Spheroplasts of plague microbe strains from the Transcaucasian uplands and their capacity for pesticin synthesis]

Spherical formations of the plague microbe strains from the Transcaucasian Upland, I plague microbe strain of the sandwort variety and I strain of the marmot variety were obtained under the effect of lithium chloride. They had the remains of the cell wall, were viable, sensitive to osmotic shock, preserved sensitivity to the specific bacteriophage and pesticins. All this was evident of isolation of the spheroplasts of the plague microbe. The spheroplasts showed a capacity for pesticin production. The pesticin synthesis by the spheroplasts of the plague causative agent from the Transcaucasian Upland increased with an increase in the content of lithium chloride in the medium. The largest inhibiiton zones were observed, when 0.7-0.8 per cent of lithium chloride were present in the medium. In the spheroplasts of the plague causative agent from the Mountain Altai (the marmot variety) the pesticin synthesis was inhibited with an increase in the content of lithium chloride in the medium. The activity spectrum of the pesticins of the spheroplasts of the plague causative agent from the Transcaucasian Upland and the spheroplasts of the strains of the marmot and sandwort varieties was broader than that of the rod-like forms of these strains. The indicator properties were found in the strains of the plague microbe of the marmot and sandwort varieties with respect to the pesticins of the spheroplasts of the sel-like producing organisms and organisms from the Transcaucasian Upland.     

Determination of genetic bases of auxotrophy in Yersinia pestis ssp. caucasica strains

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG, aroF, thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.     

Concepts of macro- and microevolution as related to the problem of origin and global expansion of the plague pathogen Yersinia pestis

The relationship between macro- and microevolutionary processes is considered with reference to the ecological scenario of the origin of the plague pathogen and its subsequent natural and anthropogenic global expansion. The macroevolutionary transformation of the ancestral pseudotuberculosis microbe clone into the initial plague microbe Yersinia pestis tarbagani occurred in Central Asia at the end of the Late Pleistocene by a “vertical” Darwinian way in an inadaptive heterothermal continual intermediate environment—the Mongolian marmot Marmota sibirica—flea Oropsylla silantiewi system—via a sequence of unstable and currently extinct intermediate forms. Its natural geographic expansion on the “oil spot” principle in the postglacial time led to the microevolutionary formation of 20–30 hostal subspecies circulating in populations of the background species of burrowing rodents and pikas in arid areas of Eurasia. The intercontinental spread of the “marmot” and “rat” pathogen subspecies in the past few centuries has been exclusively anthropogenic, with the involvement of synanthropic (ship) rats.     

Role of antibodies in protection from pseudotuberculous infection and intoxication]

Lethal doses of virulent pseudotuberculosis bacilli and antipseudotuberculosis sera of different specificity were injected to albino mice simultaneously. A high neutralizing activity of antibodies against pseudotuberculosis intoxication was demonstrated. The type-specific antibodies proved to protect the mice from the toxins of the homologous types of the microbe only. Group antibodies of plaque antiserum and serum procured from the pseudoteburculosis convalescent produced a cross antitoxic action. The antiinfectious effect from the antibody administration was weak. Apparently in pseudotuberculosis the antibodies were the principal factor of the toxin neutralization and were of auxiliary significance in the protection from the developing infection. Neutralization of pseudotuberculosis toxins with plague antiserum served as an additional confirmation of cross immunity between plague and pseudotuberculosis.     

Determination of genetic bases of auxotrophy in <Emphasis Type="Italic">Yersinia pestis</Emphasis> ssp. <Emphasis Type="Italic">caucasica</Emphasis> strains

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG aroF thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.     

Sympatric speciation of the plague microbe <Emphasis Type="Italic">Yersinia pestis</Emphasis>: Monohostal specialization in the host–parasite marmot–flea (<Emphasis Type="Italic">Marmota sibirica–Oropsylla silantiewi</Emphasis>) system

An ecological scenario of the origin of the plague microbe that is interpreted in the light of modern Darwinism (synthetic theory of evolution) is presented. It is shown that the plague microbe emerged from a clone of the psychrophilic saprozoonotic pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan time, 22000–15000 years ago, in the monohostal Mongolian marmot (Marmota sibirica)–flea (Oropsylla silantiewi) host–parasite system. It was noted that the evolutionary process described corresponds to the sympatric form of speciation by transition of the clone of migrant founders to a new, already-existing ecological niche. It was established that monohostal specialization of the plague microbe was made possible due to heterothermia (5–37°C) of marmots in the hibernation period. The factors of the speciation process—isolation, the struggle for existence, and natural selection—were analyzed.     

Quantum Speciation of <Emphasis Type="Italic">Yersinia pestis</Emphasis> Plague Microbe in a Heteroimmune Environment: In the Populations of Hibernating Tarbagan Marmots (<Emphasis Type="Italic">Marmota sibirica</Emphasis>)

This review is devoted to the ecological mechanism for the transformation of the population of the psychrophilic saprozoobiont pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b into the population of the obligate blood plague microbe Yersinia pestis in the host–parasite system of the marmot flea (Marmota sibirica–Oropsylla silantiewi). The correspondence of this mechanism to the evolutionary principle of quantum speciation has been shown. The most significant population–genetic microbial transformations took place in the populations of hibernating marmots. The main factor of rapid speciation was the heterothermic and, accordingly, heteroimmune state of host marmots during hibernation. During winter awakenings, the body temperature of marmots increases in an S-shaped manner from 5 to 37°C within a short period of time (from dozens of minutes to several hours). A drastic acceleration of metabolic and immune processes occurs in the temperature range of 20–30°C. Rapid adaptation to the “explosive” increase in the immune activity of the primary host (tarbagan marmot) during its regular winter awakenings was the essence of the process of Y. pestis speciation.     

Characteristics of the bacterial component in the dynamics of the infectious process in experimental pseudotuberculosis]

A model of experimental pseudotuberculosis caused by the administration to albino mice of virulent local pseudotuberculosis strains was used to study migration of the causative agent into the macroorganism and its tropism. Experimental results permitted to establish a number of regularities attending bacterial spread. The infectious process in pseudotuberculosis began from invasion of the causative agent into the intestinal wall and the subsequent initial bacterial reproduction at the site of invasion and penetration from the intestine into the regional mesenteric lymph nodes. This was followed by rapid reproduction of the microbes in these nodes and contamination of all the organs. With comparatively low doses of the causative agent administered and adequate resistance of mice the infectious process could either stop at the phase of regional infection with regression, or, following hematogenic dissemination, pass into the phase of decline and terminate by animal recovery. Administration of massive doses of the virulent culture led to a septicemic process and death. Experimental data concerning the localization of the microbe in the macroorganism at various phases of the infectious process confirmed the enterotropic character of the pseudotuberculosis causative agent.     

Impact of Yersinia pseudotuberculosis on the in vitro production of cytokines by whole blood cells of donors

The impact of two plasmid (47, 82 MD), single plasmid (47 MD) and non plasmid Y. pseudotuberculosis strains, Y. enterocolitica (47 MD) as well as Y. pseudotuberculosis superantigen (YPM) on the production of interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-alpha (IFN = alpha) and tumor necrosis factor alpha (TNF-alpha) by whole blood cells obtained from donors was studied. All Y. pseudotuberculosis and Y. enterocolitica strains stimulated the production of IFN-alpha, IL-1, IL-6 and TNF-alpha by whole blood cells, but considerably less than Y. pseudotuberculosis lipopolysaccharide and YPM. These data are indicative of the pathogenetic role played by 82 MD plasmid in manifestation of Y. pseudotuberculosis immunosuppressive properties. The maximum stimulation of the production of cytokines was observed under the action of YPM, which confirmed an important role played by this superantigen in the pathogenesis of Y. pseudotuberculosis.     

Ecological aspects of the origin of Yersinia pestis,causative agent of the plague: Concept of intermediate environment

Modern phylogenies of Yersinia pestis (Logh.), causative agent of the plague, constructed using molecular-genetic methods, do not receive a satisfactory functional and adaptive interpretation and are far from being ecologically valid. We have presented an ecological scenario of the origin of the causative agent of the plague through the transition of the initial pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b to a free hostal ecological niche (and a new adaptive zone) under ultracontinental climatic conditions of the Late Pleistocene (Sartan time, 22000–15000 years ago) in southern Siberia and Central Asia. An intermediate environment, i.e., the “Mongolian marmot Marmota sibirica-flea Oropsylla silatiewi” parasitic system, where the process of adaptation development of the plague microbe took place, has been characterized. A scenario based on the major principles of the modern synthetic theory of evolution opens the way to an ecological-genetic synthesis of the problem of plague origin and is an appropriate model for developing a theory of molecular evolution of pathogenic (plaguelike) microorganisms.     

Thymidine phosphorylase activity and its relationship to trimethoprim in initial strains and thymidine-, thymine-dependent and trimethoprim-resistant mutants of plague microbe]

The comparative study revealed thymidine phosphorylase activity in the initial strains of a plague microbe of the field variety and in thymidine-, thymine-dependent and trimethoprim-resistant mutants of the plague microbe of other varieties. The data fully conformed to the results of the microbiological investigation of the strains' ability to grow on the nutrient media with trimethoprim in the presence of thymine and thymidine. On the basis of these results it appeared possible to divide the initial and mutant strains of the plague microbe into four arbitrary groups: initial strains of the plague microbe of all the varieties except the field ones sensitive to trimethoprim under any temperature conditions of incubation on any medium with any supplements; initial strains of the plague microbe of the field variety resistant to trimethoprim at 28 degrees C in the presence of thymine or thymidine alone; Tmpr mutants whose resistance to trimethoprim at 28 degrees C did not depend on the presence of thymine or thymidine, purine and vitamins, but depended on the presence of these substances at a temperature of 37 degrees C.     

Antimicrobial resistance and production of toxins in Escherichia coli strains from wild ruminants and the alpine marmot

Escherichia coli strains isolated from 81 fecal samples from red deer (Cervus elaphus), roe deer (Capreoulus capreoulus), chamois (Rupicapra rupicapra) and alpine marmot (Marmota marmota) living in the Stelvio National Park, Italy, were examined for antimicrobial resistance and production of toxic factors. Direct plating of specimens on media containing antimicrobial drugs allowed us to isolate resistant strains of E. coli from 10 of 59 (17%) specimens examined by this technique. Nine of 31 specimens from red deer (29%) contained resistant strains. Different animals were likely colonized by the same resistant strain of E. coli. Conjugative R plasmids were found in four strains isolated from the marmot, roe deer and chamois. A strain from red deer produced heat-stable enterotoxin and another strain produced both hemolysin and cytotoxic necrotizing factor. A marmot isolate produced hemolysin alone. No strains were found to produce heat-labile enterotoxin or verotoxins.     

Pathogenic properties of Yersinia pseudotuberculosis in different biological models

The study of the action of Y. pseudotuberculosis introduced into the cavity of the ligated intestinal loop of a rabbit, into the stomach of 2- to 4-day-old suckling mice or injected intradermally has made it possible to establish the importance of the invasive and toxic activity of this microbe in the pathogenesis of pseudotuberculosis infection. The lesion of the small intestine develops due to the penetration of the bacteria into the mucous membrane and the combined action of the microbial cells inhabiting the stroma, and secreting into the environment of cytotoxins, toxic substances with an enterotropic action or enterotoxins and factors increasing the permeability of the vessels. Y. pseudotuberculosis strains with high and low invasiveness have been isolated.     

Molecular characterization of Corynebacterium pseudotuberculosis isolated from goats using ERIC-PCR

Corynebacterium pseudotuberculosis, the infectious agent of caseous lymphadenitis (CLA), is responsible for substantial economic losses in goat and sheep production. Molecular characterization of C. pseudotuberculosis isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR has shown promising results in genotyping strains isolated from sheep with CLA. We evaluated the genetic diversity of C. pseudotuberculosis isolates collected from the Sert?o region of the Pernambuco (PE) State, Brazil, and investigated the potential of ERIC-PCR as a tool for the molecular typing of strains of C. pseudotuberculosis isolated from goats. Thirty-two C. pseudotuberculosis strains isolated from goats in the municipalities of Floresta and Ibimirim, PE, C. pseudotuberculosis type strain ATCC 19410, the 1002 vaccine strain, and a field isolate of Rhodococcus equi were fingerprinted using the primers ERIC-1R and ERIC-2 and the primer pair ERIC- 1R+ERIC-2. Using 100% similarity as the cutoff, 8, 10, and 7 genotypes were obtained with ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively. The Hunter-Gaston discriminatory index calculated for the ERIC-1-PCR was 0.75. The index for the ERIC-2-PCR was 0.88, and the index for the ERIC-1+2-PCR was 0.79. Among goat isolates of C. pseudotuberculosis, three, two and four genotypes (found by ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively) had been previously described among sheep isolates from Minas Gerais State, Brazil. These results showed that ERIC-PCR has good discriminatory power and typeability, making it a useful tool for discrimination among C. pseudotuberculosis isolates from goats.     

Pseudotuberculosis in the Krasnodar Territory. The clinico-epidemiological characteristics of its morbidity and the biological properties of its strains]

Group cases of pseudotuberculosis, first registered at health-restoring institutions of the health resort of Gelendzhik (the Krasnodar Territory), are described. The etiology of these cases of the disease was established on the basis of clinico-epidemiological data, specific seroconversion in 66% of the examined patients and the isolation of Yersinia pseudotuberculosis strains from the feces of four patients. The combination of signs observed in the isolated strains, viz. the presence of plasmids with molecular weights of 75 and 45 MD, resistance to bactericidal factors of normal human serum and autoagglutination in cell culture medium, made it possible to consider these strains to be virulent. The cases of infection were probably caused by the use of fresh cabbage salad. The study of 3,128 rodents caught in the Krasnodar Territory, including those caught in the area of the health resort, resulted in the isolation of 105 Y. enterocolitica strains and strains of 5 other Yersinia species, but not Y. pseudotuberculosis strains. The concept on the saprozoonotic nature of pseudotuberculosis was substantiated.     

Genetic bases of methionine dependence in Yersinia pestis strains of major and non-major subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and minor subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metB, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and minor subspecies revealed that the reason for the methionine dependence of strains belonging to the major subspecies is a deletion of a single nucleotide (-G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Analysis of factors which determine the existence of Yersinia pseudotuberculosis in a saprophytic phase

Data are presented on the effects of a variety of abiotic and biotic environmental factors on the existence and changes in the numbers of Y. pseudotuberculosis. Experiments with sterile soil showed that Y. pseudotuberculosis populations were resistant over a wide range of major abiotic factors: temperature (0-30 degrees C), humidity (15-50%), pH (5.9-9.0). Although exerting some effect on the duration of different growth phases, the above abiotic factors did not influence, within the tested range, the general nature of populational dynamics of the microbe. Comparative experiments carried out in sterile and natural soil specimens using an RNA-polymerase mutant warranted the conclusion that the numbers of Y. pseudotuberculosis in soil (water) are largely controlled by the biotic components of ecosystems, including microflora and microfauna. Y. pseudotuberculosis was shown to exist in the environment (vegetable storehouses and substrate of rodent nests) in association with bacteria belonging to the family Enterobacteriaceae as well as the genera Acinetobacter and Pseudomonas. Endosymbiotic relationships are described between Y. pseudotuberculosis and the free-living infusorian Tetrahymena pyriformis which sustains microbial populations in the soil (water).     

Experimental study on the effect of low molecular DNA from salmon milt in pseudotuberculosis infection]

The effect of low molecular DNA from salmon milt (nDNA) in experimental pseudotuberculosis in mice was studied. When nDNA was admiministered orally, dissemination of the organs by Yersinia pseudotuberculosis lowered and the survival of the animals infected with 100-percent lethal dose of the bacteria increased. nDNA decreased contamination of the epithelial cells by the microbe in vitro and prevented the lethal effect of the Y. pseudotuberculosis toxins on the mice.     

The gene identification of bacterial species and serovariants by the polymerase chain reaction with universal oligonucleotides: the reidentification of earlier isolated strains of Yersinia pseudotuberculosis]

Genetic analysis of 19 standard strains belonging to 6 Yersinia species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kirstensenii, Y. frederiksenii, Y. intermedia) revealed that gene typing by the method of polymerase chain reaction (PCR) with the use of universal primers permitted the identification of species in bacterial cultures by PCR patterns and the determination of Y. pseudotuberculosis serovars within 4 hours. By this method 23 Y. pseudotuberculosis strains (serovar 1), earlier isolated in different regions of the USSR from humans and rodents, were studied. The study showed that out of 14 strains of human origin only two strains could actually be classified with serovar 1, while the remaining strains were reidentified as belonging to serovar 5. Among 9 strains isolated from rodents those of serovar 1 prevailed (8 strains). The authors suppose that strains of serovar 5 cause outbreaks and sporadic cases of pseudotuberculosis, occurring considerably more often than it is commonly believed in the USSR.     

A cyclic 3',5'-adenosine monophosphate-binding protein in the causative agent of plague

cAMP-binding protein was isolated from the plaque agent and purified to the homogeneous state. Purification process included filtration of the initial preparation through the membrane able to transmit particles with mol. weight to 300,000 Da, chromatography on cellulose "DE-52" and biogel HTP. The protein homogeneity was confirmed by electrophoresis in polyacrylamide gel and precipitation with commercial plague agglutinating serum. The protein with mol. weight of 180,000 Da consisted of two identical subunits (90,000 Da. each) which could dissociate with formation of monomers (mol. weight approximately 18,000 Da), Cu2+, Co2+, Mn2+ ions stimulated activity of cAMP-binding protein of a plague microbe while Fe3+, Ca2+, Zn2+ ions inhibited it by 30-70%. A monospecific rabbit serum to the homogeneous preparation of cAMP-binding protein was obtained. It helped finding the similar protein in the close relative bacterium Yersinia pseudotuberculosis but not in Y. enterocolitica.