Doxycycline in the prevention of experimental plague induced by plague microbe variants]

A comparative study was performed on the efficacy of doxycycline in experimental plague infection induced in albino mice by strain 231 of the plague microbe and its variant 231 Fra- deprived of the ability to produce the fraction I antigen. It was shown that the LD50 for strain 231 during animal treatment with doxycycline was significantly higher than that for variant 231 Fra-. Prophylaxis of the plague infection caused by the Fra- forms of the plague microbe required significantly higher doses of doxycycline (ED50) than that of the infection caused by the Fra+ forms. The use of the daily maximum permissible doses of doxycycline (50 to 100 mg/kg a day) for 10 days in treatment of albino mice infected with the strain Fra- did not provide animal survival at the level higher than 60 to 70 per cent while the survival rate in the animals infected with the strain Fra+ of the plague microbe and treated according to the same scheme amounted to 90-100 per cent. The lower therapeutic efficacy of doxycycline in the treatment of the infection caused by the fractionless variant of the plague microbe should be considered in development of rational schemes for prophylaxis and treatment of plague.     

Increased invasiveness as one of the manifestations of phenotype variability of the plague microbe in fleas

Experimental studies conducted on genetically connected virulent subcultures of Y. pestis showed that the death of albino mice infected by flea bite occurred earlier than in the animals infected by a syringe subcutaneously. A high invasiveness of Y. pestis subcultures isolated from fleas (in comparison with the initial strains and subcultures from the animals) persisted for 2--3 passages in their cultivation on artificial nutrient media.     

Experimental protective of fraction I of the plague microbe]

The immunogenic properties of the freeze-dried preparation of fraction I tisolated from the culture of avirulent Pasteurella pestis strain were studied in experiments on hamadryas baboons, guinea-pigs and white mice. The preparation was found to have a pronounced protective effect in hamadryas baboons and white mice, especially when the preparation was used in mixture with incomplete Freund adjuvant. In hamadryas baboons resistance to infection was shown to correlate with hemagglutionation antibody titres.     

Characteristics of the multiplication of a virulent strain of the plague microbe in Xenopsylla cheopis fleas infected parenterally

Xenopsylla cheopis fleas infected parenterally with the virulent strain of plague microbe of gerbil variant preserved the agent to the end of their lives. In the body cavity the microbes retained their ability for reproduction which was, however, limited. During the first seven days after the infection the number of microbes slightly increased and later became stabilized. Its mean indices (mean g) varied within the limits of 500 to 2000 microbe cells per 1 individual, maximum index rarely exceeded 30 000 microbe cells. Parenteral infection with plague agent did not affect essentially the longevity of fleas.     

Proline-dependent wild strains and proline-dependent mutants of the plague microbe]

It is found that the growth of Yersinia pestis wild strains, isolated from Citellus musicus Menetrié in the Central Caucasus, depends on the presence of proline in the medium. Proline can not be substituted by glutamic acid, other amino acids or vitamins. 28 proline-requiring mutants were selected from Y. pestis marmot strain 20b. Three groups of proline-requiring Y. pestis mutants are established, similar to those of Escherichia coli. The requirement of proline does not affect the virulency, pigment formation and calcium dependence.     

Antibiotic sensitivity of plague microbe strains from foreign countries]

The method of serial dilutions on the Hottinger agar was applied to comparative assay of antibiotic sensitivity in 50 strains of the plague microbe isolated abroad and in 5 strains isolated in the plague focus in the Central Caucasus. The antibiotics used in the assay were the following: streptomycin, gentamicin, doxycycline, monomycin, kanamycin, tetracycline, erythromycin, ristomycin, lincomycin and polymyxin M. Irrespective of the origin, all the isolates were resistant to erythromycin, lincomycin and polymyxin M. The levels of the sensitivity to the other antibiotics were different. The data serve as a ground for the statement that there is no tendency to development of antibiotic resistance in the plague microbe in patients treated with high doses of the antibiotics and mainly streptomycin. Along with streptomycin, such antibiotics as gentamicin, tetracycline, doxycycline and kanamycin are useful in the therapy of plague and require further investigation.     

Utilization of hemoglobin by the plague microbe

Hemin and hemoglobin are found to be adequate sources of iron for growth of Yersinia pestis. The mechanism of their assimilation is similar and consists of absorbtion of the free intact hem molecule.     

Isepamycin in prophylaxis and treatment of experimental plague due to FI+ and FI- variants of plague microbe]

The efficacy of isepamycin vs. other aminoglycosides was studied in vitro and on albino mice with experimental plague due to natural antigen valuable strains of the plague microbe and the pathogen variants deprived of the ability to produce the capsular antigen fraction I (FI- phenotype). The MICs of isepamycin for the strains of the plague microbe (20 FI+ and 20FI-) were 1.0-4.0 mg\l, that did not differ from those of streptomycin, kanamycin, amikacin and tobramycin. The ED50 of isepamycin in the prophylaxis and treatment of the experimental plague of the mice had no statistically significant differences from the ED50 of the other aminoglycosides. The efficacy index of isepamycin was > 10(4), that did not differ from that of streptomycin, amikacin and gentamicin, irrespective of the strain phenotype (Y. pestis 231 FI+ or Y. pestis 231 FI-). The same as the other aminoglycosides, isepamycin in doses equivalent to the human average daily doses, protected 80-100% of the albino mice from death when used in the prophylaxis and therapy of plague irrespective of the strain phenotype. The results of the study made it possible to consider isepamycin as an agent promising for the prophylaxis and treatment of plague.     

Constructing a test system for the identification of plague microbe based on genetic probes]

DNA probes for detection of the plague agent Yersinia pestis were made on a basis of its three typical extrachromosomal replicons. The recombinant plasmid pBS2 including pBR327 vector and SalGI-BspRI fragment of the plasmid pFra was constructed. The above fragment is connected with synthesis of Y. pestis capsular antigen and it is a 400 bp species-specific DNA probe called F1 which is suitable for identification of Y. pestis species that bears the 60 mdal plasmid. The DNA probes called P1 was made on a basis of the plasmid pPst; it is the 460 BglII-BamHI fragment of the fibrinolysin-coagulase gene suitable for species-specific detection of Y. pestis species that bears the 60 mdal plasmid. The P1 fragment was cloned into the pAT153 vector and the constructed recombinant plasmid was called pEK7. The recombinant plasmid pCL1, including the pBR325 vector and the 6th BamHI fragment of Y. pestis EV plasmid pCad was constructed. The above fragment includes the replication origin of the pCad and it is hybridized to the pCad-bearing strains of Y. pestis and Y. tuberculosis only. Thus, it may be a basis for a bi-species-specific DNA probe making. These three recombinant plasmids are considered as a test-system for detection of both typical and atypical strains of Y. pestis.     

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.     

Isolation and properties of several auxotrophic mutants of a highly virulent strain of the plague microbe]

2432 stable auxotrophic mutants were selected from high virulent Yersinia pestis strain 20b after treatment with nitroso guanidine. They were deficient in amino acids (arginine, aspartic acid, citrulline, glycine, glutamic acid, histidine, isoleucine, serine, leucine, lysine, ornithine, proline, tryptophan, tyrosine, valiney, pyrimidine and vitamins (riboflavin, thyamine, nicotinamide). Some mutants were two- and three-fold dependent. The leucine-, histidine-, purine-dependent mutants were isolated with the high frequency. All the mutants, like their original strain, grew in R-form; they were sensitive to diagnostic phages, had pesticine-fibrinolysin-coagulase sustem (fraction I) and were calcium-dependent. P+ cultures of auxotrophs were not virulent for laboratory animals.     

Synergistic action of some antibacterial agents in studies on albino mice with experimental plague caused by Fr- phenotype strain of the plague microbe]

Possible use of ciprofloxacin combinations with some other antibiotics such as rifampicin, ampicillin, cefotaxime, doxycycline and amikacin was studied on albino mice with experimental plague caused by the pathogen strain (approximately 1000 LD50) deprived of the ability to produce the capsular antigen, fraction I (Fra- phenotype). The combination of ciprofloxacin with ampicillin or doxycycline had no effect on the increase of the survival rate (t<2) evident of inexpediency of its use in the infection caused by the Fra- strains of the plague microbe. The combination of ciprofloxacin and cefotaxime used in definite doses had some effect (t=2.6). The most significant synergistic effect was observed with the use of ciprofloxacin in combination with amikacin or rifampicin (t>3.3-9.0) which made the combination most promising.     

Morphological study of the damaging effect of an EB vaccine strain of the plague microbe in inbred mice

The data obtained during the study of the morphological changes in the internal organs and blood of 6 mouse strains at the early stages of anti-plague immunity are presented. It has been established that the introduction of 5.10(3) and 1.10(5) m. b. of the EV vaccine strain resulted in the development of morphological changes typical of residual virulence. The degree of these changes depended on the haplotype of the animals and the dose used. The most resistant to injury induced by EV strain were CBA mice (haplotype H-2k) and the most sensitive were C57BL/6 mice (haplotype H-2b).