Relevance. The broad use of antimicrobials by agriculture and consumers motivation to buy antibiotic-free production form a basis to development of substitutes to antimicrobials. The goal of research was the analysis of antimicrobial resistance caused by veterinary antimicrobials administration to broilers, as well as the effect of metaprobiotic “Probiocid-Ultra” on antimicrobial resistance in dynamic.Methods. Three groups of broiler chickens were formed: I — control, who received the main diet, II — experimental, who received veterinary antibiotics enrofloxacin and colistin in addition to the diet of group I, III — experimental, who received the metaprobiotic “Probiocide-Ultra” in addition to the diet of group II. Gene expression analysis was performed using quantitative reverse transcription PCR. With inoculation to different media the antimicrobial resistance was examined with classical microbiological methods.Results. The dynamic of accumulation of antimicrobial resistance determinants caused by enrofloxacin and colistin administration to broilers was examined. Under the influence of enrofloxacin the increase in antimicrobial resistance to enrofloxacin itself, as well as to beta-lactams, tetracycline and colistin was observed. Under the influence of colistin the increase in antimicrobial resistance to colistin itself, as well as to enrofloxacin and beta-lactams was observed. The feed administration with metaprobiotic “Probiocid-Ultra” promoted noticeable decrease of the antimicrobial resistance genes amount, when analyzed with growth medium led to antimicrobial resistant enterobacteria vanished.

Применение пробиотиков позволяет нормализовать состав микрофлоры и снизить патогенную нагрузку в рубце, что приводит к улучшению зоотехнических показателей коров. Эксперимент проводили на 2 группах дойных коров в течение 90 суток. Животные контрольной группы получали основной рацион (ОР), а опытной — ОР и испытуемую кормовую добавку на основе штамма микроорганизма Bacillus sp. и органических кислот. При скармливании дойным коровам кормовой добавки отмечено увеличение, в среднем за опыт, среднесуточного удоя молока 4%-ной жирности на 2,3 кг и снижение содержания соматических клеток в молоке на 60% (P≤0,05). Результаты, полученные методом T-RFLP, показали, что важным эффектом от действия препарата явилось уменьшение в рубце числа клостридий на 4,4% по сравнению с контролем (Р≤0,05). Также в опытной группе наблюдалось увеличение содержания целлюлозолитических бактерий — лахноспир и руминококков в 1,5 и 4,2 раза соответственно по сравнению с контролем (P≤0,05). The use of probiotics allows to normalize the composition of microflora and reduce pathogenic microorganisms in the rumen, which leads to zootechnical indicators of cows improvement. The experiment was carried out on 2 groups of dairy cows for 90 days. Animals in the control group received the basic diet (BD), while the animals in the experimental group received the BD and a tested feed additive based on the strain Bacillus sp. and organic acids. When dairy cows were fed the feed additive, the average daily milk yield of 4% fat milk increased by 2.3 kg and somatic cell content in milk decreased by 60% (P≤0.05). The results obtained by the T-RFLP method showed that a significant effect of the probiotic was a 4.4% decrease in the number of clostridia in the rumen compared to the control group (P≤0.05). Additionally, an increase in cellulolytic bacteria — Lachnospira and Ruminococcus by 1.5 and 4.2 fold, respectively, was observed in the experimental group compared to the control (P≤0.05).

ПОНОМАРЕВА 1 , А.В.ДУБРОВИН 1 , Н.И.НОВИКОВА 1 , Д.А.АХМАТЧИН 1 , В.В.МОЛОТКОВ 1 , В.Х.МЕЛИКИДИ 1 , Е

В условиях интенсивного животноводства организму высокопродуктивных коров не удается адаптироваться к метаболическим вызовам, возникающим вследствие перегрузки рубца концентратами. Эксперимент проводили на коровах голштинизированной черно-пестрой породы в течение 111 суток. Методом аналогов были сформированы 2 группы: контрольная, получавшая основной рацион (ОР), и опытная, получавшая ОР и натуральную комплексную кормовую добавку. Бактериальное сообщество рубца оценивали методом NGS-секвенирования. Результаты исследования показали, что применение кормовой добавки дополнительно к ОР позволило увеличить на 90-е сутки лактации среднесуточный удой на 1,6 кг по сравнению с контролем (P≤0,05). В период последействия (1 мес) среднесуточный удой увеличился на 3,2 кг молока по сравнению с продуктивностью коров в контрольной группе (P≤0,05). В результате проведенного NGS-секвенирования в составе микрофлоры рубца исследованных животных было обнаружено 20 филумов микроорганизмов. Самыми обильно представленными (P≤0,05) оказались бактерии филума Bacteroidetes — от 47,1±3,35 до 52,1±4,81%. Показано, что у коров в период раздоя возникают дисбиотические нарушения микрофлоры рубца, которые могут подвергнуть их высокому риску развития воспалительных и инфекционных заболеваний. Так, доля бактерий филума Proteobacteria возрастала (P≤0,05) в обеих группах в конце эксперимента по сравнению с исходными данными, а число Candidatus Saccharibacteria, наоборот, снижалось (P≤0,05). Доля некоторых видов и родов патогенных и условно-патогенных бактерий (Acinetobacter spp., некоторые виды Clostridium, в том числе C. colinum, Porphyromonas spp., Mycoplasma spp., Stenotrophomonas maltophilia) в образцах от коров из опытной группы к концу эксперимента была ниже, чем в образцах от особей из контрольной группы (P≤0,05). Так, например, общая доля рода Acinetobacter составляла 0,059±0,0049% в опытной группе и 0,15±0,009% — в контрольной. В условиях интенсивного ведения животноводства использование комплексной кормовой добавки помогает восстановить микробиом рубца, нарушенный высококонцентратным кормлением, что позволяет поддерживать молочную продуктивность на более высоком уровне, а также профилактирует инфекционные заболевания.

Background. Feed-borne T-2 toxin may inhibit innate immune system function in birds.The aim. To evaluate the effect of T-2 toxin, artificially introduced with feed, on the expression level of a number of immunity-related genes in the tissues of the broiler digestive system.Materials and methods. The experiments were carried out in the vivarium of the FSC “VNITIP” RAS broilers of the Smena 8 cross from 33 to 47-day old. Experimental contamination of feed T-2 toxin was performed. The birds were divided into 4 groups of 5 animals each: I – control, receiving a diet without the introduction of T-2 toxin, II experimental – receiving a diet with the addition of T-2 toxin, III experimental – receiving a diet with the addition of T-2 toxin and the sorbent Zaslon2+, IV experimental – receiving a diet with the addition of T-2 toxin, the same sorbent Zaslon2+and Axtra Pro enzyme. The level of mRNA expression was analyzed by quantitative reverse transcription PCR.Results. The data obtained indicated the impact of T-2 toxin contamination of broiler feed on the modulation of the level of expression of genes associated with the functioning of the immune system in the cecum and pancreas. Exposure to T-2 toxin (group II) led to an increase in the expression of the pro-inflammatory gene IL-6 in the tissues of the caecum by 10.8 times and IL-8 in the pancreas by 3.89 times (p ≤ 0.05) compared with control group I. The effect of the sorbent, as well as the complex, including the sorbent and the enzyme, on the expression of broiler genes was positive. The sorbent without the enzyme showed greater efficiency than with the additional introduction of the enzyme.

The adaptive ability of reindeer to the harsh conditions of the Russian Arctic is not determined solely by the genome of the macroorganism and, of course, includes an extensive genetic and metabolic repertoire of the microbiome. The aim. To compare the taxonomic and predicted metabolic profiles of the rumen microbiome of adult reindeer living in the natural pastures of the Yamalo-Nenets and Nenets Autonomous districts of the Russian Federation. Materials and methods. Expeditions to the Yamal-Nenets and Nenets Autonomous districts of the Russian Arctic in 2017 were carried out to take samples of the rumen. The contents of the rumen were taken from clinically healthy reindeer individuals (at least 3 times repetition). To analyze the animal scar microbiota and determine metabolic profiles, 16S rRNA NGS sequencing was performed on a MiSeq device (Illumina, USA). Bioinformatic data analysis was performed using QIIME2 software ver. 2020.8. The noise sequences were filtered by DADA2. Silva 138 reference database was used for taxonomy analysis. Reconstruction and prediction of the functional content of the metagenome was carried out using the software complex PICRUSt2 (v. 2.3.0). Results. During NGS sequencing, a total of 223 768 sequences of the 16S rRNA gene of the reindeer scarring microbiome were studied. Significant (p ≤ 0.05) differences between the groups in 10 bacterial phyla and superphyla were revealed: Actinobacteriota, Spirochaetes, Chloroflexi, Verrucomicrobia, Bdellovibrionota, Synergistetes, Fusobacteriota, Myxococcota, Cyanobacteria, Campilobacterota. The results of the reconstruction and prediction of the functional content of the metagenome using the PICRUSt2 bioinformatic analysis made it possible to identify 328 potential metabolic pathways. Differences between the groups were revealed in 16 predicted metabolic pathways, among which the pathways of chlorophyllide and amino acid biosynthesis dominated.

Косвенное воздействие гербицидов глифосатов на здоровье животных и птиц через изменения в их микробиомах могут оказаться не менее важными, чем прямое воздействие на физиологию макроорганизмов. Методом NGS-секвенирования показано, что глифосат, содержащийся в загрязненных кормах для бройлеров, даже в минимальных концентрациях, которые в несколько раз ниже уровней ПДК для кормов, при хроническом воздействии может негативно влиять на микробное сообщество слепых отростков кишечника. Бройлеров кросса Росс-308 разделили на 3 группы по 40 голов в каждой: I контрольная, II опытная, получавшая рацион с добавлением глифосата в количестве 20 мг/кг корма; III опытная, получавшая рацион с добавлением глифосата, а также пробиотического штамма микроорганизма Bacillus sp. ГЛ-8. Показано, что патогенные и оппортунистические микроорганизмы, которые, вероятно, менее чувствительны или даже нечувствительны к глифосату, могут увеличивать численность, вытесняя представителей нормофлоры. Так, в группе II, по сравнению с контролем, возрастало количество микроорганизмов семейств Staphylococcaceae - в 5,0 раз и Enterobacteriaceae - в 1,5 раза (P≤0,05). Введение в рацион пробиотика на фоне присутствия в кормах глифосата оказало позитивное влияние на разнообразие и численность микроорганизмов различных таксонов. Под влиянием пробиотика в группе III по сравнению с группой II происходило снижение численности таких клостридиальных кластеров, как Clostridium_III, Clostridium_IV, Clostridium_sensu_stricto Clostridium_XlVa, Clostridium_XlVb, Clostridium_XVIII (P≤0,05). Вероятно, это связано с антимикробной активностью штамма в составе пробиотика и/или присутствием генов биодеструции ксенобиотиков, что предполагает будущие исследования в данном направлении.

The use of biological products based on beneficial microorganisms that produce siderophores, which reduce the concentration of available to pathogensiron ions, can be successfully used in agriculture.The aim of our study was the sequencingand functional annotation of the genome of Bacillus subtilis strain from the collection of BIOTROFLtd to identify genes associated with the synthesis of siderophores.Nucleotide sequences were analyzed using a MiSeq instrument (Illumina, Inc., USA) together with a MiSeq Reagent Kit v3 (300-cycle) (Illumina, Inc., USA).Functional genome annotation was carried out using the PROKKA 1.12 (https://github.com/kbaseapps/ProkkaAnnotationhttp://vicbioinformatics.com/) and RAST 2.0 (https://rast.nmpdr.org)programs.The KEGG Pathway database was used to assess the pool of genes associated with antimicrobial activity and construct a metabolic map (http://www.genome.jp/kegg/).When carrying out bioinformatic processing of whole genome sequencing data of the B. subtilis strain, several gene clusters associated with the synthesis of siderophores were identified.Аlmost all genes necessary for the implementation of the process of iron binding with the participation of bacillibactin were found.A cluster of DhbA, DhbB, DhbC genes associated with the production of enzymes 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EC 1.3.1.28),isochorismatase (EC 3.3.2.1) and isochorismates synthase (EC 5.4.4.2), which are responsible for synthesis of bacillibactin precursor was found.The cluster of genes FeuA, FeuB, FeuC, and FeuD is associated with the synthesis of substrate-binding proteins of the iron transport system.In the genome of B. subtilis, genes associated with the synthesis of other siderophores were also found -enterochelin, described for E. coli and Campylobacter coli, alsomyxochelin A, first found in Angiococcusdisciformis.It is likely that B. subtilis bacteria can synthesize several synergistic siderophores to increase their competitiveness.In our opinion, there is a potential for using the B. subtilis strain in animal husbandry and plant growing as a basis for bacterial biopreparations -producers of siderophores, in order to reduce the pathogenic load.

Today, there is great interest in the development of environmentally friendly feed additives for poultry farming as a worthy alternative to antibiotics capable of positively modulating the microbiota to control pathogenic microorganisms. However, very few studies have been devoted to comparing the effects of probiotics and antibiotics on the structure of the gut microbiome in broilers. In this study, we compared the composition of the intestinal microbiota and zootechnical parameters in chickens of the Cobb 500 cross during the starter, growth and finishing periods when a probiotic (Bacillus subtilis in the composition of Cellobacterin®-T) or an antibiotic (Stafac® 110 based on virginiamycin) was added to the diet and showed that the B. subtilis strain accelerates the formation of intestinal microflora. The probiotic also reduces the number of microorganisms of the Campylobacteriaceae family which includes many types of gastroenteritis pathogens, and also increases the digestibility of fiber. T-RFLP analysis and qPCR method were used to assess changes in the intestinal microbiota of Cobb 500 broiler chickens fed a Bacillus subtilis-based dietary probiotic and virginiamycin-based dietary antibiotic Stafac® 110. On day 14, the total counts of cecal bacteria, as compared to control, were 9.1 times higher (p ≤ 0.05) in broilers fed Stafac® 110, and 54.2 times higher (p ≤ 0.001) when fed B. subtilis preparation. This indicates rapid microbial colonization of gastrointestinal tract of the chickens fed Stafac® 110 and B. subtilis. T-RFLP analysis revealed two dominant cecal phyla, Firmicutes and Proteobacteria, while phyla Actinobacteria, Bacteroidetes, and Fusobacteriawere less abundant. The taxa are detected which ferment non-starch polysaccharides to produce short-chain fatty acids, inhibit the competing pathogens due to production of bacteriocins, and acidize the chyme as synthesize organic acids. Administration of the dietary antibiotic mostly positively influences the cecal microbiota, e.g., the cellulolytic bacteria and Clostridia forms involved in the synthesis of organic acids became more abundant (p ≤ 0.05). Similar beneficial effects, e.g., an increase in Clostridia counts (p ≤ 0.05) compared to control, occurred when the probiotic strain was administered. On day 14 of rearing, the dietary antibiotic and probiotic reduced abundance of Campylobacteriaceae family comprising gastroenteritis pathogens (p ≤ 0.05) when compared to control. An increase in bodyweight as compared to control (from 1845.8±20.9 to 1936.4±17.9 g, p = 0.046) occurred in 36-day-old chickens fed Stafac® 110 but not the probiotic strain but not the probiotic strain, despite recovery of gut microbiota in the chickens fed B. subtilis. A 7.1 % increase infiber digestibility (p = 0.0027) occurred in broilers fed dietary probiotic and a 2.3 % increase (p = 0.047) in those fed the dietary antibiotic, which may be due to the action of cellulolytic microorganisms. Therefore, a dietary B. subtilis-based probiotic which promotes recovery of gut microbiota and increases fiber digestibility in feeds for broiler chickens can be an effective alternative to the virginiamycin-based antibiotic Stafac® 110.