PECULIARITIES OF INTERACTION BETWEEN CITELLOPHILUS TESQUORUM ALTAICUS (IOFF, 1936) FLEA AND YERSINIA PESTIS WITH VARIOUS PLASMID COMPOSITION

Objective of the study is in vitro investigation of mutual relations between Citellophilus tesquorum altaicus and Yersinia pestis with various plasmid composition: influence of the strain on flea alimentary activity and mortality rate, frequency and dynamics of biofilm formation.Materials and methods. C. tesquorum altaicus were infected by three Yersinia pestis strains: virulent triple-plasmid I-3230 isolated in Mongolia, referential for the Tuva focus I-2638 carrying four plasmids (pYT, pYV, pYP, pTP 33) and also selected from it avirulent isogenic clone I-3480 that lost two plasmids (pYV, pYP). Peculiarities of interaction between fleas and Y. pestis strains were estimated through the lens of specimens with «conglomerates» and “blocks” for feeding, the period from infection prior to the beginning of conglomerates’ formation, alimentary activity, and mortality rate of the infected fleas.Results and conclusions. It was revealed that alimentary activity of the infected insects was higher than that of the control group, and the highest – in fleas infected with I-2638 strain. Greater numbers of dead fleas at feeding was noted in specimens inoculated with I-3230 strain. Predominant significance of I-2638 strain was established in C. tesquorum altaicus biofilm formation both as «conglomerates» and “blocks”. I-3480 strain also formed the conglomerates in fleas more actively than I-3230 lacking pTP33 plasmid. Thus, four-plasmid I-2638 strain surpassed triple-plasmid I-3230 and two-plasmid I-3480 strains in reference to all tested indicators except flea mortality rates. It may testify to co-adaptation of Y. pestis and C. tesquorum altaicus from the Tuva plague focus and to the possibility of a pTP33 functional role in enhancement of a biofilm formation in vivo

Taxonomic Position of Entomoparasitic Nematodes Isolated from Fleas in Gorno-Altai High-Mountain Natural Plague Focus

Objective of the study was to investigate entomoparasitic nematodes from rodent fleas, collected in Gorno-Altai high-mountain natural plague focus in 2016–2017, with identification of their taxonomic status. Materials and methods. Given are the results of morphological and genetic analysis of entomoparasitic nematodes – parasites of fleas Amphipsylla primaris primaris living on flat-headed high-mountain voles Alticola strelzovi, fleas Rhadinopsylla li transbaicalica of Daurian pika Ochotona dauurica, fleas Rhadinopsylla dahurica, collected at the burrows of the rodents. Results and conclusions. Identified have been nucleotide sequences of ITS2 spacer region of ribosomal operon in entomoparasitic nematodes of three flea species: Amphipsylla primaris primaris, Rhadinopsylla li transbaicalica, and Rhadinopsylla dahurica. Based on comparative phylogenetic analysis and identified high percentage of homology of the stated nucleotide sequences (92–99 %), appurtenance to Rubzovinema spp. species and close relation to earlier proposed by us multi-host species Rubzovinema polyxenica from C. tesquorum, A. rossica, and C. secundus fleas from Volga-Ural steppe plague focus has been established. For the first time ever separate branch of evolution of entomoparasitic nematodes – flea parasites, represented by Rubzovinema ssp., has been identified. Determined has been wide spread of Rubzovinema ssp. nematodes in biocenoses of steppe zone of Russia across Precaspian territory and Altai Mountains

Optimization of cultivation medium for the production of Bacillus intermedius 3-19 glutamyl endopeptidase | Optimizatsiia sredy kul'tivirovaniia dlia produktsii glutamiléndopeptidazy Bacillus intermedius 3-19.

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase

Optimization of cultivation medium for the production of Bacillus intermedius 3-19 glutamyl endopeptidase

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase. © 2002 MAIK "Nauka/Interperiodica"

Synthesis and secretion of proteinases by Bacillus intermedius in the late stages of sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites

Synthesis and secretion of proteinases by Bacillus intermedius in the late stages of sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites

The Infestation of Fleas of the Long-Tailed Souslik with Entomoparasitic Nematodes in Tuva Mountain Natural Plague Focus

Entomoparasitic nematodes are supposed to be a link between parts of Yersinia pestis population in the environment and the flea vector. The aim of the study was to assess the prevalence and intensity of infestation in the fleas of the long-tailed souslik with entomoparasitic nematodes on the territory of Mongun-Taiginsky station in the Tuva natural plague focus. Materials and methods. Fleas were collected during the scheduled epizootiological surveys in 2019–2021. In the course of taxonomic identification the presence of parasitic nematodes was registered. In order to evaluate the intensity of nematode invasion, a total of 190 fleas were dissected. The number of adult parasitizing females and presence of larvae was recorded. Statistical processing of the data was performed with the help of conventional methods using the Excel software. The criterion χ2 was applied; the influence of various factors (species, gender of fleas) on the studied parameters was assessed through single- and two-factor analysis of variance. Results and discussion. During three years of observations, entomoparasitic nematodes were found in six species of fleas: Citellophilus tesquorum, Frontopsylla elatoides, Rhadinopsylla li transbaikalica, Frontopsylla hetera, Oropsylla alaskensis, and Neopsylla mana. The differences in infestation with nematodes between the species are presented. The highest invasion rate – 25.1–25.6 % – is observed in Rh. li transbaikalica. The gender of leas does not influence their infestation. It is established that invaded fleas are more often found in the nest than in the fur of animals, they are less actively migrate to the burrow entrance compared to not invaded ones. Evaluation of infestation prevalence has revealed that fleas Rh. li transbaikalica are the hosts for nematodes of mono- or oligoxenic species, which do not occur in other fleas

The role of neurohumoral factors in the persistence of aseptic bone inflammation in patients with diabetic neuroosteoarthropathy

BACKGROUND: Diabetic neuroosteoarthropathy (DNOAP, Charcot foot) is a relatively rare complication of diabetes mellitus (DM), which can lead not only to impaired support function of the lower limb in such patients, but also to high amputation. DNOAP is characterized by persistent aseptic inflammation of the bone structures of the foot, which creates significant ­difficulties in planning therapeutic measures. In the medical literature, there are data demonstrating the role of individual ­cytokines and neurohumoral factors in the prolongation of the inflammatory process in diabetes, however, there are currently very few studies that determine reliable markers of aseptic inflammation in DNOAP.AIM: To study the effect of neurohumoral factors and advanced glycation end products on the activity of aseptic inflammation in the bone structures of the foot in patients with type 2 diabetes mellitus (DM2) and diabetic neuroosteoarthropathy.MATERIALS AND METHODS. The study included 88 patients with type 2 diabetes (45 men, 43 women). Group 1 consisted of patients with DM2 and inactive DNOAP (n= 43), group 2 (n= 45) consisted of patients with DM2 and distal diabetic neuropathy without osteoarticular pathology. The diagnosis of diabetic neuropathy was based on the analysis of the clinical picture and indicators of peripheral sensitivity. Diagnosis of DNOAP and determination of its stage was based on clinical data, the results of infrared thermometry and radiology tests of the foot bones. General clinical assessment was used, radiology tests (X-ray, MRI), evaluation of CRP, calprotectin, copeptin, glutathione peroxidase 1 (GP1).RESULTS. According to the results of examination and palpation of the feet, as well as the analysis of the temperature gradient of the skin of the affected and contralateral limb (infrared thermometry), DNOAP was detected and the stage of this complication was determined. The diagnosis of the chronic stage of DNOAP was confirmed by the results of MRI and the clinical picture (no difference in skin temperature on the symmetrical areas of the feet). According to the results of laboratory analysis, a statistically significant difference in copeptin values was revealed — in group 1 — 0.232 µg/ml [0.147; 0.342], in group 2 — 0.115 µg/ml [0.065; 0.203] (p>0.05) and CRP — in group 1 — 7.113 mg/l [2.453; 16.505], in group 2 — 2.187 mg/l [1.131; 5.567] (p>0.05), leukocyte levels in the groups did not differ significantly: group 1 — 7.86 [6.40; 9.00]*10^9, group 2 — 7.00 [6.00; 8.15] (p>0.05). There was a trend towards an increase in the level of calprotectin and glutathione peroxidase-1 in the DNOAP group, however, the differences were not significant. calprotectin — in group 1 — 1.948 [1.229; 2.969], in group 2 — 1.692 [1.16; 2.514] μg/ml and glutathione peroxidase-1 in group 1 — 24.72 [20.1; 31.82], in group 2 — 22.98 [18.94; 31.2] ng/ml.CONCLUSION. In the study, statistically significant differences were obtained in the levels of copeptin and C-reactive protein: in patients with DNOAP, their values were significantly higher, which indicates the persistence of the aseptic inflammatory process in the bone tissue of patients even in the chronic stage of DNOAP. These data may help in deciding whether to use one or another method of unloading the affected joints, which will affect the clinical prognosis. The study of neurohumoral markers of arthropathy in the blood serum of patients with DM2 is carried out for the first time, and therefore it is difficult to compare with the results of other authors. It can be assumed that copeptin and CRP are significant markers of persistent inflammation of the osteoarticular structures of the foot in DNOAP

Immunohistochemical features of the bone tissue of the lower extremities in patients with type 2 diabetes mellitus

Background: Diabetic neuroosteoarthropathy is a serious disabling complication of diabetes mellitus, which, in the absence of timely correct treatment, can lead to high amputations of the affected limb. At present, the reasons and mechanism of the development of Charcot’s foot are not completely clear. It is extremely important to determine the pathophysiological mechanisms of DNOAP formation and to search for reliable markers-predictors of this pathology.Aim: To study the immunohistochemical characteristics of the bone tissue of the lower extremities in patients with diabetic neuroosteoarthropathy in comparison with patients with diabetes mellitus without this pathology.Materials and methods: During the foot surgery, a bone fragment of the foot was harvested for immunohistochemical study of receptor markers for PINP, PIIINP, and RAGE in the group of patients with DNOAP compared with the control group.Results: The study included 20 patients with type 2 diabetes mellitus and were divided into 2 groups: 10 patients with DNOAP made up group 1, 10 patients without DNOAP — group 2.Patients in both groups were comparable in AGE, experience with type 2 diabetes, and glycemic control.During the immunohistochemical study, a significant increase in the staining intensity of receptor markers for PINP, PIIINP, and AGE was recorded in the group of patients with DNOAP compared with the control group (p <0.05).Conclusion: For the first time, an immunohistochemical study of markers of bone resorption and AGE was carried out in persons with DNOAP. The results obtained indicate impaired collagen formation and, as a consequence, impaired bone formation and bone resorption in patients with DNOAP: in group 1, a statistically significant increase in the expression of PINP, PIIINP, and RAGE was revealed

Characteristics of an antioxidant activity of Yersinia pestis with different plasmid spectrum

Experimental data concerning complex study of antioxidant activity of Y. pestis with different plasmid spectrum (wild-type Yersinia pestis subsp. pestis, Yersinia pestis subsp. altaica and their isogenic variants) are represented in the article. Superoxide dismutase activity of the tested Y. pestis strains was from 6.0 to 9.0 x 109 microbe cells. Significant differences between the Y. pestis strains with different plasmid composition were not detected by this parameter. Our results, consistent with the data of other authors, tell that superoxide dismutase activity is a thermo-inducible feature and does not depend on a plasmid spectrum. High peroxide destroying activity was also detected in all tested Y. pestis strains. The differences between the strains regarding their common peroxide destroying activity were found. This parameter of the plague microbe strains lacking pYP plasmid was at least 3 times lower than common peroxide destroying activity in strains with this plasmid in the genome. In our opinion these revealed differences were caused by characteristics of plasmid spectrum. The common peroxide destroying activity's degree of Y. pestis strains can be associated with the presence of pYP plasmid in the genome. The isogenic variants of Y. pestis strains lacking one of the plasmids had smaller pathogenic activity. This fact points to the need for further study of these strains