Abstract Bacillus subtilis has been widely used in animal husbandry as a potential alternative to antibiotics due to its excellent bacteriostasis and antioxidant activity. This study aims to investigate the effects of Bacillus subtilis on the protection of ducks from Escherichia coli infection and its mechanism. The four experimental groups include the negative control group, positive control group, antibiotic group and Bacillus subtilis group. Ducks in positive, antibiotic and Bacillus subtilis groups are orally administered with Escherichia coli and equivalent saline solution for the negative group. The results show that supplements with Bacillus subtilis enhances the performance and health status of the infected ducks. Moreover, Bacillus subtilis alleviates the increase in globulin, LPS and MDA, and the decrease in albumin, T-AOC and T-SOD in the serum caused by Escherichia coli infection. Bacillus subtilis also attenuates injury in the intestine and partially reverses the increase in ROS production and the depletion of ATP in the jejunum. These effects are accompanied with the change of related genes of the ribosome (13.54%) and oxidative phosphorylation (6.68%). Collectively, Bacillus subtilis alleviates the damage caused by Escherichia coli infection in ducks by activating ribosome and oxidative phosphorylation signaling to regulate antioxidant and energy metabolism. Keywords: Bacillus subtilis, pekin duck, Escherichia coli, oxidative stress, ribosome, oxidative phosphorylation 1. Introduction Oxidative stress caused by an imbalance between the excessive production of free radicals and the antioxidant defense is an important component of biological damage and also the main source of serious diseases [[38]1]. It was reported that the endotoxin produced by pathogenic Escherichia coli (E. coli), such as lipopolysaccharide (LPS), can cause excessive production of reactive oxygen or nitrogen species, which exceeds the defense capacity of the host and increases the level of oxidative stress products such as MDA, ultimately causing a series of oxidative injuries [[39]2,[40]3]. What is more, severe oxidative stress caused by E. coli infection leads to impairment of energy metabolism through interference with oxidative phosphorylation pathways, ultimately compromising host antioxidant capacity, immune response and productive performance [[41]1]. Ducks and chickens are the main victims of avian pathogenic E. coli. The infected individuals show progressive debilitation, which results in damage to organs throughout the body and eventual death due to functional failure [[42]4,[43]5]. Antibacterial drugs and vaccines are the main treatments for E. coli infections; however, the multidrug-resistant and complicated pathogenicity of E. coli continues to increase and attracts endless attention [[44]6]. The challenges outlined above have prompted a global search for alternatives to antibiotics. Probiotics are widely used in humans and have gained acceptance as an animal feed additive to reduce the use of antibiotics gradually [[45]7,[46]8]. In the poultry industry, probiotics have been shown to stimulate innate immunity and overall health by preventing pathogen infections, thereby improving growth performance, promoting intestinal morphology and other functions [[47]9,[48]10,[49]11,[50]12]. Bacillus subtilis (B. subtilis) is a kind of probiotic that produces subtilisin, polymyxin, nystatin, short bacitracin and other active substances. It has been widely used in animal husbandry as a potential alternative to antibiotics due to its excellent bacteriostasis, antioxidant, immune and growth improvement functions [[51]13,[52]14,[53]15]. However, B. subtilis has been less studied in ducklings, especially the ducklings exposed to E. coli. In recent years, we have been concerned about the negative effect of pathogenic E. coli on waterfowl and have successfully established a model of E. coli infection in Pekin ducklings [[54]16]. Based on previous work, we compared the effects of B. subtilis L6 and virginiamycin on growth performance, antioxidation function and the intestinal health of ducks challenged with E. coli in the current study. In addition to the physiological and biochemical analyses, the RNA-Seq was also used to determine possible molecular mechanisms by which B. subtilis relieved intestinal oxidative stress caused by E. coli infection in Pekin ducklings. 2. Materials and Methods 2.1. Preparation of E. coli O88 and B. subtilis L6 The strain of pathogenic E. coli O88 was obtained from China Veterinary Culture Collection Center (CVCC). The probiotic B. subtilis L6 in microcapsules (viable count ≥1.0 × 10^10 CFU/g, powder state) was provided by Challenge Biotechnology Co., LTD (Beijing, China). The viable B. subtilis count in feed was determined based on Nikoskelainen (2003) [[55]17]. 2.2. Experimental Design The experiment was conducted in Nankou pilot base of the Chinese Academy of Agricultural Sciences. The methods for animal experiments were set out by the National Institute of Animal Health and research reporting follows the guidelines of ARRIVE [[56]18]. A total of 192 newly hatched, male lean Pekin ducklings were randomly allocated into 4 treatment groups with 6 replicates of 8 ducks each replicate. The 4 treatment groups were negative control group (NC), positive control group (PC), 30 mg/kg virginiamycin group (ANT) and 2.5 × 10^9 CFU/kg B. subtilis L6 group (BS), respectively. The basal diets meet the nutritional requirements of the ducks as determined by the National Research Council (NRC, 1994) and the Nutrient Requirements of Meat-type Duck published by the Ministry of Agriculture of the People’s Republic of China, NY/T 2122-2012 ([57]Supplementary Table S1). The experiment lasted for 28 days. 2.3. Oral Challenge The infection model was established based on our previous protocol [[58]16]. Briefly, the frozen E. coli O88 was thawed and cultured in Luria-Bertani (LB) broth to activate three times (37 °C, 12 h). Bacteria were resuspended in sterilized 0.9% saline solution and counted by plate cultivation. On day 7, the ducks in PC, ANT and BS groups were orally administered with 0.2 mL E. coli (3 × 10^9 CFU/mL) twice, 8 h apart and equivalent volumes of 0.9% sterile saline solution for the NC group. The workflow is shown in [59]Figure 1. Figure 1. [60]Figure 1 [61]Open in a new tab Workflow for the experiment. NC–negative group, basal diet without E. coli challenge. PC–positive group, basal diet with E. coli challenge. ANT–antibiotic group, basal diet + virginiamycin (30 mg/kg) with E. coli challenge. BS–basal diet + 2.5 × 10^9 CFU/kg B. subtilis with E. coli challenge. 2.4. Sampling On days 9, 14 and 28, after fasting 6 h, all ducks were weighed and the feed intake was measured on a per cage basis. Average daily feed intake (ADFI), average daily gain (ADG) and the feed intake/weight gain (F/G) ratio were calculated. After a 6 h fast, one duck (close to the average BW) from each replicate was selected and euthanized by electric stunning, and then the blood samples (2.5 mL) were taken from the wing vein into an anticoagulant-free vacuum test tube (5 mL), centrifuged at 3000× g for 10 min and stored at −20 °C. The ducks were opened longitudinally, both ceca were ligated and aseptically removed from the gastrointestinal tract for cecal E. coli analysis. The middle portion of jejunum (1.5 cm) was cut off and flushed residual digesta with ice-cold phosphate-buffered saline (PBS), and then fixed in 10% neutral formalin for intestinal histomorphology analysis [[62]19]. The mucosa of jejunum segments was gently scraped and snap-frozen in liquid nitrogen and then transferred to a −80 °C freezer till analyzed. 2.5. Cecal E. coli Cecal contents were obtained from the ligated ceca on days 9, 14 and 28. The viable counts of E. coli were analyzed by the method of Manafi [[63]20]. Briefly, the cecal contents of each bird were pooled and serially diluted. E. coli was counted on Eosin Methylene Blue agar plates after incubation 24 h at 37 °C. The colony-forming unit was defined as distinct colony at least one mm in diameter. 2.6. Serum Indices The serum albumin, total protein and LPS levels were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The catalog numbers were A028-1-1, A045-3-2 and H255, respectively) by colorimetric method (UV-2550, Shimadzu, Japan). Because the total protein in serum mainly consists of globulin and albumin, the globulin content was obtained by subtracting the albumin value from that of the total protein [[64]21]. Serum total antioxidant capacity (T-AOC), activity of total superoxide dismutase (T-SOD) and malonaldehyde (MDA) concentration were determined by the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The catalog numbers were A015-2-1, A001-1-2 and A003-2-2, respectively) with an automated fluorescence instrument (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA). 2.7. Intestinal Morphology The middle portion of jejunum was fixed in formalin for 24 h, embedded in paraffin, deparaffinized, dehydrated and stained, observed the jejunum morphological structure by Motic Panthera Moticam 5 trinocular microscope BA210LED (Motic Incorporation Ltd., Hong Kong, China) and analyzed by Moticam digital imaging system (Motic images Software Plus 2.0, Motic Incorporation Ltd., Hong Kong, China). Villus height and crypt depth were measured at least 10 well-oriented villus and then calculated the villus height/crypt depth ratio (V/C) ratio. The measurement has referred to the method of Lin [[65]22]. 2.8. ROS Production and ATP Level of Jejunum The reactive oxygen species (ROS) production of jejunum was measured by using the ROS commercial assay kits (S0033S. Beyotime Institute of Biotechnology, Haimen, China) and 2,7-dichlorofluorescein diacetate as fluorescence probe. The specific process was carried out as the method of Zhang [[66]23,[67]24]. The adenosine triphosphate (ATP) level was determined by the commercial ATP assay kits (BC0305. Solarbio, Beijing, China). 2.9. RNA-Seq Analysis of the Jejunum The RNA-Seq analysis was performed on 12 jejunum samples from PC and BS groups. The middle portion of jejunum was collected on day 14. Total RNA in jejunum tissues was extracted by Trizol kits (Invitrogen, Carlsbad, CA, USA) and treated by DNase I to avoid DNA contamination. RNA concentration was determined by Nanodrop 2000. Total RNA was analyzed qualitatively and quantitatively using Agilent 2100 (Agilent Technologies, Inc., Palo Alto, CA, USA). RNA-seq libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). Libraries were sequenced on a HiSeq2500 platform (Illumina Inc., San Diego, CA, USA) to generate Paired-end 100 bp raw reads. The adaptor sequences and low-quality sequence reads were removed from the data sets using fastx-toolkit tool. These clean reads were then mapped to the reference genome sequence (Anas_platyrhynchos: ASM874695v1). Gene expression levels were quantified as fragments per kilo base of transcript per million fragments (FPKM) mapped from different jejunum samples [[68]25]. Differential expression analysis was performed using the DESeq2 R package (1.6.3) [[69]26]. DEGs with |fold change (FC)| ≥ 2 and false discovery rate (FDR) < 0.05 were considered as differentially expression. The differentially expressed genes (DEGs) were annotated and enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG, [70]http://www.kegg.jp/kegg/pathway.html, accessed on 13 September 2022) using the clusterProfiler R package (3.10.1). 2.10. RT-qPCR Nine DEGs associated with “ribosome” and “oxidative phosphorylation” pathways, including Ribosomal protein lateral stalk subunit P2 (RPLP2), Mitochondrial ribosomal protein L23 (MRPL23), NHP2 ribonucleoprotein (NHP2), NADH-ubiquinone oxidoreductase core subunit V1 (NDUFV1), Cytochrome c oxidase subunit 7B mitochondrial (COX7B), ATP synthase membrane subunit F (ATP5MF), Ribosomal protein L22 like 1 (RPL22L1), ATPase H^+ transporting V1 subunit A (ATP6V1A), NADH-ubiquinone oxidoreductase subunit A6 (NDUFA6) were selected and using RT-qPCR to confirm the accuracy and reliability of RNA-Seq results. The total RNA from the jejunum tissues was isolated by TRI-zol reagent (TIANGEN, Beijing, China) and reversely transcribed into complementary DNA (cDNA) pursuant. The concentration of total RNA was determined by a spectrophotometer (Ultrospec 2100 pro, GE Healthcare, Chicago, IL, USA) and purified by agarose gel electrophoresis. Then 500 ng total RNA was reversely transcribed into cDNA using the primescript of Fast Quant RT Kit (TIANGEN, Beijing, China). The qPCR was conducted by the Biosystems Bio-Rad Real-Time PCR system (Bio-Rad, Carlsbad, CA, USA) with the Brilliant SYBR Green qPCR Master Mix (Stratagene, La Jolla, CA, USA). The primers used are listed in ([71]Supplementary Table S2). The beta-actin (β-actin) was used to normalize the expression of the targeted genes. The mRNA level of the relative genes was calculated using the method of 2^−ΔΔCt [[72]27]. All samples were analyzed in triplicate and the geometric mean of internal references.