Screening method of key bacteria influencing poultry grain-saving character, probiotics and application

Abstract

The invention belongs to the field of biology, and discloses a method for screening key bacteria influencing the grain-saving character of poultry, which comprises the following steps: step 1, constructing two poultry groups with different feed utilization rates, one group being named as a low feed utilization rate group, and the other group being named as a high feed utilization rate group; step 2, preparing a bacterial liquid by taking excrement of the high-feed-utilization group as a raw material, and implanting the bacterial liquid into intestinal tracts of the low-feed-utilization group through an excrement transplantation technology to construct an excrement transplantation group; step 3, taking the information of the cecum microbiota of the low feed utilization rate group and the information of the IgA + microbiota as contrasts, and screening to obtain first difference information and second difference information; and finding out coexisting bacteria from the first difference information and the second difference information, wherein the coexisting bacteria are the key bacteria influencing the poultry feed utilization rate. According to the method disclosed by the invention, a high / low feed utilization rate group and an excrement transplantation group are constructed, and cecum microbiome detection and IgA + microbiome detection are adopted, so that key bacteria playing an obvious role in the poultry grain-saving character can be quickly and accurately screened; meanwhile, the invention also discloses a key bacterium and application thereof.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a screening method for key bacteria affecting poultry grain-saving traits, probiotics and uses. Background Art

[0002] Feed costs account for about 70% of the total cost of poultry production, and low feed utilization is one of the bottlenecks restricting the development of my country's poultry industry. As the second genome of the host, intestinal microorganisms can affect the feed utilization efficiency of poultry by participating in nutrient metabolism, but its effect and mechanism are still unclear.

[0003] In the prior art, most of the strains that can affect poultry feed utilization are screened from a single perspective, such as analyzing the dominant flora in the intestines of a certain type of chicken to further obtain strains. However, this screening method has the problems of low efficiency and poor accuracy.

[0004] In the prior art, some people use biomolecular technology for screening, such as publication number CN115725709A, the subject of which is a method for using 16S rRNA gene sequencing technology to detect the intestinal flora of Licha black pigs to assist breeding. It uses 16SrRNA gene sequencing technology to detect the intestinal flora of Licha black pigs for assisting breeding, which includes: extracting and amplifying the total DNA of Licha black pig feces, building a library and sequencing, and analyzing the relative abundance information of Prevotella, Treponema, Ruminococcus and Lactobacillus related to feed utilization and the α-diversity index of intestinal microorganisms to assist Licha black pig breeding. This method also screens a single-state population and has certain limitations.

[0005] Therefore, the technical problem solved by the present invention is: how to screen key bacteria that affect poultry's grain-saving traits. Summary of the invention

[0006] The purpose of the present invention is to provide a method for screening key bacteria for poultry grain-saving traits. The method of the present invention constructs a high / low feed utilization rate group and a feces transplantation group, uses cecal microbiome detection and IgA + Microbiome testing can quickly and accurately screen out key bacteria that play a significant role in poultry's grain-saving traits.

[0007] At the same time, the invention also discloses a key bacterium and a use thereof.

[0008] To achieve the above objectives, this application discloses:

[0009] A method for screening key bacteria affecting poultry grain-saving traits comprises the following steps:

[0010] Step 1: construct two groups of poultry with different feed utilization rates, one of which is named the low feed utilization rate group and the other is named the high feed utilization rate group;

[0011] Step 2: Use the feces of the high feed utilization group as raw material to prepare bacterial liquid, and use fecal transplantation technology to implant the bacterial liquid into the intestines of the low feed utilization group to construct a fecal transplant group;

[0012] Step 3: Obtain information on the cecal microbiota at the genus level in the low feed utilization group, high feed utilization group, and fecal transplant group, as well as IgA at the genus level in the cecal microbiota in the low feed utilization group, high feed utilization group, and fecal transplant group + Information about the microbiome; the IgA + The microbiome is a microbiome that can bind to IgA;

[0013] Information on cecal microbiota and IgA in the low feed utilization group + The information of the microbiome is used as a control to screen and obtain the first difference information and the second difference information;

[0014] Among them, the first difference information is the information difference of the cecal microbiota between the high feed utilization group and the low feed utilization group, IgA + The diversity of microbiome information is aggregated;

[0015] The second difference information is the difference in information of cecal microbiota between the fecal transplant group and the low feed utilization group, IgA + The diversity of microbiome information is aggregated;

[0016] The bacteria that exist simultaneously in the first difference information and the second difference information are the key bacteria that affect the utilization rate of poultry feed.

[0017] Immunoglobulin A (IgA) is the most secreted antibody in the intestine, and plays an important role in microbial colonization and maintaining intestinal homeostasis. On the one hand, intestinal IgA can directly bind to symbiotic bacteria to promote their colonization in the intestine, and on the other hand, it can encapsulate pathogens to promote their removal from the intestine. The present invention uses humans and mice as research objects, and finds that IgA can regulate the composition of intestinal flora, affect microbial and host functions, maintain metabolic homeostasis, etc. by binding to microorganisms. Recent studies have found that in malnourished children, the binding of IgA to microorganisms changes, resulting in adaptive changes in the host's utilization of nutrients. Therefore, from the perspective of IgA binding to microorganisms, a new idea is provided for screening probiotics that improve the utilization rate of livestock and poultry feed.

[0018] Over the past decade, we have used residual feed intake (RFI) as a breeding index and successfully constructed a genetic resource population of Huiyang bearded chickens with significant differences in feed utilization through 15 generations of continuous breeding selection, providing a unique biological resource for studying the effects of intestinal microorganisms on feed utilization. The present invention systematically compared the developmental laws and composition differences of the cecal microbiome and IgA-bound microbiome of chickens with high feed utilization efficiency (H-FE) and low feed utilization efficiency (L-FE); clarified the effects of intestinal microorganisms on feed utilization efficiency traits through fecal microbial transplantation (FMT); integrated the information of cecal microorganisms and IgA-bound microbiome, and accurately excavated Blautia as a key candidate bacterium affecting feed utilization efficiency traits; further verified its effect on improving feed utilization in chickens and mice; finally, combined with in vitro cell and microbial co-culture experiments, the mechanism of action of the bacteria in promoting B cell activation to produce IgA was preliminarily analyzed.

[0019] The research of the present invention provides a new idea for exploring the core microorganisms for improving the feed utilization efficiency of chickens from the perspective of IgA binding microorganisms.

[0020] In the above method, the method for obtaining the information of the IgA+ microbiome is: determining it through the IgA-SEQ detection method.

[0021] In the above method, the poultry is one of chicken, duck and goose.

[0022] In the above method, the poultry is bearded chicken.

[0023] In the above method, in step 2, after 5 to 35 days of infusion of bacterial solution, a successfully constructed fecal transplant group is obtained.

[0024] At the same time, the present invention also discloses a key bacterium that affects the grain-saving trait of poultry, which is obtained by screening using any of the above methods.

[0025] Finally, the present invention also discloses the use of probiotics of the genus Blautia, especially Blautia coccoides, in preparing probiotics for improving the utilization rate of poultry feed.

[0026] This application has at least the following beneficial effects:

[0027] 1. Based on the long-term selection of high / low feed utilization genetic resource populations, the present invention systematically compares the developmental laws and composition differences of intestinal microorganisms and IgA-binding microorganisms of the two chicken groups; through fecal microbial transplantation (FMT) technology, it is clarified that intestinal microorganisms are an effective strategy to improve chicken feed utilization; integrating the cecal microbiome and IgA-binding microbiome information, accurately mining the key candidate bacteria Blautia that affects chicken feed utilization, and preliminarily analyzing its mechanism of action that may regulate feed utilization by promoting B cell activation and secretion of IgA. The present invention provides an important reference for mining the core microorganisms that regulate the traits of chicken feed utilization efficiency from the perspective of IgA-binding microorganisms.

[0028] 2. By integrating the cecal microbiome and IgA combined microbiome information, the present invention accurately screened out Blautia as a key candidate bacterium for improving feed utilization efficiency traits, and further revealed the potential mechanism of action of the bacterium in improving chicken feed utilization by inducing B cell activation and IgA production through animal experiments and in vitro cell and microbial co-culture experiments. In summary, the present invention provides an important reference for accurately screening core strains for improving livestock and poultry feed utilization efficiency traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A The feed conversion rate diagrams of the low feed utilization rate group and the high feed utilization rate group were tested from 49 to 70 days of age (n≥70), and the statistical significance was analyzed by unpaired two-tailed Student's t test;

[0030] Figure 1B The average daily feed intake of the low feed utilization rate group and the high feed utilization rate group was tested from 49 to 70 days of age (n≥70). The statistical significance was analyzed by unpaired two-tailed Student's t test.

[0031] Figure 1C Shannon diversity diagrams of low feed utilization group and high feed utilization group at different ages, Shannon diversity of cecal microbiome (n≥9);

[0032] Figure 1D Table of bacterial abundance of Gram-negative bacteria, potential pathogenic Gram-positive bacteria, and stress-tolerant bacteria in the low feed utilization group. Red indicates a higher relative abundance in L-FE chickens, while blue indicates a higher relative abundance in H-FE chickens.

[0033] Figure 1E This is a flow chart of fecal microbial transplantation (FMT) trials (n≥15);

[0034] Figure 1F Feed conversion rate diagrams of the low feed utilization rate group, high feed utilization rate group, and feces transplantation group;

[0035] Figure 1G This is the weight gain chart of the low feed utilization group, high feed utilization group, and fecal transplant group;

[0036] Figure 1H Prediction of cecal microbiome and IgA in low feed utilization group, high feed utilization group, and fecal transplant group for BugBase + Relative abundance plots of Gram-negative bacteria and potential pathogens in the microbiome;

[0037] Fig. 1I The figure is the result of Spearman correlation analysis between the relative abundance of IgA-binding bacteria Blautia and FCR;

[0038] Figure 2A The body weight of chickens at day 70 in the low feed utilization rate group, high feed utilization rate group, and Blautia gavage group;

[0039] Figure 2B The feed intake graphs of the low feed utilization rate group, high feed utilization rate group, and Blautia gavage group from 49 to 70 days of age;

[0040] Figure 2C This is the feed conversion rate chart of the low feed utilization rate group, high feed utilization rate group, and Blautia gavage group from 49 to 70 days of age;

[0041] Figure 2D Detection of IgA in chicken cecal contents by flow cytometry + proportion of bacteria (n=15);

[0042] Figure 2E Flow cytometry was used to detect CD19 in mouse mesenteric lymph nodes. + B cell ratio result diagram (n=15);

[0043] Figure 2F Flow cytometry was used to detect IgA in mouse mesenteric lymph nodes + Cells, IgA + Cells and CD19 + IgA + Cell ratio result diagram (n=15);

[0044] Figure 2G Flow cytometry was used to detect the expression of CD19 in B cell lines after 3 hours of co-culture with Blautia coccoides in vitro - CD138 + Proportion of plasma cells (n=6);

[0045] Figure 2HFlow cytometry was used to detect IgA in B cell lines after co-culture with Blautia coccoides for 3 hours in vitro + Proportional graph of cells (n=6);

[0046] Fig.2I The expression level of Blimp1, a gene related to B cell activation and IgA secretion, after 3 hours of co-culture (n=6);

[0047] Figure 2J The expression level of Xbp1s, a gene related to B cell activation and IgA secretion, after 3 hours of co-culture (n=6);

[0048] Figure 2K The expression level of Igha1, a gene related to B cell activation and IgA secretion, after 3 hours of co-culture (n=6);

[0049] Figure 2L The concentration of IgA in the supernatant after co-culture was determined using enzyme-linked immunosorbent assay (ELISA); * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001;

[0050] Figure 3A This is the OTU level diagram of cecal microorganisms at different ages in the low feed utilization group;

[0051] Figure 3B This is the OTU level diagram of cecal microorganisms at different ages in the high feed utilization group;

[0052] Figure 3C The common and unique cecal microbial OTU levels of the high feed utilization group and the low feed utilization group at different ages;

[0053] Figure 3D The observed species diversity diagrams for the low feed utilization group and the high feed utilization group at different ages;

[0054] Figure 3E Chao index diversity diagram of low feed utilization rate group and high feed utilization rate group at different ages;

[0055] Figure 3F The bar graphs show the composition of cecal microbial phyla at different ages in the low feed utilization rate group and the high feed utilization rate group;

[0056] Figure 3G The bar graphs show the composition of cecal microorganisms at the genus level in the low feed utilization rate group and the high feed utilization rate group at different ages.

[0057] Figure 4AThe relative abundance diagram of lipopolysaccharide biosynthesis-related microorganisms in the low feed utilization group and the high feed utilization group at 1 day and 70 days of age;

[0058] Figure 4B This is the principal component analysis diagram of cecal microbial functions in the low feed utilization group and the high feed utilization group predicted by PICRUSt at 70 days of age;

[0059] Figure 4C This is the difference analysis diagram of cecal microbial functions between the low feed utilization group and the high feed utilization group predicted by PICRUSt at 70 days of age;

[0060] Figure 5A The observed species diversity diagram of IgA-bound microorganisms in the low feed utilization group and the high feed utilization group at different ages;

[0061] Figure 5B The principal component analysis diagram of IgA binding microorganisms in the low feed utilization rate group and the high feed utilization rate group at different ages;

[0062] Figure 5C The bar graphs show the composition of IgA combined with intestinal microbial phylum levels in the low feed utilization rate group and the high feed utilization rate group at different ages;

[0063] Figure 5D The relative abundance of potential pathogens in the cecal microbiome and IgA-bound microbiome of the low feed utilization group, high feed utilization group and different age groups were analyzed for BugBase;

[0064] Fig. 6A This is the cecal microbiome diversity analysis diagram of the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0065] Figure 6B This is the IgA-bound microbiome diversity analysis diagram for the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0066] Figure 6C This is the composition diagram of cecal microorganisms at the phylum level in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0067] Fig.6D This is the IgA-binding microbial phylum level composition diagram of the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0068] Fig. 6E The genus composition of cecal microorganisms in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0069] Fig. 6FFigure 2 shows the composition of IgA-bound microorganisms at the genus level in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0070] Fig. 7A Screening of common key candidate bacteria in the cecal microbiota of the high feed utilization group and fecal transplantation group for linear discriminant analysis;

[0071] Figure 7B Screening of common key candidate bacteria in IgA-binding microorganisms of high feed utilization group and fecal transplantation group for linear discriminant analysis;

[0072] Fig. 8A Relative abundance diagram of common key candidate bacteria screened for the cecal microbiome;

[0073] Figure 8B Relative abundance diagram of common key candidate bacteria screened for IgA binding microbiome;

[0074] Fig.9A This is the area diagram of the bursal lymph follicles in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0075] Fig. 9B The bursal Bu-1 of the low feed utilization group, high feed utilization group, and fecal transplantation group + B cell ratio chart;

[0076] Fig. 9C Flow chart of the experiment to verify the efficacy of the key candidate bacterium Blautia coccoides in chickens;

[0077] Fig.9D Flow chart of the experiment to verify the efficacy of the key candidate bacterium Blautia coccoides in C57BL / 6j mice;

[0078] Fig.9E This is the weight of mice after oral administration of the key candidate bacteria Blautia coccoides;

[0079] Fig.9F This is the feed conversion rate diagram of mice after oral administration of the key candidate bacteria Blautia coccoides;

[0080] Figure 9G This is the ratio of IgA tuberculosis bacteria in the cecum of mice after oral administration of the key candidate bacterium Blautia coccoides;

[0081] Fig. 10A This is a graph showing the cell activity of primary chicken B cells after they were co-cultured with Blautia coccoides in vitro for 3 hours;

[0082] Fig. 10B IgA was produced after chicken primary B cells were co-cultured with Blautia coccoides for 3 hours in vitro + Proportional diagram of B cells;

[0083] Fig. 10C This is a graph showing the concentration of IgA in the supernatant after chicken primary B cells were co-cultured with Blautia coccoides for 3 hours in vitro.

[0084] Specific embodiment

[0085] Below in conjunction with embodiments of the present invention, the present invention is clearly and completely described. In the description of the present invention, it should be noted that, in the embodiments, those not indicating specific conditions, are carried out according to the conditions of normal conditions or manufacturer's suggestions. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained by commercial purchase. In the case of not making special instructions, the parts used in the embodiments of the present invention are all parts by weight.

[0086] Part I Construction of low feed utilization group and high feed utilization group

[0087] Feed conversion rate (FCR) is one of the key indicators to measure feed efficiency. After 15 generations of breeding and selection, the FCR and average daily feed intake (ADFI) of low feed efficiency (L-FE) chickens of the present invention were significantly higher than those of high feed efficiency (H-FE) chickens (p<0.001, Figure 1A , Figure 1B ), the group composed of the above L-FE chickens is a low feed utilization group, and the group composed of the above H-FE chickens is a high feed utilization group.

[0088] Figure 1A The feed conversion rate diagrams of the low feed utilization rate group and the high feed utilization rate group were tested from 49 to 70 days of age (n≥70), and the statistical significance was analyzed by unpaired two-tailed Student's t test;

[0089] Figure 1B The average daily feed intake of the low feed utilization rate group and the high feed utilization rate group was tested from 49 to 70 days of age (n≥70). The statistical significance was analyzed by unpaired two-tailed Student's t test.

[0090] Among them, there were 117 L-FE chickens in the low feed utilization rate group and 77 H-FE chickens in the high feed utilization rate group.

[0091] The cecal contents of L-FE chickens and H-FE chickens at different growth stages were sequenced by 16S rRNA.

[0092] At the five sampling time points, there were 284 and 233 operational taxonomic units (OTUs) in L-FE and H-FE chickens, respectively ( Figure 3A , Figure 3B ).

[0093] The number of OTUs shared by the two groups increased with age ( Figure 3C ), and α diversity increased with age from 1 to 49 days of age. From the 49th day of age, the Chao index and the number of observed species remained relatively stable. Among them, the number of observed species and Chao index of L-FE chickens at 1 day of age were significantly higher than those of H-FE chickens ( Figure 3D , E), and at 9 days of age, the Shannon diversity (a commonly used indicator for measuring biodiversity, which combines information on species richness and species evenness) of L-FE chickens was also higher than that of H-FE chickens ( Figure 1C ). However, at 49 and 70 days of age, the Shannon diversity of H-FE chickens was significantly higher than that of L-FE chickens ( Figure 1C ).

[0094] Figure 1C Shannon diversity diagrams of low feed utilization group and high feed utilization group at different ages, Shannon diversity of cecal microbiome (n≥9);

[0095] The microbial composition changed with age and showed significant differences between high and low lines ( Figure 3F , Figure 3G ). Firmicutes was the only dominant phylum at 1 and 9 days of age, while the relative abundance of Bacteroidetes increased significantly from 49 to 140 days of age and subsequently became the first dominant phylum ( Figure 3F It is worth mentioning that the relative abundance of Proteobacteria (Gram-negative bacteria, including many pathogenic bacterial genera) in 1-day-old L-FE chickens was significantly higher than that in H-FE chickens (4.2% vs. 0.0059%, p = 0.0001).

[0096] The phenotypes of microorganisms were predicted by BugBase, and the results showed that L-FE chickens had a higher relative abundance of Gram-negative bacteria and potential pathogens. Conversely, H-FE chickens showed a higher abundance of Gram-positive bacteria and stress-tolerant bacteria ( Figure 1D ). The results of microbial function prediction based on PICRUSt showed that the relative abundance of bacteria related to lipopolysaccharide (LPS) biosynthesis in the cecum of L-FE chickens was significantly higher than that in H-FE chickens. LPS is the main component of the outer membrane of most Gram-negative bacteria and can induce host inflammatory responses ( FIG. 4A to FIG. 4C The relative abundance of Gram-negative bacteria and LPS biosynthesis-related bacteria in L-FE chickens was higher, suggesting that their intestinal flora may be disturbed.

[0097] Figure 1D Table of bacterial abundance of Gram-negative bacteria, potential pathogenic Gram-positive bacteria, and stress-tolerant bacteria in the low feed utilization group. Red indicates a higher relative abundance in L-FE chickens, while blue indicates a higher relative abundance in H-FE chickens.

[0098] Figure 3A This is the OTU level diagram of cecal microorganisms at different ages in the low feed utilization group;

[0099] Figure 3B This is the OTU level diagram of cecal microorganisms at different ages in the high feed utilization group;

[0100] Figure 3C The common and unique cecal microbial OTU levels of the high feed utilization group and the low feed utilization group at different ages;

[0101] Figure 3D The observed species diversity diagrams for the low feed utilization group and the high feed utilization group at different ages;

[0102] Figure 3E Chao index diversity diagram of low feed utilization rate group and high feed utilization rate group at different ages;

[0103] Figure 3F The bar graphs show the composition of cecal microbial phyla at different ages in the low feed utilization rate group and the high feed utilization rate group;

[0104] Figure 3G The bar graphs show the composition of cecal microorganisms at the genus level in the low feed utilization rate group and the high feed utilization rate group at different ages.

[0105] Figure 4A The relative abundance of microorganisms related to lipopolysaccharide biosynthesis in the low feed utilization group and the high feed utilization group at 1 day and 70 days of age;

[0106] Figure 4B This is the principal component analysis diagram of cecal microbial functions in the low feed utilization group and the high feed utilization group predicted by PICRUSt at 70 days of age;

[0107] Figure 4C This is an analysis of the differences in cecal microbial functions between the low feed utilization group and the high feed utilization group predicted by PICRUSt at 70 days of age.

[0108] Part II IgA + Determination of the microbiome

[0109] IgA binding (IgA) in high / low feed utilization chickens was determined using IgA-SEQ + ) microbiome.

[0110] The IgA-SEQ assay method is to use fluorescence activated cell sorting (FACS) technology to sort IgA-binding bacteria in the cecal samples of high / low feed utilization chickens and collect IgA + The microorganisms were analyzed by 16S rRNA sequencing.

[0111] The results showed that IgA + Microbiome α diversity increased with age. At 140 days of age, IgA + The observed species of microorganisms were significantly higher than those in L-FE chickens ( Figure 5A ). PCA results showed that IgA in chickens with high / low feed utilization + The microbial composition was significantly different ( Figure 5B ).

[0112] Very importantly, IgA + The abundance of Firmicutes is not entirely determined by their relative abundance in the cecal microbiota. For example, at 49 days of age, Firmicutes are the most abundant IgA + The first dominant phylum of microorganisms, and the dominant bacteria of cecal microorganisms are Bacteroidetes ( Figure 5C In addition, at 49 and 70 days of age, the relative abundance of potential pathogens in the cecal microbiota of L-FE chickens was higher, and IgA + However, at 140 days of age, although there were no significant differences in potential pathogens in the cecal microbiota between high and low feed utilization chickens, IgA + The relative abundance of potential pathogens was significantly higher in L-FE chickens ( Figure 5D ).

[0113] Figure 5A The observed species diversity diagram of IgA-bound microorganisms in the low feed utilization group and the high feed utilization group at different ages;

[0114] Figure 5B The principal component analysis diagram of IgA binding microorganisms in the low feed utilization rate group and the high feed utilization rate group at different ages;

[0115] Figure 5C The bar graphs show the composition of IgA combined with intestinal microbial phylum levels in the low feed utilization rate group and the high feed utilization rate group at different ages;

[0116] Figure 5DThe relative abundance diagrams of potential pathogens in the cecal microbiome and IgA-binding microbiome of the low feed utilization group, high feed utilization group and different age groups were analyzed for BugBase.

[0117] Part III Screening of key bacteria affecting poultry grain-saving traits

[0118] 3.1 Construction of fecal transplantation group

[0119] In order to clarify the regulatory effect of intestinal microorganisms on feed utilization efficiency, this part collected feces from H-FE chickens to prepare bacterial liquid, and conducted FMT experiments on L-FE ( Figure 1E ).

[0120] The method for constructing the fecal transplant group is as follows: fecal samples for fecal microbiota transplantation (FMT) were collected from the high feed utilization group. Fresh feces were obtained in the morning, the white part of the feces was removed, and it was stored in liquid nitrogen. These samples were mixed evenly with 0.9% sterile saline in a volume ratio of 1:2, and then filtered through sterile gauze. Next, sterile glycerol was added to the suspension at a ratio of 1:9 (1 ml of sterile glycerol was added to 9 ml of suspension), and the resulting microbial suspension was stored at -80°C for subsequent use. Before use, the stored suspension was thawed and methylene blue staining was used to count the viable bacteria. The suspension was then diluted with sterile saline to a concentration of 1×10 3 ~1×10 8 Colony-forming units (CFU) / ml. Fecal microbial transplantation was performed on one-day-old chicks with low feed efficiency (the low feed efficiency resource group obtained through 15 generations of breeding in the first part). Chickens in the fecal microbial transplantation group were continuously or intermittently gavaged with 0.1-1 ml of fecal microbial suspension (1×10 3 ~1×10 8 CFU / mL) were administered orally for 5 to 35 days, and body weight gain and feed intake were recorded from day 49 to day 70 to calculate feed conversion rate (FCR).

[0121] The results showed that FMT significantly improved feed utilization (reduced FCR value) and growth performance (weight gain) of L-FE chickens ( Figure 1F , Figure 1G ), demonstrating that microbiota intervention is an effective strategy to improve feed utilization efficiency traits in poultry.

[0122] Figure 1E This is a flow chart of fecal microbial transplantation (FMT) trials (n≥15);

[0123] Figure 1F Feed conversion rate diagrams of the low feed utilization rate group, high feed utilization rate group, and feces transplantation group;

[0124] Figure 1G This is the weight gain chart of the low feed utilization group, high feed utilization group, and fecal transplant group;

[0125] 3.2 Changes in cecal microbial composition in the fecal transplant group

[0126] This section further explored whether FMT would change intestinal microbiota and IgA + The composition of microorganisms.

[0127] The results showed that FMT significantly affected the composition and diversity of cecal microorganisms in L-FE chickens, making them close to the composition structure of H-FE chickens ( Fig. 6A , Figure 6B ).

[0128] The relative abundance of Gram-positive bacteria Actinobacteria in H-FE chickens and FMT chickens was significantly higher than that in L-FE chickens (p<0.05), while the relative abundance of Gram-negative bacteria Proteobacteria decreased significantly after FMT ( Figure 6C ). IgA + The microbiome results showed that the relative abundance of Bacteroidetes, Synergistetes, and Tenericutes in the H-FE and FMT groups were significantly higher than those in the L-FE group ( Fig.6D ). BugBase analysis showed that the relative abundance of Gram-negative bacteria and potential pathogens in the cecal microbiota of L-FE chickens was significantly higher than that of H-FE and FMT chickens. + In microorganisms, no significant differences were observed among the three groups ( Figure 1H ). This result suggests that the IgA of L-FE chickens may have a lower ability to bind to pathogens.

[0129] Figure 1H Prediction of cecal microbiome and IgA in low feed utilization group, high feed utilization group, and fecal transplant group for BugBase + Relative abundance of Gram-negative bacteria and potential pathogens in the microbiome.

[0130] Fig. 6A This is the cecal microbiome diversity analysis diagram of the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0131] Figure 6B This is the IgA-bound microbiome diversity analysis diagram for the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0132] Figure 6C This is the composition diagram of cecal microorganisms at the phylum level in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0133] Fig.6D This is the IgA-bound microbial phylum level composition diagram of the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0134] 3.3 Screening of key bacteria affecting poultry grain-saving traits

[0135] In order to accurately explore the key candidate bacteria that affect feed utilization, this section analyzes the cecal microbiome and IgA after FMT. + The microbiome was comprehensively analyzed.

[0136] Taking L-FE as the control, the dominant bacteria shared by FMT and H-FE were considered as core functional bacteria. At the genus level, the abundance of Peptococcus, Dorea and Blautia in the cecal microbiota of FMT and H-FE chickens was significantly higher than that of L-FE chickens, while the relative abundance of Coprococcus and Sutterella was lower ( Fig. 6E ). Similarly, IgA in FMT and H-FE chickens + Among the bacteria, Prevotella, YRC22, Enterococcus, and Blautia were more abundant, while Megamonas and Coprococcus were less abundant ( Fig. 6F ).

[0137] Fig. 6E The genus composition of cecal microorganisms in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0138] Fig. 6F (Figure 2) shows the composition of IgA-bound microbial genus levels in the low feed utilization rate group, high feed utilization rate group, and fecal transplantation group.

[0139] Subsequently, this part conducted a linear discriminant analysis (LDA) to mine biomarkers related to feed utilization (threshold ≥ 2). The results showed that Peptococcus, Dorea, and Blautia were biomarkers of the cecal microbiome, and Blautia was also an IgA + Biomarkers screened by microbiome Fig. 7A and Figure 7B ). In summary, Blautia is the cecum and IgA + Common biomarkers screened by microbiome Fig. 8A and Figure 8B ). In addition, IgA + There was a significant negative correlation between the relative abundance of Blautia and FCR ( Fig. 1I ). Therefore, the integration of cecum and IgA +Based on the microbiome information, we precisely screened out Blautia as the key candidate bacteria affecting feed utilization.

[0140] Fig. 7A Screening of common key candidate bacteria in the cecal microbiota of the high feed utilization group and fecal transplantation group for linear discriminant analysis;

[0141] Figure 7B Screening of common key candidate bacteria in IgA-binding microorganisms of high feed utilization group and fecal transplantation group for linear discriminant analysis;

[0142] Fig. 8A Relative abundance diagram of common key candidate bacteria screened for the cecal microbiome;

[0143] Figure 8B Relative abundance diagram of common key candidate bacteria screened for IgA binding microbiome.

[0144] Fig. 1I Figure 2 is the result of Spearman correlation analysis between the relative abundance of IgA-binding bacteria Blautia and FCR; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001; L-FE indicates low feed utilization group; H-FE indicates high feed utilization group

[0145] Since the B cells that produce IgA are mainly derived from the bursa of Fabricius in birds, the size of the bursa lymphoid follicles and Bu-1 + The results showed that FMT significantly increased the levels of these two key indicators, bringing them closer to the H-FE group ( Fig.9A , Fig. 9B ).

[0146] Fig.9A This is the area diagram of the bursal lymph follicles in the low feed utilization group, high feed utilization group, and fecal transplantation group;

[0147] Fig. 9B The bursal Bu-1 of the low feed utilization group, high feed utilization group, and fecal transplantation group + B cell ratio chart.

[0148] Part 4 Verification of the effect of the screened strains on feed utilization

[0149] Blautia is a potential probiotic with prebiotic function. It can improve metabolic disorders by metabolizing tryptophan to indole-3-acetic acid and relieve inflammation by upregulating intestinal regulatory T cells. However, whether it can affect animal feed utilization and its mechanism are still unclear. Therefore, this part of the study was conducted on newly hatched chicks and weaned C57BL / 6j mice orally administered with Blautiacoccoides (a probiotic that plays an important role in regulating host energy metabolism and mucus immunity) ( Fig. 9C , Fig.9D ), forming the Blautia gavage group, with a gavage dose of 1×10 3 ~1×10 9 CFU / mL, by continuous or alternate-day gavage for 3 to 5 weeks.

[0150] Fig. 9C Flow chart of the experiment to verify the efficacy of the key candidate bacterium Blautia coccoides in chickens;

[0151] Fig.9D Flow chart of the experiment to verify the efficacy of the key candidate bacterium Blautia coccoides in C57BL / 6j mice.

[0152] The results showed that B. coccoides had no significant effect on the body weight of chickens and mice ( Figure 2A , Fig.9E ), but reduced feed intake in L-FE chickens ( Figure 2B In addition, B. coccoides has the potential to improve feed utilization in L-FE chickens and mice ( Figure 2C , Fig.9F ). The FCR value of L-FE chickens decreased from 3.86 to 3.75. After B. coccoides treatment, IgA in the cecal contents of chickens and mice + The number of bacteria increased significantly ( Figure 2D , Figure 9G In addition, CD19 in the mesenteric lymph nodes of mice treated with B. coccoides + Cells, IgA + Cells and CD19 + IgA + The proportion of cells increased significantly ( Figure 2E , F). The above results indicate that B. coccoides has the potential to improve feed utilization and enhance B cell activation.

[0153] Figure 2A The body weight of chickens on day 70 in the low feed utilization rate group, high feed utilization rate group, and Blautia gavage group;

[0154] Figure 2BThe feed intake of chickens from day 49 to 70 in the low feed utilization rate group, high feed utilization rate group, and Blautia gavage group;

[0155] Figure 2C This is the feed conversion rate chart of the low feed utilization rate group, high feed utilization rate group, and Blautia gavage group from 49 to 70 days of age;

[0156] Figure 2D Detection of IgA in chicken cecal contents by flow cytometry + proportion of bacteria (n=15);

[0157] Figure 2E Flow cytometry was used to detect CD19 in mouse mesenteric lymph nodes. + B cell ratio result diagram (n=15);

[0158] Figure 2F Flow cytometry was used to detect IgA in mouse mesenteric lymph nodes + Cells, IgA + Cells and CD19 + IgA + Cell ratio result diagram (n=15);

[0159] Fig.9E This is the weight of mice after oral administration of the key candidate bacteria Blautia coccoides;

[0160] Fig.9F This is the feed conversion rate diagram of mice after oral administration of the key candidate bacteria Blautia coccoides;

[0161] Figure 9G This is the proportion of IgA tuberculosis bacteria in the cecum of mice after oral administration of the key candidate bacterium Blautia coccoides.

[0162] To further explore its mechanism of action, this part conducted a co-culture experiment of B cells (Ramos line) and B. coccoides in vitro. The results showed that B. coccoides can induce B cell activation and increase CD19 - CD138 + Plasma cells and IgA + The proportion of cells ( Figure 2G , H), and the expression of genes related to B cell activation (Blimp1 and Xbp1s) and IgA production (Igha1) were significantly upregulated ( Fig.2I -K), the secretion level of IgA also increased significantly ( Figure 2LWe further successfully isolated primary B cells from the bursa of 49-day-old chickens and conducted a co-culture experiment with primary chicken B cells and B. coccoides. The results showed that IgA + The proportion of B cells increased and the level of IgA secretion increased significantly ( FIG. 10A to FIG. 10C ), the results are consistent with the above.

[0163] Figure 2G Flow cytometry was used to detect the expression of CD19 in B cell lines after 3 hours of co-culture with Blautia coccoides in vitro - CD138 + Proportion of plasma cells (n=6);

[0164] Figure 2H Flow cytometry was used to detect IgA in B cell lines after 3 hours of co-culture with Blautia coccoides in vitro + Proportional graph of cells (n=6);

[0165] Fig.2I The expression level of Blimp1, a gene related to B cell activation and IgA secretion, after 3 hours of co-culture (n=6);

[0166] Figure 2J The expression level of Xbp1s, a gene related to B cell activation and IgA secretion, after 3 hours of co-culture (n=6);

[0167] Figure 2K The expression level of Igha1, a gene related to B cell activation and IgA secretion, after 3 hours of co-culture (n=6);

[0168] Figure 2L The concentration of IgA in the supernatant after co-culture was determined using enzyme-linked immunosorbent assay (ELISA); * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001;

[0169] Fig. 10A This is a graph showing the cell activity of primary chicken B cells after they were co-cultured with Blautia coccoides in vitro for 3 hours;

[0170] Fig. 10B IgA was produced after chicken primary B cells were co-cultured with Blautia coccoides for 3 hours in vitro + Proportional diagram of B cells;

[0171] Fig. 10C This is a graph showing the concentration of IgA in the supernatant after chicken primary B cells were co-cultured with Blautia coccoides for 3 hours in vitro.

[0172] Note: The raw data of 16S rRNA gene sequencing of the cecal microbiome and IgA-binding microbiome of the present invention have been uploaded to the Sequence Read Archive (SRA) database, with the accession number PRJNA994595 (https: / / www.ncbi.nlm.nih.gov / sra / ?term=PRJNA994595). The main data and code have been uploaded to the Github website, the URL is: https: / / github.com / cylinqueen / chicken-micro.

[0173] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that the equivalents falling within the claims be included. All changes in the meaning and scope of the elements are encompassed by the present invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for screening key bacteria affecting poultry grain-saving traits, characterized in that: The steps include: Step 1: construct two groups of poultry with different feed utilization rates, one of which is named the low feed utilization rate group and the other is named the high feed utilization rate group; Step 2: Use the feces of the high feed utilization group as raw material to prepare bacterial liquid, and use fecal transplantation technology to implant the bacterial liquid into the intestines of the low feed utilization group to construct a fecal transplant group; Step 3: Obtain information on the cecal microbiota at the genus level in the low feed utilization group, high feed utilization group, and fecal transplant group, as well as IgA at the genus level in the cecal microbiota in the low feed utilization group, high feed utilization group, and fecal transplant group + Information about the microbiome; the IgA + The microbiome is a microbiome that can bind to IgA; Information on cecal microbiota and IgA in the low feed utilization group + The information of the microbiome is used as a control to screen and obtain the first difference information and the second difference information; Among them, the first difference information is the information difference of the cecal microbiota between the high feed utilization group and the low feed utilization group, IgA + The diversity of microbiome information is aggregated; The second difference information is the difference in information of cecal microbiota between the fecal transplant group and the low feed utilization group, IgA + The diversity of microbiome information is aggregated; The bacteria that exist simultaneously in the first difference information and the second difference information are the key bacteria that affect the utilization rate of poultry feed.

2. The method according to claim 1, characterized in that The method for obtaining the information of the IgA+ microbiome is: determining it through an IgA-SEQ detection method.

3. The method according to claim 1, characterized in that The poultry is one of chicken, duck and goose.

4. The method according to claim 3, characterized in that The poultry is bearded chicken.

5. The method according to any one of claims 1 to 4, characterized in that: In the step 2, after 5 to 35 days of infusion of the bacterial solution, a successfully constructed fecal transplant group is obtained.

6. A key bacterium affecting poultry grain-saving traits, characterized in that: The method is screened by any one of claims 1 to 5.

7. Use of probiotics of the genus Blautia to prepare probiotics for improving the utilization rate of poultry feed.

8. The use according to claim 7, characterized in that The probiotic bacteria of the genus Blautia is Blautiacoccoides bacteria.

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