B. velezensis Y01 improves the growth performance of Langya chickens

Dietary supplementation with B. velezensis Y01 resulted a 7.01% increase in the final body weight, a 7.27% increase in WG, and a 7.24% increase in ADG of Langya chickens at 42 days of age in the BVT group when compared to the CON group (p < 0.05, Table 2). Furthermore, the final body weight was increased by 5.88%, WG by 6.18%, and ADG by 6.19% in the BVT group when compared to the ANT group (p < 0.05, Table 2). However, no significant differences were found in the final body weight, WG, and ADG between the CON group and the ANT group. The F/G ratio and ADFI had no significant differences within these three groups at 42 day (p > 0.05, Table 2).

Table 2 Effects of dietary B. velezensis Y01 supplementation on the growth performance of 1 to 42 d Langya chickens B. velezensis Y01 affects the blood parameters of Langya chickens

To investigate the effects of B. velezensis Y01 on the blood parameters of Langya chickens, the hematological and serum biochemical indexes of Langya chickens were evaluated. The hematology results were shown in Table 3. The hematological indexes of Langya chickens at 42 days of age, including RBC, HGB, MCV, LYM, WBC, NEU, MONO, and PLT levels, had no significant differences within these three groups (p > 0.05).

Table 3 Effects of dietary B. velezensis Y01 supplementation on the hematological indexes of 1 to 42 d Langya chickens

The serum biochemistry results were shown in Table 4. Compared to the CON group, the ANT group with aureomycin resulted in a 26.10% decrease in LDH, and the BVT group with B. velezensis Y01 resulted in a 26.80% decrease in LDH, a 52.92% decrease in UA, a 20.70% decrease in CH, and a 40.84% decrease in urea in Langya chickens at 42 days of age (p < 0.05). In addition, the BVT group with B. velezensis Y01 resulted in a 31.72% decrease in urea in Langya chickens at 42 days of age in comparison to the ANT group (p < 0.05). While no significant differences were found in other serum biochemical characteristics of Langya chickens at 42 days of age within these three groups, including ALB, GLB, TP, A/G ratio, BIL, GT, ALT, AST, ALP, AMY, CK, GLU, calcium and phosphorus levels (P > 0.05).

Table 4 Effects of dietary B. velezensis Y01 supplementation on the serum biochemical indexes of 1 to 42 d Langya chickens B. velezensis Y01 increases the immune function of Langya chickens

To investigate the effects of B. velezensis Y01 on the immune performance of Langya chickens, the immunoglobulins and immune cytokines in the serum of Langya chickens were evaluated. The serum immunoglobulin results were shown in Table 5. Compared to the CON group, the ANT group with aureomycin resulted in a 19.15% decrease in the serum IgE levels (p < 0.05), and the BVT group with B. velezensis Y01 resulted in a 36.09% increase in the serum IgG levels and a 56.08% increase in the serum IgM levels at 42 day (p < 0.05). In addition, the BVT group with B. velezensis Y01 showed significantly higher levels of serum IgE (higher by 17.64%, p < 0.05), IgG (higher by 24.13%, p < 0.05), and IgM (higher by 20.69%, p < 0.05) at 42 day than the ANT group with aureomycin. However, no significant differences were observed in the serum IgA levels within the three groups at 42 day (P > 0.05).

Table 5 Effects of dietary B. velezensis Y01 supplementation on the immune performance of 1 to 42 d Langya chickens

The serum immune cytokines results were shown in Table 5. Compared to the CON and ANT group, B. velezensis Y01 in the BVT group resulted in a 24.71% increase and a 29.61% increase in the serum IL−2 levels at 42 day, respectively (p < 0.05). However, aureomycin had no significant effects on the serum IL−2 levels at 42 day as compared to the CON group (P > 0.05). Moreover, no significant differences were found in the serum IL-1, IL-6, and TNF-α levels within the three groups at 42 day (P > 0.05).

B. velezensis Y01 alters the cecal microbial composition of Langya chickens

To investigate the effects of B. velezensis Y01 on the cecal microbial composition in Langya chickens, we conducted metagenomic sequencing of the cecal content. The relative abundance of cecal microbiota in Langya chickens was presented at the phylum, order, family, and genus levels (Fig. 1). At the bacterial phylum level, Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes, and Euryarchaeota were the top five most abundant bacterial phyla in the cecal microbiota within the three groups (Fig. 1A). Bacteroidales, Eubacteriales, unclassified Firmicutes, Campylobacterales, and Lactobacillales were the top five most abundant bacterial orders in the cecal microbiota within the three groups (Fig. 1B), and at the bacterial family level, the top five dominant bacterial families were Bacteroidaceae, Oscillospiraceae, Clostridiaceae, Lachnospiraceae, and Rikenellaceae (Fig. 1C). Bacteroides was the most abundant genus in the cecal microbiota within the three groups, followed by Clostridum, Alistipes, Phocaeicola, and Lachnoclostridium (Fig. 1D).

Fig. 1

Distribution of the cecal microbiota of 1 to 42 d Langya chickens at the phylum, order, family, and genus levels. (A) Relative abundance of predominant bacteria at the phylum level; (B) Relative abundance of predominant bacteria at the order level; (C) Relative abundance of predominant bacteria at the family level; and (D) Relative abundance of predominant bacteria at the genus level. CON: the control group was fed with a corn-soybean-based diet; ANT: the antibiotic-treated group was fed with the corn-soybean-based diet supplemented with 50 mg/kg aureomycin; and BVT: the B. velezensis-treated group was fed with the corn-soybean-based diet supplemented with 2.0 × 109 CFU/kg B. velezensis Y01

There were no differences in the alpha diversity analysis based on shannon index, simpson index, and invsimpson index among the CON, ANT and BVT groups (Fig. 2A), while the three groups were clearly separated in the beta diversity analysis based on PCA (Fig. 2B). In addition, although the CON and ANT groups were much more similar to each other than they were to the BVT group based on NMDS analysis, the ANT and BVT groups as well as the CON and BVT groups were clearly separated (Fig. 2C). Finally, the LEfSe analysis (LDA > 3) was performed to investigate which microbes in the cecal microbiota was mostly impacted by B. velezensis Y01. As shown in Fig. 2D, the LEfSe results revealed that Ligilactobacillus, Bacteroidales, unclassified Bacteroidales, unclassified Lentisphaerae, Gemmiger, Mediterranea, Methanobrevibacter, and Lentisphaerae were significantly enriched in the BVT group, while Tyzzerella, Akkermansia, Methanocorpusculum, Bilophila, Cloacibacillus, Chlamydia, and Budvicia were more abundant in the CON group, as well as Bacteroides, Megamonas, Oscillibacter, and Anaeromassilibacillus were more abundant in the ANT group.

Fig. 2

B. velezensis Y01 alters the cecal microbial composition of 1 to 42 d Langya chickens. (A) Shannon index, simpson index, and invsimpson index in alpha diversity analysis of the cecal microbial communities; (B) Principal component analysis (PCA) of the cecal microbial communities; (C) Non-metric multidimensional scaling (NMDS) analysis of the cecal microbial communities; and (D) Linear discriminant analysis with effect size (LEfSe) analysis of the cecal microbial communities via histogram based on the linear discriminant analysis (LDA) score. CON: the control group was fed with a corn-soybean-based diet; ANT: the antibiotic-treated group was fed with the corn-soybean-based diet supplemented with 50 mg/kg aureomycin; and BVT: the B. velezensis-treated group was fed with the corn-soybean-based diet supplemented with 2.0 × 109 CFU/kg B. velezensis Y01

B. velezensis Y01 alters the metabolic potential of cecal microbial communities in Langya chickens

To investigate the effects of B. velezensis Y01 on the metabolic potential of cecal microbial communities, the KEGG and CAZy databases were adopted to predict the gene functional profiling of cecal microbiota. As shown in Fig. 3A, the third-level KEGG pathway analysis exhibited that a total of 23 KEGG pathways were enriched in the BVT group by comparison with the observation for the CON and ANT groups, including amino acid biosynthesis (phenylalanine, tyrosine, tryptophan, lysine), amino acid metabolism (glycine, serine, threonine, glyoxylate, dicarboxylate), organic acid metabolism (pyruvate, butanoate, fatty acid), carbohydrate metabolism (glycolysis, citrate cycle, amino sugar, nucleotide sugar, fructose, mannose, galactose), vitamin metabolism (nicotinate, nicotinamide, thiamine), vitamin biosynthesis (pantothenate, folate), and nitrogen base metabolism (pyrimidine, purine). Meanwhile, a total of 11 KEGG pathways were enriched in the ANT group by comparison with the observation for the CON and BVT groups, including carbohydrate metabolism (starch, sucrose, pentose), amino acid metabolism (cysteine, methionine, alanine, aspartate, glutamate), O-antigen nucleotide sugar biosynthesis, quorum sensing, oxidative phosphorylation, and ABC transporters. While the top 18 more abundant KEGG pathways in the CON group than the observation for the ANT and BVT groups were glycerophospholipid, methane and propanoate metabolism, DNA replication, aminoacyl-tRNA biosynthesis, ribosome, protein export, quorum sensing, oxidative phosphorylation, peptidoglycan biosynthesis, bacterial secretion system, two-component system, as well as mismatch, base excision, and nucleotide excision repair.

Fig. 3

The functional genes annotation analysis of the cecal microbial communities of 1 to 42 d Langya chickens. (A) The third-level KEGG pathway analysis in predicted metabolic pathway of cecal microbiota; (B) The carbohydrate-active enzymes annotation analysis using the CAZy database in predicted metabolic function of cecal microbiota; (C) The virulence genes annotation analysis using the VFDB database in predicted the pathogenicity of cecal microbiota; and (D) The antibiotic resistance genes annotation analysis using the CARD database in predicted the antibiotic resistance of cecal microbiota. CON: the control group was fed with a corn-soybean-based diet; ANT: the antibiotic-treated group was fed with the corn-soybean-based diet supplemented with 50 mg/kg aureomycin; and BVT: the B. velezensis-treated group was fed with the corn-soybean-based diet supplemented with 2.0 × 109 CFU/kg B. velezensis Y01

The gene functional annotation analysis using the CAZy database showed that a total of 25 carbohydrate-active enzymes were enriched in the BVT group, including 19 GHs (glycoside hydrolases, GH33, GH68, GH30, GH18, GH63, GH31, GH57, GH65, GH38, GH29, GH130, GH20, GH26, GH66, GH3, GH95, GH5, GH8, GH43), 5 GTs (glycosyl transferases, GT30, GT26, GT19, GT3, GT2), and 1 CE (carbohydrate esterase, CE1), as well as 20 carbohydrate-active enzymes in the ANT group, including 10 GHs (GH101, GH121, GH32, GH4, GH88, GH23, GH13, GH77, GH105, GH51), 5 GTs (GT4, GT35, GT39, GT1, GT5), 2 PLs (polysaccharide lyases, PL8, PL11), 2 CBMs (carbohydrate-binding modules, CBM48, CBM50), and 1 CE (CE10); while only 5 carbohydrate-active enzymes in the CON group, including 4 GTs (GT28, GT51, GT9, GT66) and 1 CBM (CBM6) (Table 6; Fig. 3B). In addition, the differential carbohydrate-active enzymes that analyzed using Kruskal-Wallis analysis showed that the abundance of 10 carbohydrate-active enzymes was significantly increased in the BVT group in comparison to the CON and ANT groups. These enzymes include GT19, GH95, GH31, GH3, GT25, GT8, GT30, GT21, GH66, and GH30 (Fig. 4A).

Table 6 The functional description of CAZy of cecal microbial community in Langya chickensFig. 4

The differential carbohydrate-active enzymes and antibiotic resistance genes of 1 to 42 d Langya chickens using Kruskal-Wallis analysis. (A) The differential carbohydrate-active enzymes using Kruskal-Wallis analysis; and (B) The differential antibiotic resistance genes using Kruskal-Wallis analysis. CON: the control group was fed with a corn-soybean-based diet; ANT: the antibiotic-treated group was fed with the corn-soybean-based diet supplemented with 50 mg/kg aureomycin; and BVT: the B. velezensis-treated group was fed with the corn-soybean-based diet supplemented with 2.0 × 109 CFU/kg B. velezensis Y01

B. velezensis Y01 decreases the pathogenicity and antibiotic resistance of cecal microbiota in Langya chickens

To investigate the effects of B. velezensis Y01 on the pathogenicity and antibiotic resistance of cecal microbiota, the VFDB and CARD databases were adopted to predict the virulence genes and antibiotic resistance genes of cecal microbial communities. As shown in Fig. 3C, the virulence genes annotation analysis using the VFDB database showed that only 1 virulence gene such as wbtL was enriched in the BVT group, 39 virulence gene were enriched in the CON group, including fliI, GBS_RS06585, flaG, flgL, cheA, Cj0883c, cheV, flgC, flhB, kpsD, flgS, pseC, hldD, kpsT, ptmA, flgE2, flgE, pseB, flhA, fliD, hddA, hldE, flhF, flgB, pseI, pflA, flgI, fliS, fliF, flgG2, cps4L, rfbC, fliR, cheY, ciaB, flgK, Cj1419c, gmhA2, and motA, as well as 11 virulence gene were enriched in the ANT group, including ugd, fliN, cps4J, groEL, hasC, gmhA, wbtE, ciaC, tufA, kpsS, and flgK. These results indicated that dietary B. velezensis Y01 supplementation considerably reduced the virulence genes of cecal microbial communities, thereby decreasing the pathogenicity of cecal microbiota in Langya chickens.

The antibiotic resistance genes annotation analysis using the CARD database revealed that 13 antibiotic resistance genes were enriched in the BVT group, including aac6-I, ant9-I, aph2-II, aph3-VII, CfxA2, ermF, ermT, lnuA, lnuB, macA, mefA, tet40, and class A beta-lactamase coding genes, 22 antibiotic resistance genes were enriched in the CON group, including aadA, OXA-61, TEM-1, catD, cat, vatB, cmeA, cmeB, emrB, qacB, qacEdelta1, sul1, tet44, tetA, tetL, tetO, tetX, vanR, vanS, vanU, bifunctional aminoglycoside N-acetyltransferase and aminoglycoside phosphotransferase coding genes, and tetracycline resistance protein coding genes, as well as 25 antibiotic resistance genes were enriched in the ANT group, including aadE, aph2-IV, aph2-Ie, aph3-I, aph3-III, aph6-I, bacA, TEM-1, cat, fosB, ermB, ermX, ermG, vatB, emrA, sul2, tet32, tetM, tetQ, tetW, metallo-beta-lactamase coding genes, chloramphenicol exporter coding genes, class A beta-lactamase coding genes, tetracycline resistance protein coding genes, and 16 S rRNA methylase coding genes (Fig. 3D). As shown in Fig. 4B, the differential antibiotic resistance genes that analyzed using Kruskal-Wallis analysis manifested that the abundance of 5 antibiotic resistance genes was significantly reduced in the BVT group when compared to the CON group, including aph3-III, tetX, tet44, qacEdelta1, and ermB. Meantime, compared to the ANT group, the abundance of 7 differential antibiotic resistance genes was significantly reduced in the BVT group. These genes include ermG, aph3-III, tet44, qacEdelta1, ermB, fosB, and 16 S rRNA methylase coding genes (Fig. 4B). These results illustrated that dietary B. velezensis Y01 supplementation obviously reduced the antibiotic resistance genes of cecal microbial communities, thereby decreasing the antibiotic resistance of cecal microbiota in Langya chickens.