of Y. pseudotuberculosis, recultivated by passage through the axenic culture of unfusoria (Tetrahymena pyryformis), were also studied with the use of electron microscopy. The revertants were found to be at different stages of restoration of their typical morphological characteristics and antigenic properties were partially retained. The fine structure of Y. pseudotuberculosis cells in the initial culture was shown to be similar to that of the revertants of dormant forms, morphological criteria of the dormant cell ultrastructure were established. The cyclic processes of reversible transition from vegetative forms to dormant ones in bacterial populations under the influence of hydrobios is regarded as an adaptive mechanism of their existence in the environment.

The dynamics of vegetative and dormant (noncultivable) of S. typhimurium cells of two isogenic strains in association with microalgae Scenedesmus quadricauda and under the action of the exometabolites of the algae at different stages of their growth was studied using in parallel bacteriological method and PCR. The study revealed that at the stage of active growth green algae and their metabolic products maintain the survival of salmonellae (strain TR = 1) vegetative forms in water at an optimum temperature. Low temperatures induced their gradual (3 weeks) transition to the dormant state. The exometabolites of old dying algae induced the rapid (several hours) and complete transition of the bacterial population (TR = 1) to noncultivable state. In our experiments the insertional mutation in gene pqi (strain PhoA = 8), inducing the defect of transmembrane protein and disturbances in the transition of salmonellae to dormant state, led to stable existence (lasting 7 months, i.e. the whole term of observation) of vegetative cells. The natural inducers tried in our experiments did not lead to the formation of the dormant forms of salmonellae in this mutant strain.

Research, carried out with the use of bacteriological methods and polymerase chain reaction, revealed that the transformation of Y. pseudotuberculosis, associated with blue-green algae Anabaena variabilis, into resting (noncultivable) forms took shorter time than in soil extract containing no algae. The exometabolites of "old" cultures of these algae sharply accelerated the formation of resting Y. pseudotuberculosis forms. The influence of the algae and the products of their metabolism was manifested far more intensively at 22 degrees C than at 4 degrees C. After passage through infusoria resting Y. pseudotuberculosis forms, preserved in the mucous covering of cyanobacteria, partially reverted into vegetative forms, capable of growing on solid culture media. The revertants essentially differed from the initial vegetative forms by having lower enzymatic activity, agglutinability and cytopathogenicity, as well as by the loss of plasmid p45. The probable role of blue-green algae, widely spread in soils and water reservoirs, in the processes of reversible transformation of Y. pseudotuberculosis vegetative and resting forms, closely connected with seasonal changes of temperature conditions.

The currently available data on different variations in cholera vibrio habiting in water reservoirs are summarized. The pattern of variations of the main signs of vibrions is discussed in the context of "typical-atypical-inactive-resting (uncultivable) forms". Emphasis is placed on the reversible pattern and adaptive essence of variation whose basis is clonal and selective processes in the heterogeneous bacterial population. Possible populational mechanisms responsible for the development of epidemic types of cholera vibrions in the aqueous ecosystems as a prerequisite for human morbidity are discussed. The efficiency of ecological and molecular genetic studies for the epidemiology of cholera is recognized.

A short history of the concept of natural focal infections is presented: the idea put forward by D. K. Zabolotnyĭ, E. I. Pavlovskiĭ's teaching, 3 stages of its development. A number of fundamental questions and the modern content of the concept are considered. The natural foci of infections are a combination of surface, soil and/or water ecosystems, including the population of the causative agent of infection. In contrast to surface ecosystems, in soil and water ecosystems the hosts of the causative agents of sapronotic infections ae soil invertebrates and hydrobios, in which these agents may circulate in biocenotic trophic chains. The circulation of the causative agents in natural foci is a discrete process; the mechanisms and forms of the existence of pathogenic bacteria during seasonal and prolonged periods between epidemics is considered. Special attention is given to latent (nonculturable) forms of bacteria. The complex character of the status of the causative agents of natural focal infections is discussed.

E. N. Pavlovskiĭ's concept of natural focality of diseases and the development of general knowledge about natural foci and their structural (components), functional (mechanisms of pathogen maintenance), and ecosystem-related organization (assortment and interrelations of ecosystems) are reviewed from principal (in authors' opinion) aspects. The 60-year history of this theory includes three stages at which its scope and contents differed. At the first stage, it concerned transmissible zoonoses. It had been assumed that structurally, natural foci necessarily include the pathogen-vector-host triad, and the functioning of the focus is provided for by only pathogen circulation in terrestrial ecosystems. At the second stage, it became clear that vector is not a necessary structural component of any focus (an example of nontransmissible diseases), although the functioning of foci remained to be unequivocally attributed to the continuous pathogen circulation among animals of terrestrial ecosystems. The third stage is characterized by an understanding that, in general, the presence of a warm-blooded host in the focus is also unnecessary for pathogen survival, and natural foci can be represented by soil and aquatic ecosystems. The only necessary and specific component of any natural focus is the pathogen population. In this context, modern views on natural focality of diseases are reviewed, and the essence of the terms "natural focus" and "epizootic process" is defined. It is proposed to distinguish the phases of pathogen reservation and epizootic spread (circulation) in ecosystems of any type. The current state of this concept provides evidence that, in general biological terms, studies on natural focality of diseases belong to one of the fields of symbiotology.

The dynamics of the transition of Y.pseudotuberculosis to the latent (noncultivable) state in sterile soil extraction and in soil extraction, containing Scenedesmus algae or their exometabolites, was evaluated by the bacteriological method and with the use of polymerase chain reaction. The formation of Y.pseudotuberculosis latent forms achieved its highest rate under the action of algal exometabolites (on day 11), while in the presence of algal cells such formation was delayed to 3-5 months. The partial reversion of Y.pseudotuberculosis nonculativable forms to vegetative, bacteriologically detectable forms occurred under the action of fetal serum, as well as live or killed infusoria. Transmission electron microscopy demonstrated that noncultivable Y.pseudotuberculosis had a round form, diminished cell size, denser cytoplasm and enlarged cytoplasmic space. The reversible transition of bacteria to the latent state is regarded as their adaptation to unfavorable factors during their existence in soils and water reservoirs.

The parallel study of soil specimens from the burrows of great gerbils, taken in the natural focus of infection at the period between epidemics, by the bacteriological method and with the use of polymerase chain reaction (PCR) demonstrated that, simultaneously with the negative results of inoculations into nutrient media, PCR revealed the presence of Y. pestis in 4 out of 72 soil specimens. The serological study (in the indirect hemagglutination test) revealed the presence of Y. pestis capsular antigen (fraction l) in 16 out of 75 soil specimens. The transition of Y. pestis (strain EV) into the nonculturable state was experimentally shown in sterile soil extract and in association with protozoa, algae and Euglena. Bacteriologically detected Y. pestis disappeared or were represented by a few cells after day 7, while PCR could register the presence of Y. pestis at a concentration of 10(4)-10(5) microbial cells per ml until the end of the term of observation (30 days). After the enrichment of the nutrient medium with fetal serum the complete reversion of Y. pestis nonculturable forms was observed: the concentration of bacteria grown on agar was 10(5) colony-forming units per ml. Transition into the nonculturable state was also observed in distilled water. The results of this investigation are discussed with special emphasis to the possibility of the preservation of Y. pestis in the nonculturable from during the period between epidemics as applied to the problem of plague epizootic.

The analysis of the dynamics of the amount of L. monocytogenes in soil extract in association with green algae and in their absence, carried out in parallel by the bacteriological method and in polymerase chain reaction (PCR), revealed the mass transition of L. monocytogenes into the nonculturable state. The proportion of vegetative (bacteriologically detected) forms in the bacterial population rapidly decreased, while its total amount remained unchanged due to the formation of inactive (nonculturable) L. monocytogenes forms. Algae or their metabolites considerable accelerated this process: by day 26 L. monocytogenes could not be isolated, but in PCR they could be registered at their initial concentration: 10(6) microbial cells/ml. Inactive L. monocytogenes forms were shown to revert into the vegetative (culturable) state under the action of fetal serum, live and killed infusoria, auxin. The concentration of revertants grown on a solid culture medium was high: 10(4)-10(5) colony-forming units/ml.

Soil samples taken from the natural focus of pseudotuberculosis, characterized by the persistent presence of Y. pseudotuberculosis, were studied. Bacteria were detected by means of polymerase chain reaction with the use of 2 test systems; one of them was specific to the chromosomal invasiveness gene (inv) and the other, to gene yopA, localized on plasmid pCad. In 10 out of 24 sample of genetic material of the total soil microflora, Y. pseudotuberculosis gene inv was detected, two of these samples being also containing gene yopA. The calculated concentration of Y. pseudotuberculosis varied 10(2)-10(4) microbial cells per 1 g of soil. Bacteriological studies yielded negative results, which is indicative of the existence of Y. pseudotuberculosis in the soil of the natural focus in the nonculturable form.

The possibility for Listeria monocytogenes to penetrate into plants from the soil via the root system was experimentally proved. Listeria were shown to continuously persist for 30 days (the term of observation) in the vegetative organs of wheat (roots, stems and leaves). The concentration of Listeria was 10(9) CFU/g in the environment (soil extract) and the roots of wheat, 10(6)-10(7) CFU/g in stems, 10(8)-10(9) CFU/g in leaves. Six days later the dissociation of colonies in the S-form into small (up to 1 mm) and large (3-4 mm) was observed; in contrast to Listeria in large colonies, those in small colonies had high catalase activity and pronounced cytopathogenic action. The problem of the possible role of plants as the natural reservoir of bacteria, pathogenic for humans and animals, is discussed.

Complex probiotic sporolact created on the basis of lactobacteria and aerobic sporeforming bacteria has been studied for its effect on microflora of the gastrointestinal tract of laboratory animals with expressed dysbacteriosis. It has been stated that sporolact inhibits pathogenic microorganisms and renewal of quantitative characters of obligate representatives of the microbiocenosis of the gastrointestinal tract up to the normal level. This complex probiotic has also a curing effect on acute gastrointestinal infection of calves induced by pathogenic bacteria.

The results of the study of interaction between V. cholerae of different virulence and T. pyriformis are presented. The study has revealed the heterogeneity of V. cholerae population: alongside easily phagocytized vibrios, there are vibrios resistant to the digestive action of T. pyriformis. An increase in the number of V. cholerae in association with T. pyriformis has been evaluated, taking into account the selective multiplication of vibrios resistant to phagocytosis. The data on changes in the agglutinative, phagolytic and virulent properties of V. cholerae cultivated together with T. pyriformis are presented. The suggestion has been made that protozoa can function as hosts of pathogenic vibrios supporting their existence in water.

On the basis of the polymerase chain reaction (PCR) a test system permitting the detection of Y.pseudotuberculosis in different environmental objects was developed. On a model approximating natural conditions the dynamics of the amount of these bacteria in sterile soil extract were studied. The comparison of the amount of Y.pseudotuberculosis, determined in terms of colony forming units and by the detection of their DNA marker fragment with the use of PCR, showed the possibility of the transition of Y.pseudotuberculosis cells into the noncultivated state during their persistence in the aqueous soil extract.

On the basis of literature data and the results of experimental studies the concept of the universal character of the main pathogenicity factors of bacteria (adhesion, enzymes, toxins, resistance to phagocytosis) as the adaptive mechanisms of bacterial populations under different ecological conditions, including their existence in the environment, has been put forward. Resistance of bacteria to phagocytosis as the mechanism of their protection from predators (protozoa, etc.), necessary for the existence bacteria in the ecological systems of water and soil, is considered in detail.

The interaction between Y. pseudotuberculosis and different representatives of the aqueous and, partially, soil biocenosis was studied. The study revealed that invertebrates of the plankton and benthos (water fleas, cyclops, mollusks, Oligochaeta, larvae of insects), as well as fish and higher plants, ensured the prolonged existence of Yersinia in water. Thus hydrobiotic organisms were to be regarded as potential natural hosts of Y. pseudotuberculosis. A suggestion was made that fish, the final link of some alimentary chains, could present epidemic danger as a food product for human consumption.

The experimental study of the interaction of Tetrahymena pyriformis with different microorganisms of the genus Pseudomonas, isolated from the soil, was made. The study revealed that T. pyriformis phagocytosed some Pseudomonas pigment-forming species (P. cepacia, P. putida, P. fluorescens, P. pirkettii). The most pronounced cytopathogenic effect was produced by P. cepacia. The dynamic observations of the ultrastructural features of interaction between P. cepacia and protozoa were made. Even at early stages of this interaction some types of parasitiferous phagosomes containing both intact bacteria capable of multiplication by binary division and Pseudomonas cells exhibiting different degrees of destruction were registered. In several phagosomes morphologically intact bacteria differing in their cell-wall profiles and the density of their cytoplasm and nucleotide were present simultaneously. More dense cells with sinuous cell-wall membranes were more virulent. By hour 18 one giant parasitiferous vacuole was formed by fusion of smaller phagosomes, which subsequently broke up, liberating a new generation of bacteria. In infected cells disturbances in the structure of their mitochondria and macronucleus appeared. During the first 2 days of the joint cultivation of P. cepacia and T. pyriformis the accumulation of bacteria occurred due to the selection and multiplication of digestion-resistant bacterial cells, which ensured the resistance of this Pseudomonas population in association with protozoa.

Y. enterocolitica and Y. pseudotuberculosis have been experimentally found to penetrate into plants (cabbage, salad, pea, oat) from infected soil and water and to remain in different plant organs (roots, seeds, leaves) up to 30 days (the term of observation). The processes of S-R dissociation in Yersinia cultures isolated from plants, as well as changes in some enzymatic properties, have been established. In the test on Infusoria the cytotoxic action of strains isolated from plants has been noted. The possible role of plants in carrying pathogenic bacteria from the soil to the surface ecological systems and the subsequent infection of animals and humans is discussed.

Selective changes in Y. enterocolitica virulence have been made in experiments on eukaryotic cells, Infusoria, and peritoneal macrophages, used as models. In parallel experiments with the passage of Yersinia through Infusoria and mammalian macrophages an analogy to the processes of phagocytosis has been established.

In this work the morphological features of the interaction of L. pneumophila virulent strain and T. pyriformis have been studied on the submicroscopic level in the time course of the process. The study has shown the process of the destruction of the bacterial population and the penetration of individual intact Legionella cells from the phagosome into the endoplasm of T. pyriformis after 6-9 hours of interaction in the form of the budding of the phagosome and further multiplication of Legionella in the endoplasm. As revealed in this study, T. pyriformis have two types of phagosomes characterized by different variants of the destruction of Legionella. In T. pyriformis lysosomes-like granules, mitochondria and the granular endoplasmatic network take part in the process of interaction. The process of interaction has been found to end by day 7 in the death of all protozoal cells taking part in interaction.

L. pneumophila avirulent strains have been shown to lose their capacity for multiplication in T. pyriformis, while the concentration of the virulent strain increases 1000-fold. The loss of the hemolytic activity of L. pneumophila virulent strain leads to the loss of its capacity for multiplication in infusorians.

The mechanisms of interaction between the populations of Yersinia and T. pyriformis have been analyzed on the cellular and subcellular levels. As shown in this investigation, Yersinia, when phagocytized by T. pyriformis, may undergo morphological changes, remain unchanged and also multiply, destroying the host cell in the process.

Field experiments with the use of radioisotope models have shown that urine containing leptospirae is regularly excreted in the same "infected spots" of the territory under study. For this reason, it is an increase in the frequency of Leptospira excretion in each "infected spot", rather than an increase in the number of such spots, which occurs in the course of the epizootic. To detect the "infected spots" on the experimental ground with the use of radioisotope models, the registration of such spots for 5-7 days is sufficient. The excretion of urine by animals in the "infected spots" has been shown to decrease the acidity of the soil, thus creating conditions favorable for the survival of leptospirae in the soil.

The decisive role of biotic factors in the preservation of the viability of Y. pseudotuberculosis is shown. The results of experiments suggest that protozoa may serve as reservoir in the spread of Y. pseudotuberculosis infection.

The number of mast cells has been established to increase sharply during the metamorphosis of Bufo viridis tadpoles and during the period of postnatal ontogenesis of rats. The data obtained are under discussion to represent mast cell as a system-forming element of thyroid microregion.

The number of L. hebdomadis and L. grippotyphosa in the body of tundra voles (Microtus oeconomicus) has been shown to be approximately 500-800 million; about 100 million of them are daily eliminated from the body of a vole with urine. About 70% of the total volume and separate portions of urine contain leptospires, a single portion of urine containing about 2 million microbial cells. These data have proved to be the same for both L. hebdomadis and L. grippotyphosa.

Leptospires released with a carrier's urine were found to inhabit the upper layer of the soil 1 cm deep at a radius of 1-2 cm from the place of their penetration into the soil. A sharp drop in the number of leptospires in the "infected spot" was shown to occur; in 6-12 hours their concentration became no different from that in the intact soil. Most of such spots retained their infective capacity for 6 hours (up to 2 days), though the presence of leptospires at low concentration was registered for 10 days (the term of observation). The infective capacity was probably maintained by the regular addition of leptospires with carriers' urine.

Siberian lemmings and Middendorf's voles were found to be susceptible to infection caused by Leptospira of the Grippotyphosa serogroup after the intraperitoneal injection of Leptospira culture, the application of the culture or infected urine to the skin, as well as after Leptospira-carrying animals were placed together with the animals to be infected. The infectious sensitivity of these animals to Leptospira was not high: leptospiruria was observed for 1-3 weeks; in some of the voles leptospiruria was slightly pronounced, whereas other voles had a great number of Leptospira in urine. Antibodies appeared in the blood on day 5 after infection, and their titers increased till days 61-63. In no case could Leptospira be isolated from the kidneys of the animals killed on days 61-83 of the experiment.

The radioisotope modeling of the processes of the release of leptospires into the soil and the daily measurements of the radiation doses revealed that leptospires were regularly added by individual carriers, as well as by a group of carriers, into common infected spots on the territory of the infection focus. The frequency with which leptospires were added by a carrier into different infected spots varied: during 7 days leptospires were added 7 times and more into 16% of the spots, up to 5 times into 49% of the spots; into 35% of the spots no leptospires were added. The microscopic and bacteriological methods of investigation demonstrated that the regular release of leptospires into the infected spots resulted in maintaining the concentration of leptospires at such spots and their infective capacity. The number and distribution of these spots determined the stability of the epizootic potential of the territory.

The study of the immunological structure of the population in respect of intestinal yersiniosis at territories with different hydrothermal conditions has revealed the presence of a greater immune stratum (23.9% with the average agglutination titer being 1.8 +/- 0.09 log D) in humid areas, such as the region of the Upper Volga, in comparison with dry areas, such as the region of the Middle Volga, where the level of the immune stratum has proved to be twice as low (10.6% with the average agglutination titer being 0.8 +/- 0.1 log D). At both territories no essential differences in the risk of infection between the inhabitants of rural and urban areas, as well as between different professional and age groups have been revealed, with the exception of persons over 51 years of age, among whom the occurrence of seroconversions has proved to be lower.

The mass radioisotope (32P) labelling of all tundra voles excreting Zeptospira, infected urine was carried out over the area of 1 ha, and the results of the experiment analyzed by the method of planar coherent graphs, showed the irregular (spotted) distribution of "infected spots", i. e. soil patches contaminated by Zeptospira. The borders of individual small foci of infection did not coincide with the outlines of soil and vegetational divisions of the area. The spatial structure of the "extraorganismic" part of the Zeptospira population was originally formed by the carriers and thus determined by their mobility, their manner of using the territory. Later this structure was changed by selective elimination under the influence of living conditions for Zeptospira in the soil. The "infected spots", subjected to such selection, seem to be capable of constantly sustaining Zeptospira; the presence of these organisms is also due to the fact that they are regularly added into the soil with urine excreted by the carriers visiting the same spots of their territory.