Metagenetic analysis of the bacterial diversity of Kazakh koumiss and assessment of its anti-Candida albicans activity.

Koumiss, a five-thousand-year-old fermented mare's milk beverage, is widely recognized for its beneficial nutrient and medicinal properties. The microbiota of Chinese and Mongolian koumiss have been largely characterized in recent years, but little is known concerning Kazakh koumiss despite this drink historically originates from the modern Kazakhstan territory. In addition, while koumiss is regarded as a drink with therapeutic potential, there are also no data on koumiss anti-Candida activity. In this context, the aims of the present study were to investigate the bacterial diversity and anti-Candida albicans activity of homemade Kazakh koumiss samples as well as fermented whey and cow's milk, derived from koumiss and propagated for several months. Koumiss bacterial communities were largely dominated by lactic acid bacteria including Lactobacillus sensu lato spp. (69% of total reads), Streptococcus (8.0%) and Lactococcus (6.1%), while other subdominant genera included Acetobacter (2.6%), Enterobacter (2.4%), and Klebsiella (1.5%). Several but not all koumiss samples as well as fermented whey and cow's milk showed antagonistic activities towards C. albicans. Linear discriminant effect size (LEfSe) analysis showed that their bacterial communities were characterized by a significantly higher abundance of amplicon sequence variants (ASV) belonging to the genus Acetobacter. In conclusion, this study allowed to identify the key microorganisms of Kazakh koumiss and provided new information on the possible underestimated contribution of acetic acid bacteria to its probiotic properties.

Antagonistic and protective activity of Lactobacillus plantarum strain 17 M against E. amylovora.

The subject of the study was to identify the antagonism of the Lactobacillus plantarum strain 17 M against the causative agent of fire blight, the bacterium Erwinia amylovora, and to evaluate its protective capabilities on apple blossoms against this disease. For comparison 9 strains of lactic acid bacteria from the LLP "SPC of Microbiology and Virology" collection were included in the study. Strain 17 M appeared to be superior in limiting the growth of the pathogen on all 3 liquid media tested. Its maximum inhibitory activity was proved on MRS Broth medium. The analysis of the secondary metabolites produced by strain 17 M in liquid medium revealed that it consisted mainly of acetic acid (53.2 ± 4.3%), lactic acid (16.3 ± 2.3%) and 2,3-butanedione (14.84 ± 4.1%). The presence of other organic compounds was also detected but in a smaller amount. Study on influence of those compounds on growth of E. amylovora showed that lactic acid at concentration of 5% showed inhibitory activity but it was not toxic to apple flowers. The effectiveness of strain 17 M culture liquid diluted with sterile water and added to E. amylovora inoculum at 10% or 20% was 76.7 ± 5.8% and 88.3 ± 12.6%, respectively. This study confirms the potential use of strain 17 M as an active microbial agent to combat fire blight of fruit crops in Kazakhstan.

Occurrence of Spot Blotch in Spring Barley Caused by Bipolaris sorokiniana Shoem. in South-Eastern Kazakhstan.

In Kazakhstan, barley (Hordeum vulgare L.) is the second most important cereal crop after wheat, with an annual production of approximately 1.9 million tons. The study aimed to characterize Bipolaris sorokiniana isolates obtained from barley fields surveyed. A total of 21 diseased leaves showing spot blotch symptoms were collected from experimental plots located close to the Kazakh Research Institute of Agriculture and Crop Production, where the spring barley Arna cultivar was planted in June 2020. The overall strategy for control of spring barley blotch in the Almaty region of Kazakhstan should include the determination of the aggressiveness of the pathogen isolates to better understand the biology of the diseases and ultimately proper control strategy. Pathogenicity of B. sorokiniana isolates was made on barley seedlings in vitro. Inoculated seedlings showed clear symptoms of B. sorokiniana, and therefore, Koch's postulates were fulfilled by reisolating the pathogen from artificially inoculated seedlings and identifying it based on standard morphology criteria. Further investigation is needed to understand the impact of B. sorokiniana on barley production in Kazakhstan.

Comparative Analysis of the Methanogen Microbiota Associated to Pasture and Stall Housing in Kazakh Cattle.

The microbial community of cattle rumen (archaea) are the key players in methane emissions. Methane pollutes the atmosphere and leads to the loss of feed energy. The aim of this study was to comparatively investigate the cattle microbiota, with a particular focus on archaea, in relation to the type of housing: pasture versus stall. A 16S metabarcoding analysis of the intestinal contents of cattle was carried out. Аlpha - diversity of grazing animals showed to be higher compared to animals in the stall period (p=0.002). Beta - diversity confirmed a difference in methanogens and microbiota between animals kept on pasture and those in a stall. Differential abundance analysis showed that the relative abundance of the Methanobacteriacea family in animals in the pasture period was significantly higher compared to animals in the stall period (FDR p = 0.00122). In conclusion, this study demonstrates that the concentration of methanogens in the fecal contents of animals during pasture period was higher than that in animals during the stall period. We recommend feeding grazing animals with concentrates in the evening in order to mitigate methane emissions.

Prokaryotic Diversity of Ruminal Content and Its Relationship with Methane Emissions in Cattle from Kazakhstan.

In this study, we analyzed the microbial composition of the rumen contents of cattle from Kazakhstan. Specifically, samples of the liquid and solid fractions of the rumen were collected to determine the quantitative and qualitative composition of methanogenic archaea. Cattle were six steers receiving hay-concentrate feeding. Methane emission was determined by repeated measurements for each animal. Rumen samples were then taken from fistulas and analyzed using 16S metabarcoding via Next-Generation Sequencing (NGS). The difference between the rumen fractions was investigated, resulting in differential distribution of the families Streptococccaceae, Lactobacillaceae, Desulfobulbaceae, and Succinivibrionaceae, which were more abundant in the liquid fraction, while Thalassospiraceae showed a higher presence in the solid fraction. These differences can be explained by the fact that fibrolytic bacteria are associated with the solid fraction compared to the liquid. A relationship between methane emission and methanogenic microbiota was also observed. Steers producing more methane showed microbiota richer in methanogens; specifically, most Mathanobacteriaceae resided in the liquid fraction and solid fraction of animals 1 and 6, respectively. The same animals carried most of the Methanobrevibacter and Methanosphaera genera. On the contrary, animals 2, 3, and 5 hosted a lower amount of methanogens, which also agreed with the data on methane emissions. In conclusion, this study demonstrated a relationship between methane emission and the content of methanogenic archaea in different rumen fractions collected from cattle in Kazakhstan. As a result of the studies, it was found that the solid fraction of the rumen contained more genera of methanogens than the liquid fraction of the rumen. These results prove that taking rumen contents through a fistula is more useful than taking it through a probe. The presented data may be of interest to scientists from all over the world engaged in similar research in a comparative aspect.

Study of cattle microbiota in different regions of Kazakhstan using 16S metabarcoding analysis.

Methane (CH4) is an important greenhouse gas (GHG). Enteric methane emissions from farmed ruminant livestock account for approximately 15% of global GHG emissions, with approximately 44% of livestock emissions in the form of methane. The purpose of the research is to study the influence of feeding types and regional characteristics of Kazakhstan on the microbiota of feces and the number of methane-forming archaea of beef and meat-and-dairy cattle productivity. For this purpose, fecal samples were taken rectally from 37 cattle heads from four regions of Kazakhstan (Western, Southern, Northern and Southeast). The taxonomic composition of the community in all samples was determined by 16S metabarcoding; additionally alpha and beta diversities were calculated. The dominant phyla were: Firmicutes (57.30%), Bacteroidetes (17.00%), Verrucomicrobia (6.88%), Euryarchaeota (6.49%), Actinobacteria (4.77%) and Patescibacteria (3.38%). Significant differences with regard to methanogens bacteria were found: Euryarchaeota were less present in animals from Western Kazakhstan (2.40%), while Methanobacteriales and Methanobrevibacter were prevalent in Southeast, and less abundant in Western region. Western Kazakhstan differs from the other regions likely because animals are mainly grazed in the pasture. Thus, grazing animals has an impact on their microbiota thus leading to a decrease in methane emissions.

Validation of suitable reference genes for quantitative expression analysis by qPCR in bovine terminal ileum and ileocecal valve.

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. These sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by Mycobacterium avium subsp. paratuberculosis, agent of paratuberculosis. We employed ten PCR assays for commonly used reference genes belonging to various functional classes and then determined their expression stability. The most stable genes were RPL13A and HMBS, followed by TFRC and B-ACT. NormFinder analysis provided similar results with B-ACT as the best reference gene, followed by RLP13A and TFRC. This validated gene panel may be useful for studies on paratuberculosis aiming to identify genes differentially expressed by qRT-PCR.

Polymorphisms associated to bovine paratuberculosis: investigation of their role in DNA-protein interactions and transcriptional regulation.

Previous studies led to identify SNPs in putative regulatory regions of the SLC11A1 and CARD15 genes with association to paratuberculosis in cattle. Aim of this study was to investigate the role of these mutations at the regulatory level by DNA-protein interaction analyses and transcriptome comparison between wild-type and mutated animals. Gene regions carrying the SNPs of interest were analysed by bioinformatic tools to predict allele-dependent binding sites for transcription factors (TFBS). Putative TFBS were in vitro explored by Electrophoretic Mobility Shift Assays (EMSA). EMSA did not show specific gel shifts for any allele indicating that these SNPs may eventually influence gene transcription without altering TFBS. Whole transcriptome expression analysis was performed on intestinal tissues of wild-type and mutated cattle by RNA-Seq. Differential regulation of five genes involved in innate immune system was detected. Specifically, ULBP3 was down-regulated, while S100A8, S100A12, LOC510860, and IFI27 were up-regulated. In previous studies, ULBP3, S100A8, and S100A12 resulted differentially expressed in cattle affected by paratuberculosis, suggesting a possible implication in the pathogen response. Further investigations are needed to elucidate the functional role of these SNPs and to understand the gene network involved in the interactions between non-coding SNPs and other genome regions.