No abstract

Within barrier facilities, autoclaved diet and bedding are used for husbandry of laboratory rodents. Bacillus spp. are ubiquitous in nature and some of them are known as probiotics. Inactivation of the Bacillus spores and reduction of the diet nutritional value due to autoclavation could be especially critical for immunodeficient mice. We studied the effect of the autoclaved and non-autoclaved diets on the reproductive performance and the age of prolapse manifestation in Muc2−/− mice with impaired gut barrier function and, therefore, sensitive to change of microbiota. We found that the non-autoclaved diet led to enhancement of the fertility index of Muc2−/− and Muc2+/− female mice. The non-autoclaved diet affected the prolapse of Muc2−/− mice that occurred later in comparison with females eating the autoclaved diet. We showed that Bacillus spp. was present in the non-autoclaved diet and feces of mice on the non-autoclaved diet. Bacterial strains of the non-autoclaved diet and feces belonged to B. amyloliquefaciens, B. thuringiensis, B. subtilis, Lysinibacillus macrolides, B. cereus, and other representatives of Bacillus spp. Moreover, autoclavation of the diet affected on the percent of the blood and spleen immune cells, the bacterial composition of the intestine, and increased the level of methionine in the thigh muscle of mice. Enhanced reproductive performance and delayed prolapse manifestation in Muc2−/− mice could be due to improved digestion, as Bacillus spp. from diet and feces had enzymatic activity.

The results of assessing the effect of Bacillus thuringiensis ssp. aizawai strain on fi ve species of lepidopteran pests (Lepidoptera) belonging to different families are presented. The mortality of larvae infected with B. thuringiensis strain varied signifi cantly depending on the species of insect. In four species: the cabbage white butterfl y (Pieris brassicae L.), the cabbage moth (Mamestra brassicae L.), the greater wax moth (Galleria melonella L.) and the black-veined white (Aporia crataegi L.), death of individuals was noted on the second day of the experiment. However, mortality rate was signifi cantly different between species and increased over time. The diamondback moth (Plutella xylostella L.) was more susceptible to the pathogen. Signifi cant mortality of the larvae of this phytophage was observed already on the fi rst day after infection; while on the second day of the experiment all individuals died. Lethal time LT90 of more resistant species (black-veined white) and more sensitive ones (diamondback moth) differed sevenfold. Semi-lethal concentrations of B. thuringiensis strain for the tested insect species were determined: on the third day of the experiment, lethal concentration LC50 for the cabbage moth larvae, the greater wax moth and blackveined white ranged from 1.7 to 4.5 × 108 spores/ ml. For the cabbage white butterfl y, it was much lower (1.8×107 spores/ml). For diamondback moth caterpillars, LC50 was 4.25×107 spores/ml already one day after infection. It was noted that the specifi city of B. thuringiensis strain is associated with various mechanisms of insect protection from pathogens, and is due to individual characteristics of the species. In particular, the pH of excrement of intact caterpillars of the cabbage white butterfl y, a species sensitive to B. thuringiensis, was 2 times higher than that of caterpillars of the greater wax moth – 8.9 and 4.3, respectively, which is one of the reasons for the susceptibility (or resistance) of the species to bacterial entomopathogen.

The development of the nutriculture medium composition and the optimal concentrations of its constituent components to increase the bacteriocin-like activity of the strain Bacillus thuringiensis ssp. Dakota are presented. The study was carried out using a multifactor experiment with further processing of statistical data in order to optimize the basic nutriculture medium and maximize the activity of the target product. Sources of nitrogen (peptone and yeast extract) and carbon (glycerin and glucose) were used as optimization factors. The control growth medium was medium "A", traditionally used for the cultivation of bacteria of the genus Bacillus. The degree of impact of the studied factors on the effective feature was determined using the model of multiple linear regression of the first order, whereby the optimal ratio of the components was calculated on the basis of the quadratic model. The ability of the Bacillus thuringiensis ssp. dakota strain to produce a bacteriocin-like substance (BLIS) was revealed. The dependence of BLIS synthesis on the cultivation medium was established: on the carbohydrate-free medium, the antimicrobial activity of BLIS was 1.5 times lower than that on the medium containing glucose and glycerin. To obtain maximum BLIS activity, the carbohydrate medium was optimized using a multifactor experiment performed by the method of orthogonal Latin rectangles. Mathematical models of linear regression of the first and second order were constructed depending on the concentration of nutrient medium components. The optimal concentration of the components was determined on the basis of a second-order regression model that takes into account the effects of the interaction of factors and the nonlinearity of the process. As a result of optimization of the nutrient medium, the quantitative composition of the components of the culture medium was determined: peptone – 9 g/l; yeast extract – 2.6; glycerol – 5.6 g/l, on which the antimicrobial activity of BLIS increased by 60% compared with the activity on the initial medium. It was shown that the synthesis of BLIS, unlike the synthesis of delta-endotoxin, is not regulated by catabolite repression of carbon.

The gut is inhabited by a trillion bacteria that produce up to 60% of the host’s metabolites. The gut microbiome plays an important role in regulating host immune function. A lot of research concerned the effect of probiotic on the pathologies associated not only with dysbiosis and metabolic disorders, but there is breakthrough in the treatment of inflammation, oncology and neurodegenerative disorders. Animals with mutation of the genes leading to pathology used to assay probiotic effect. To understand direct action of probiotics, cells derived from control mice or cell culture of tumor genesis in vitro studies are used. However, there is little research of the probiotic effect on cells derived from mice with pathology. In this study, we assessed the phenotypes of dendric cells derived from Muc2-/- mice with chronic inflammation and assessed the effect of L. johnsonii on the dendric cells. It is known that the key features of IBD models are thinning of mucin layer and changes in the intestinal microbiome. We compared the efficiency of maturation and activation of dendric cells derived from the bone marrow of Muc2-/- mice and dendric cells obtained from healthy C57BL/6 mice free from specific species pathogens. We evaluated the expression of co-stimulatory molecules, the proliferative index, and the ability to trigger the T regulatory response of dendric cells, which were stimulated with the probiotic L. johnsonii. Markers of dendritic and T cells were assessed by flow cytometry using antibodies to extra- and intracellular proteins. The proliferative activity of splenocytes was assessed using the WST test. It was shown that dendric cells derived from the Muc2-/- had an immature phenotype. Dendric cells of Muc2-/- mice could not effectively stimulate the proliferation of allogeneic and syngeneic T cells. L. johnsonii was able not only to stimulate the maturation of dendric cells derived from Muc2-/- mice, but also to increase the expression of FoxP3 on CD25+ T cell that were co-cultured with DCs. Thus, we believe that this probiotic bacterium can reduce signs of inflammation and reduce pathological processes in animals of an experimental model of IBD in vivo.