Intestinal Microbiom in Necrotic Enteritis Infection of Broiler and Comparison of Treatment Alternatives

Intestinal Microbiom in Necrotic Enteritis Infection of Broiler and Comparison of Treatment Alternatives | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intestinal Microbiom in Necrotic Enteritis Infection of Broiler and Comparison of Treatment Alternatives Ozge YILMAZ CAGIRGAN, Serol KORKMAZ, Serdar DİKER This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6190428/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Clostridium perfringens is the primary causative agent of necrotic enteritis (NE), a gastrointestinal disease that leads to substantial economic losses in poultry. This study aims to characterize the intestinal microbiome of chickens and assess the effects of Bacillus velezensis on gut microbiota and recovery from necrotic enteritis, comparing its efficacy to antibiotic treatment. The experiment involved five groups, each consisting of 16 chickens. The first group, the start-of-challenge (DB) group, included 1-day-old chicks. The second group, the post-challenge control (DS) group, was reared until the end of the trial. The third group was infected with C. perfringens (NE group). The fourth group received both C. perfringens and B. velezensis (BV group), while the fifth group was treated with C. perfringens and amoxicillin (AB group). All chickens were euthanized via cervical dislocation following the experimental infection. Fecal samples collected from the cecum underwent 16S rRNA gene-based metagenomic analysis, and the resulting data were statistically evaluated. Macroscopic examination after euthanasia revealed pathological changes in the intestines of chickens in the NE group, which had received only C. perfringens . Their intestines appeared swollen, with slight mucosal bleeding. In contrast, no macroscopic lesions were observed in the DB, DS, BV, or AB groups. Microbiome analysis showed a decline in microbial diversity within the NE group. The BV group exhibited a microbial composition most similar to that of healthy animals, followed by the AB group. The study concludes that B. velezensis could serve as an alternative to prophylactic antibiotics in mitigating the adverse effects of necrotic enteritis on the gut microbiome. B. velezensis C. perfringes microbiome challenge broiler Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Necrotic enteritis (NE), a gastrointestinal disease that results in major financial losses for poultry, is caused by C. perfringens . This disease, particularly affecting broiler chickens, results in reduced feed conversion rates, high mortality rates, and severe deterioration of animal welfare (M’sadeq et al., 2015 ; Opengart & Songer, 2013 ). NE, forecasted to impose an annual financial burden of $ 6 billion on the global poultry industry, has become a widespread and challenging problem to control (Wade & Keyburn, 2015 ). With the ban on antimicrobial feed additives, the incidence of this disease has increased, further emphasizing the need for alternative control strategies (Van Immerseel et al., 2004 ). The spore-forming, anaerobic, Gram-positive bacterium C. perfringens is frequently found in the environment. It proliferates primarily in the small intestine, causing necrotic enteritis through toxin production and mechanical mucosal degradation in the intestines. While healthy chickens have C. perfringens concentrations of 10²–10⁴ CFU/g in their intestines, concentrations of 10⁷–10⁹ CFU/g can cause disease (Shojadoost et al., 2012 ). The bacterium produces several virulence factors, such as alpha-toxin and NetB toxin, which cause severe damage to the intestinal epithelium (Keyburn et al., 2008 ). The NetB toxin is particularly recognized as an essential virulence factor in the NE pathogenesis (Keyburn et al., 2008 ; Prescott et al., 2016 ). The gene encoding NetB toxin is carried on a conjugative plasmid, enabling the bacterium to easily spread its pathogenic genetic traits (Bannam et al., 2011; Parreira et al., 2012). Additionally, dietary components, physical damage to the intestinal mucosa, and environmental factors are predispositional contributors to the disease (Moorea, 2016). Prolonged antibiotic use for managing and efforts to treat NE have facilitated the rise of antibiotic resistance in C. perfringens (Gillings et al., 2017 ). This situation has increased interest in alternative therapeutic methods, Including probiotics, prebiotics, organic acids, and plant-derived compounds (Ramlucken et al., 2020c). Probiotics have been shown to regulate intestinal microbiota, inhibit toxin production, and support the host immune system by competing with pathogenic bacteria (Pan & Ye, 2014; Amara & Shibl, 2015 ). Among probiotics, B. velezensis stands out for its ability to colonize the gut, produce antimicrobial metabolites, and support gut health (Ye et al., 2018; Grady et al., 2019 ). Studies have proven that Bacillus species improve villus morphology and feed conversion rates by colonizing the intestinal surface (Ramlucken et al., 2020b ). A healthy gut microbiome is vital for both host well-being and immune responses, and metabolic processes. It supports diverse functions, involving nutrient digestion and absorption, exclusion of pathogens, and regulation of immune responses (Kogut et al., 2017 ). Maintaining a balanced gut microbiota contributes to preventing diseases like NE by restricting the growth of harmful bacteria (Wilson et al., 2018). However, factors such as antibiotic use, poor nutrition, and stress can disrupt the gut microbiome, creating a predisposition to disease (Pan & Yu, 2014 ). Regulating the microbiome is essential to enhancing animal welfare and preventing diseases (Kogut et al., 2017 ). This research focuses on evaluating the effects of B. velezensis in regulating necrotic enteritis triggered by C. perfringens. The study investigates the probiotic's impact on the gut microbiome, its potential to inhibit toxin production, and its role in promoting resistance to the disease. The findings are expected to contribute to sustainable and effective control strategies that reduce the need for antibiotics. Materials and Methods Materials Daily chicks with similar body weights (~ 45 g) were used in the study. Eighty chicks in total were allocated into five distinct groups: Trial Baseline Group (DB), Post-Trial Control Group (DS), C. perfringens -exposed Group (NE), C. perfringens + B. velezensis Group (BV), and C. perfringens + Amoxicillin Group (AB). For the isolation and culture of C. perfringens, Tryptose Sulfite Cyclocerine (TSC) Agar, blood agar, Fluid Thioglycollate (FTG) Medium, and Cooked Meat Medium (CMM) were used. The C. perfringens NCTC 8239 strain was used for the challenge application. The B. velezensis strain was obtained from the culture collection of Aydın Adnan Menderes University Department of Microbiology. The chicks received a corn-soy-based diet during the first 13 days. As of day 14, all groups other than the control group received specific experimental treatments were fed a diet containing 30% fish meal. Methods Preparation of C. perfringens The C. perfringens strain was incubated under anaerobic conditions on blood agar for 18 hours. The resulting colonies were sequentially transferred to minced meat broth and FTG media, and their culture concentration was increased with 12–15 hour incubations. As a result, a bacterial culture with a density of 2–5×10⁸ CFU/ml was prepared for use in the challenge. Challenge The chicks were fed a corn-soy-based diet from day 1 to day 13. Starting on day 14, all groups except the control group were switched to a diet containing 30% fish meal. Throughout the trial, water was made available to the chicks ad libitum, and sawdust was used as bedding material. From days 21 to 25, C. perfringens culture (1.25 FTG–1.5 feed [v/w]), previously prepared, was administered twice daily along with the feed to the NE, BV, and AB groups. From days 26 to 30, the AB group was given 20 mg/kg of amoxicillin diluted in drinking water once daily orally. In the BV group, the lyophilized form of B. velezensis (10¹¹ spores/g sucrose-filled) was used. The lyophilized powder was freshly diluted in distilled water to a dose of 10⁸ spores and administered orally to each chick twice daily. After the B. velezensis and antibiotic applications, euthanasia was performed on day 32 via cervical dislocation for all groups, including the control group. Cecal contents were collected from the chickens for molecular analysis and stored at -20°C. Microbiome Analysis Metagenomic analysis targeting the 16S rRNA gene was performed using previously defined workflows (Cusco et al., 2018 ). The primer pair used for amplicon library preparation targeted approximately 1400 bp covering the V1–V9 regions of the 16S rRNA gene (Zeng et al., 2013 ; Klindworth et al., 2013 ). Oxford Nanopore Technologies’ Nanopore barcode DNA sequences were added to the 5’ ends of the target-specific primer pairs. The target-specific primer-connector sequences for 16S rRNA were as follows: forward primer 5’-TTTCTGTTGGTGCTGATATTGC-AGRGTTTGATYHTGGCTCAG-3’ and reverse primer 5’-ACTTGCCTGTCGCTCTATCTTC-TACCTTGTTAYGACTT-3’. The initial PCR was performed using a Proof Reading DNA Polymerase 2x Reaction Mix with 200 nm of each primer. The thermal cycling program was as follows: 95°C for 3 minutes; 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds; and a final extension at 72°C for 5 minutes. The PCR product was electrophoresed on an agarose gel to confirm its size (~ 1450 bp) and purified using a PCR Product Purification Kit. For amplicon library preparation, the Ligase Sequencing Kit 1D (SQK-LSK108; Oxford Nanopore Technologies) was used, and the library was loaded onto the MinION™ (Oxford Nanopore Technologies) device. A mixture containing 1–1.5 µg DNA with 45 µl of barcoded DNA and 5 µl of lambda phage DNA as a positive control was loaded onto the device. The NEBNext End Repair/dA-tailing Module (New England Biolabs) kit was used for DNA end repair and dA-tailing. Purification was performed using the Agencourt AMPure XP beads (Beckman Coulter) kit. For adapter ligation, 0.2 pmol of DNA ends were mixed with 50 µl of Blunt/TA Ligase Master Mix (New England Biolabs) and incubated at room temperature for 10 minutes with the addition of 20 µl of adapter mix. The final purification of the DNA library was completed using the Adapter Bead Binding Buffer (provided in the SQK-LSK108 kit) and 0.5X Agencourt AMPure XP beads (Beckman Coulter). The sequencing mix (14 µl DNA library), loading beads (25.5 µl), and running buffer mixture (35.5 µl) were prepared and loaded onto the primed R9.4 flow cell. A 48-hour sequencing protocol was performed using MinION™ control software, MinKNOW™ version 0.46.1.9 (R9.4). Read data were generated by base-calling with the Guppy v3.1.5 software and demultiplexed. Barcode and adapter sequences were trimmed using the Porechop v0.2.3 software. Reads with lengths of 1350–1550 bp were filtered, and other reads were excluded from the analysis. Filtered reads were analyzed with the Mothur v.1.39.5 platform using a customized workflow. The sequences were de-chimerized, aligned, and clustered into operational taxonomic units (OTUs) based on > 99% similarity. OTUs were taxonomically annotated using the RDP 16S rRNA database, and statistical results were obtained by grouping similar OTUs. Bioinformatic Analysis The similarities of bacterial communities in the cecal contents collected from the groups (DB, DS, NE, BV, AB) were evaluated using Principal Coordinate Analysis (PCA) applied to the Bray-Curtis distance matrix obtained from the 'vegan' R package. The first two principal components, PC1 and PC2, were visualized in two dimensions. Visualizations were performed using the 'factoextra' and 'ggplot2' packages in R. Relative abundance (%) graphs were created using Excel 2013 (Microsoft Office, USA). Statistical analyses were performed using SPSS version 22.0 (IBM Corp., USA). Differences in phylum, family, and genus levels between groups were evaluated using the Kruskal-Wallis test and one-way ANOVA for independent samples. The significance of differences was determined using the post-hoc Tukey test, with a significance level of p < 0.01. Results Clinical Observations and Pathological Changes During the trial, no clinical signs or mortalities were observed in any group. Post-trial necropsy revealed pathological alterations observed exclusively in the intestines of chickens from the C. perfringens group (NE). The intestines were swollen, and the mucosa displayed mild hemorrhages. No macroscopic findings were observed in the Baseline group (DB), Post-trial Control group (DS), C. perfringens + B. velezensis group (BV), or C. perfringens + amoxicillin group (AB). Microbiome Analysis The read counts obtained from the V1-V9 regions of the 16S rRNA gene ranged from 33,517 to 35,437 in the DB group, 28,945 to 38,920 in the DS group, 13,998 to 15,518 in the NE group, 41,781 to 49,590 in the BV group, and 37,469 to 44,506 in the AB group. Cecal Microbiome of the DB Group In the DB group, the most dominant phylum was Firmicutes (56.21%), followed by Bacteroidetes (27.38%), Proteobacteria (15.20%), Fusobacteria (0.9%), and Actinobacteria (0.31%). No bacteria belonging to the Synergistetes or Tenericutes phyla were detected. The number of bacterial families identified in each of the 16 chickens in the DB group is presented in Supplementary Data 1. At the family level, Oscillospiraceae (23.42%) and Enterobacteriaceae (14.11%) were the most abundant, followed by Porphyromonadaceae (11.09%), Lachnospiraceae (10.28%), Clostridiaceae (9.78%), and Rikenellaceae (8.57%). Families with a prevalence below 1% included 22 additional families. Relative changes at the family level were consistent among the 16 chickens. At the genus level, Enterobacteriaceae gen. (14%), Porphyromonas (11%), and Faecalibacterium (8.5%) were the most abundant, followed by Clostridium (6.7%), Coprococcus (5.1%), and Alistipes (4.9%). Genera with less than 1% abundance included 32 genera. The relative distribution of bacterial genera was consistent across the group. Cecal Microbiome of the DS Group The dominant phylum in the DS group was Firmicutes (72.64%), followed by Bacteroidetes (22.41%), Proteobacteria (4.04%), Actinobacteria (0.48%), Synergistetes (0.38%), and Fusobacteria (0.05%). No bacteria from the Tenericutes phylum were detected. Family-level bacterial compositions in the DS group are provided in Supplementary Data 3. The most abundant families were Oscillospiraceae (38.54%), Lachnospiraceae (14.68%), Clostridiaceae (13.57%), Rikenellaceae (9.17%), and Bacteroidaceae (11.96%). Families with less than 1% abundance totaled 30. The 16 chickens in the group exhibited a similar relative abundance of bacterial families. At the genus level, the DS group had a similar pattern, with Faecalibacterium (21.94%) being the most abundant, followed by Bacteroides (11.18%), Ruminiclostridium (9.62%), and Clostridium (7.93%). Cecal Microbiome of the NE Group In the NE group, which was exposed to C. perfringens , the dominant phyla were Firmicutes (52.30%) and Proteobacteria (44.98%). These were followed by Bacteroidetes (1.13%), Fusobacteria (0.90%), Actinobacteria (0.60%), Synergistetes (0.06%), and Tenericutes (0.03%). The most abundant family was Enterobacteriaceae (38.70%), followed by Clostridiaceae (23.70%), Erysipelotrichaceae (17.80%), Oscillospiraceae (6%), and Desulfovibrionaceae (5.8%). Families with less than 1% abundance included 19 additional families. The distribution of bacterial families remained stable across the 16 chickens in the group. At the genus level, the most dominant genera in the NE group were Enterobacteriaceae gen. (38.7%), Clostridium (20.3%), and Erysipelatoclostridium (16.1%). Other significant genera included Desulfovibrio (4.3%), Clostridiaceae incertae sedis (3.4%), and Bilophila (1.5%). Cecal Microbiome of the BV Group In the BV group, which received C. perfringens and B. velezensis , Firmicutes (71.48%) was the dominant phylum, followed by Bacteroidetes (20.37%), Proteobacteria (5.07%), Synergistetes (1.53%), Actinobacteria (1.48%), and Fusobacteria (0.08%). No bacteria from the Tenericutes phylum were detected. Oscillospiraceae (33.21%) was the most abundant family, followed by Lachnospiraceae (15.82%), Clostridiaceae (15.80%), Porphyromonadaceae (8.53%), and Rikenellaceae (5.39%). Families with less than 1% abundance totaled 26. The relative abundance of bacterial families was consistent among the 16 chickens in the group. At the genus level, Faecalibacterium (20.25%) was the most abundant genus in the BV group, followed by Porphyromonas (8.54%), Clostridium (8.41%), and Clostridiaceae incertae sedis (7.19%). Cecal Microbiome of the AB Group In the AB group, which received C. perfringens and amoxicillin, Firmicutes (49.95%) was the dominant phylum, followed by Bacteroidetes (43.27%), Proteobacteria (5.90%), Actinobacteria (0.52%), Synergistetes (0.19%), Tenericutes (0.16%), and Fusobacteria (0.03%). Oscillospiraceae (32.34%) was the most abundant family, followed by Lachnospiraceae (4.91%), Clostridiaceae (7.15%), and Rikenellaceae (19.62%). Families with less than 1% abundance totaled 23. The relative abundance of bacterial families was consistent among the 16 chickens in the group. At the genus level, Faecalibacterium (21.06%) was the most abundant genus in the AB group, followed by Bacteroides (17.72%), Rikenella (13.73%), and Ruminiclostridium (7.66%). Evaluation of Next-Generation Sequencing Results Taxonomic Diversity/Relative Abundance A significant variation between groups at the phylum level was identified through statistical analysis (p < 0.01). (Table 1 ). Relative abundance diagrams were used to visualize these differences: Table 1 Results of Statistical Analysis Between Groups at the Phylum Level Phylum Groups p Value DB DS NE BV AB Mean SD Mean SD Mean SD Mean SD Mean SD Firmicutes 55,93 1,38b 72,75 3,35a 52,29 0,95c 71,49 1,68a 49,96 2,53d < 0.01 Bacteroidetes 27,24 0,79b 22,31 3,37c 1,13 0,06d 20,37 1,88c 43,24 2,45a < 0.01 Proteobacteria 15,12 1,05b 4,03 0,49e 44,99 0,91a 5,07 0,57d 5,91 0,68c < 0.01 Actinobacteria 0,31 0,03b 0,48 0,11b 0,60 0,03b 1,47 0,8a 0,52 0,11b < 0.01 Synergistetes 0,00 0,00 0,38 0,08b 0,06 0,02d 1,53 0,13a 0,19 0,03c < 0.01 Fusobacteria 0,90 0,06a 0,05 0,02bc 0,89 0,06a 0,08 0,03b 0,03 0,01c < 0.01 Tenericutes 0,00 0,00 0,00 0,00 0,03 0,03b 0,00 0,00 0,16 0,04a < 0.01 Firmicutes was most prevalent in the BV group (71.48%), followed closely by the DS group (72.64%). It was significantly lower in the AB group (49.95%) and further reduced in the NE group (52.30%). Bacteroidetes showed higher prevalence in the AB (43.27%) and BV (20.37%) groups compared to the DS group (22.41%), while its abundance was significantly lower in the NE group (1.13%). Proteobacteria was most abundant in the NE group (44.98%) and least in the DS group (4.04%). Other phyla such as Actinobacteria, Synergistetes, and Fusobacteria showed varying trends across the groups (Fig. 1 ). A comparison of the taxonomic relative abundances of bacterial communities at the family level and genus level across groups is presented in Fig. 2 and Fig. 3 , respectively. Taxonomic Richness A quantitative assessment of bacterial taxonomic variations across phylum, class, order, family, and genus levels indicated that the BV group exhibited the highest similarity to the DS group in taxonomic richness. Conversely, the NE and AB groups elicited reduced taxonomic richness compared to the DS group (Fig. 4 ). Common Taxa Between Groups At the phylum level, six shared phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Synergistetes, and Fusobacteria) were identified in the DS and BV groups. The NE and AB groups contained these six phyla as well as Tenericutes. The DB group shared five phyla with the DS group, excluding Synergistetes. At the family level, the BV group shared 34 families with the DS group, the highest among the groups. The DB group shared 32 families, the AB group shared 28 families, and the NE group shared 21 families with the DS group. At the genus level, the BV group shared 72 genera with the DS group, the highest among the groups. The DB group shared 50 genera, the AB group shared 55 genera, and the NE group shared 36 genera with the DS group. Venn Diagram Analysis Venn diagrams were utilized to visualize the overlap of bacterial taxa between groups at the family and genus levels (Figs. 5 and 6 ). The BV group showed the greatest similarity to the DS group in terms of both family and genus richness, followed by the AB group. The NE group was the most distinct from the DS group. At the family level, the similarity percentages between the DS group and other groups were as follows: DS vs. BV: 73.9%, DS vs. AB: 70.09%, DS vs. NE: 45.7%, DS vs. DB: 84.21%. At the genus level, the similarity percentages between the DS group and other groups were as follows: DS vs. BV: 78.3%, DS vs. AB: 60.9%, DS vs. NE: 39.2%, DS vs. DB: 63.29% Alpha Diversity Alpha diversity analysis of cecal microbiota was performed to compare OTU (Operational Taxonomic Unit) richness across the five experimental groups. The diversity indices revealed: Lower diversity in the DB and NE groups. Significantly higher diversity in the DS, BV, and AB groups. The results are shown in Fig. 7 . Statistical analysis confirmed significant differences in alpha diversity across the groups (Kruskal-Wallis chi-squared = 74.475, df = 4, p-value = 2.573e-15). Beta Diversity Beta diversity analysis demonstrated significant compositional differences among the groups. The observed variations explained 30.4% of the total variance (Fig. 8 ). Among the groups, BV was the closest to the healthy controls (DS group), with AB ranking next in similarity. The NE group showed the lowest beta diversity, suggesting the most significant microbial community disruption. Discussion NE is a serious intestinal disorder that can profoundly impact the composition of the gut microbiota (Stanley et al., 2014 ). Various predisposing factors contribute to NE, with fishmeal being one of the most commonly used triggers. As previously reported, fishmeal diets increase intestinal mucus secretion and viscosity, creating an environment conducive to pathogen proliferation and leading to NE infections (Shojadoost et al., 2012 ). C. perfringens exerts its effects through the production of alpha toxin, NetB toxin, and various enzymes (Takehara et al., 2016). In animal health, the host microbiota plays a key role by improving nutrient uptake, promoting growth and metabolism, safeguarding against pathogenic bacteria, and influencing immune function. This study demonstrated the beneficial impact of B. velezensis on gut microbiota. The bacterium likely improves the intestinal barrier function, inhibits endotoxins and pathogens from entering the bloodstream, and produces metabolites that enhance antimicrobial activity. Furthermore, B. velezensis enhances immune responses by promoting lymphocyte activation, boosting immunoglobulin levels, and improving both cellular and humoral immunity (Deng et al., 2012 ). In our study, the cecal microbiota of the DS group was predominantly composed of the phyla Firmicutes, Bacteroidetes, and Proteobacteria, aligning with previous findings in normal chickens (Qu et al., 2008 ; Wei et al., 2013 ; Oakley et al., 2014 ; Yang et al., 2019 ). The BV group exhibited a microbial composition similar to that of the DS group. In contrast, the NE group showed a significant reduction in Firmicutes and Bacteroidetes, accompanied by an increase in Proteobacteria levels. Meanwhile, in the AB group, Firmicutes levels declined, whereas Bacteroidetes abundance doubled. Bacteria within the Firmicutes (Anaerotruncus, Anaerostipes, Faecalibacterium, Megasphaera, Oscillibacter, Subdoligranum, and Butyrivibrio) and Bacteroidetes (Alistipes, Bacteroides, Parabacteroides, Paraprevotella, Prevotella, Tannerella) phyla contribute to the synthesis of short-chain fatty acids (SCFAs) such as butyrate and propionate (Polansky et al., 2016 ). SCFAs are taken up through passive diffusion across the cecal epithelium and participate in various metabolic pathways. Additionally, these SCFAs influence intestinal blood circulation, promote enterocyte growth and proliferation, regulate mucin secretion, and play a role in intestinal immune responses (Tellez et al., 2006 ; Pan & Yu, 2014 ). The reduction of Firmicutes and Bacteroidetes in the NE group likely weakened intestinal immunity, leading to the proliferation of Proteobacteria. The latter group includes pathogenic Gram-negative bacteria that produce lipopolysaccharides, triggering inflammatory responses in the host. The altered Firmicutes/Bacteroidetes ratio in the AB group appears to be due to the selective pressure of antibiotics. Members of the Lachnospiraceae family adhere to the intestinal epithelium and influence the host immune system (Thompson et al., 2013 ). The presence of fishmeal leads to a reduction in butyrate-producing strains of Oscillospiraceae. Likewise, fishmeal decreases the abundance of butyrate-producing bacteria within the Lachnospiraceae family. These microbial alterations suggest that the gut microbiota's immunoregulatory effects are crucial in counteracting the necrotic effects of C. perfringens. The colonization of butyrate-producing bacteria is vital for reducing inflammation and preserving gut integrity. In our study, Oscillospiraceae and Lachnospiraceae levels were similar in the DS and BV groups but significantly reduced in the NE group. In the AB group, Oscillospiraceae levels were similar to the DS group, whereas Lachnospiraceae levels decreased significantly. The Enterobacteriaceae family was most abundant in the NE group, consistent with its role as an enteric pathogen that colonizes the gut and triggers disease (Mora et al., 2010 ). The genus Faecalibacterium was the most abundant in the DS group (21.94%) and was similar in the BV (20.25%) and AB (21.06%) groups. However, this genus was absent in the NE group. Faecalibacterium is known to contribute to gut health through SCFA production, immune modulation, and anti-inflammatory properties. The genus Bacteroides, another key SCFA producer, showed increased abundance in the AB group but was reduced in the BV group compared to the DS group. Interestingly, Lactobacillus was entirely absent in the NE group, despite its documented ability to produce lactic acid, lower intestinal pH, and inhibit pathogenic bacterial growth (Belenguer et al., 2007 ; Sengupta et al., 2013 ). Other studies have reported varying impacts of C. perfringens on Lactobacillus populations, suggesting that these interactions may depend on host diet and immune system differences. Conclusion Probiotics are increasingly used in poultry farming to promote growth and improve animal health by protecting against enteric pathogens, particularly as antibiotic growth promoters have been banned in many countries. Bacillus species are favored among probiotics for their resilience against environmental stressors, including heat, UV radiation, extended storage, low pH, and the harsh conditions of the gastrointestinal tract. B. velezensis exhibits antimicrobial properties through the synthesis of metabolites and volatile organic compounds, including surfactin, fengycin, bacillibactin, difficidin, bacillaene, macrolactin, and acetoin (Rabbee et al., 2019). This study, for the first time, highlighted the significant role of B. velezensis supplementation in regulating gut microbiota in chickens affected by necrotic enteritis. The microbial diversity and richness in the BV group were statistically similar to those in the DS control group, suggesting that B. velezensis may act as a biological antagonist, preventing microbial dysbiosis by strengthening mucosal immune responses and enhancing epithelial barrier function. The results indicate that B. velezensis supplementation may help alleviate the detrimental effects of necrotic enteritis on gut microbiota. These findings emphasize its potential as an alternative to prophylactic antibiotics in reducing the negative impact of necrotic enteritis on the microbiome. The data from this study offer new perspectives on the prevention and management of necrotic enteritis in poultry. Further research is needed to assess the long-term effects of B. velezensis on gut microbiota and its possible contributions to enhancing poultry health and productivity. Declarations Data availability The data and material generated and/or analysed during the current study are available from the corresponding author on a reasonable request. Funding This thesis was supported by the Aydın Adnan Menderes University Scientific Research Projects Unit under project number VTF-190002. Contributions “OYÇ and SD contributed to the study conception and design. Material preparation, data collection and analysis were performed by OYÇ, SD and SK. The first draft of the manuscript was written by OYÇ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.” Ethics declarations Ethics approval This research was carried out with the authorization of the Ethics Committee of İzmir/Bornova Veterinary Control Institute, dated 25/07/2018, with decision number 71705440-170-2228. Consent to participate Not applicable. Consent to publish Not applicable. Competing interests The authors have no relevant financial or non-financial interests to disclose. References Amara, A. A., Shibl, A. (2015). Role of probiotics in health improvement, infection control and disease treatment and management. Saudi Pharmaceutical Journal , 23(2), 107-114. doi: 10.1016/j.jsps.2013.07.001 Belenguer, A., Duncan, S.H., Holtrop, G., Anderson, S.E., Lobley, G.E., Flint, H.J. (2007). 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Characterization and complete genome analysis of the surfactin-producing, plant-protecting bacterium Bacillus velezensis 9D-6. BMC Microbiology , 19, 5. doi: 10.1186/s12866-018-1380-8 Keyburn, A. L., Boyce, J. D., Vaz, P., Bannam, T. L., Ford, M. E., Parker, D., … Moore, R. J. (2008). NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathogens , 4(2), e26. doi: 10.1371/journal.ppat.0040026 Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, J., Horn, M., Glöckner, F.O. (2013). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research, 41(1): e1. doi: 10.1093/nar/gks808 Kogut, M. H., Yin, X., Yuan, J., Bloom, L. (2017). Gut health in poultry. CAB Reviews , 12(031). doi: 10.1079/PAVSNNR201712031 M’sadeq, S. A., Wu, S., Swick, R. A., Choct, M. (2015). Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. Animal Nutrition , 1, 1-11. doi: 10.1016/j.aninu.2015.02.004 Mora, A., Herrera, A., Mamani, R., López, C., Alonso, M.P., … Blanco, J. (2010). Recent emergence of clonal group O25b:K1:H4-B2-ST131 ibeA strains among Escherichia coli poultry isolates, including CTX-M-9-producing strains, and comparison with clinical human isolates. Applied and Environmental Microbiology , 76(21), 6991-7. doi: 10.1128/AEM.01112-10 Oakley, B.B., Lillehoj, H.S., Kogut, M.H., Kim, W.K., Maurer, J.J., … Pedroso, A. (2014). The chicken gastrointestinal microbiome. FEMS Microbiology Letters , 360(2), 100–12. https://doi.org/10.1111/1574-6968. Opengart, K., Songer, J.G. (2013). Clostridial Diseases. In D.E. Swayne (Ed.), Diseases of Poultry (13th ed., pp. 949). Iowa: Blackwell Publishing Ltd. Pan, D., Yu, Z. (2014). Intestinal microbiome of poultry and its interaction with host and diet. Gut Microbes , 5(1), 108-119. doi: 10.4161/gmic.26945 Polansky, O., Sekelova, Z., Faldynova, M., Sebkova, A., Sisak, F., Rychlik, I. (2016). Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota. Applied and Environmental Microbiology , 82(1), 1569-1576. doi: 10.1128/AEM.03473-15 Prescott, J. F., Parreira, V. R., Gohari, I. M., Lepp, D., Gong, J. (2016). The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review. Avian Pathology , 45(3), 288-294. doi: 10.1080/03079457.2016.1139688 Qu, A., Brulc, J.M., Wilson, M.K., Law, B.F., Theoret, J.R., … Joens, L.A. (2008). Comparative metagenomics reveals host-specific metavirulomes and horizontal gene transfer elements in the chicken cecum microbiome. PLoS One , 3(8), e2945. https://doi.org/10.1371/journal.pone.0002945 Rabbee, M. F., Ali, M., Choi, J., Hwang, B. S., Jeong, S. C., Baek, K. (2019). Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes. Molecules , 24(6), 1046. doi: 10.3390/molecules24061046 Ramlucken, U., Ramchuran, S. O., Moonsamy, G., Lalloo, R., Thantsha, M. S., Jansen van Rensburg, C. (2020b). A novel Bacillus-based multi-strain probiotic improves growth performance and intestinal properties of Clostridium perfringens challenged broilers. Poultry Science , 99(1), 331-341. doi: 10.3382/ps/pez496 Sengupta, R., Altermann, E., Anderson, R.C., McNabb, W.C., Moughan, P.J., Roy, N.C. (2013). The role of cell surface architecture of lactobacilli in host-microbe interactions in the gastrointestinal tract. Mediators of Inflammation , 2013, 237921. https://doi.org/10.1155/2013/237921 Shojadoost, B., Vince, A. R., Prescott, J. F. (2012). The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review. Veterinary Research , 43, 74. doi: 10.1186/1297-9716-43-74 Stanley, D., Hughes, R. J., Moore, R. J. (2014). Microbiota of the chicken gastrointestinal tract: influence on health, productivity and disease. Applied Microbiology and Biotechnology , 98, 4301-4310. doi: 10.1007/s00253-014-5646-2 Takehara, M., Takagishi, T., Seike, S., Ohtani, K., Kobayashi, K., … Nagahama, M. (2016). Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation. Scientific Reports , 6, 28192. https://doi.org/10.1038/srep28192 Tellez, G., Higgins, S., Donoghue, A., Hargis, B. (2006). Digestive physiology and the role of microorganisms. Journal of Applied Poultry Research , 15(1), 136-144. doi: 10.1093/japr/15.1.136 Thompson, C.L., Mikaelyan, A., Brune, A. (2013). Immune-modulating gut symbionts are not “Candidatus Arthromitus”. Mucosal Immunology , 6, 200–201. Van Immerseel, F., Buck, J. D., Pasmans, F., Huyghebaert, G., Haesebrouck, F., Ducatelle, R. (2004). Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathology , 33(6), 537-549. doi: 10.1080/03079450400013162 Wade, B., Keyburn, A. (2015). The true cost of necrotic enteritis. World Poultry , 31, 16-17. Wei, S., Morrison, M., Yu, Z. (2013). Bacterial census of poultry intestinal microbiome. Poultry Science , 92, 67183. doi: 10.3382/ps.2012-02822 Wilson, M.M., Anderson, D.E., Bernstein, H.D. (2015). Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms. PLoS One , 10, e117732. https://doi.org/10.1371/journal.pone.0117732 Yang, W.Y., Lee, Y., Lu, H., Chou, C.H., Wang, C. (2019). Analysis of gut microbiota and the effect of lauric acid against necrotic enteritis in Clostridium perfringens and Eimeria side-by-side challenge model. PLoS ONE , 14(5): e0205784. doi: 10.1371/journal.pone.0205784 Ye, M., Wei, C., Khalid, A., Hu, Q., Yang, R., Dai1, B., … Wang, Z. (2020). Effect of Bacillus velezensis to substitute in-feed antibiotics on the production, blood biochemistry and egg quality indices of laying hens. BMC Veterinary Research , 16(1), 400. doi: 10.1186/s12917-020-02570-6 Zeng, H., Ishaq, S.L., Zhao, F., Wright, A.G. (2013). Comparative metagenomics of the rumen microbiome associated with different diets in dairy cattle. Microbial Ecology, 65 (3), 528-538. doi: 10.1007/s00248-012-0158-9 Supplementary Data Supplemental Data files are not available with this version. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 24 Mar, 2025 Reviewers invited by journal 23 Mar, 2025 Editor assigned by journal 20 Mar, 2025 First submitted to journal 17 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6190428","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432706449,"identity":"2a8161b5-f377-435d-b2ba-bd7f8b29de69","order_by":0,"name":"Ozge YILMAZ 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Phylum Level\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/6f712ddca8537622db13755a.png"},{"id":79801627,"identity":"e69e84e0-3f48-4965-8581-d2134d1e5a30","added_by":"auto","created_at":"2025-04-03 04:06:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1106850,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the Taxonomic Relative Abundances of Bacterial Communities at the Family Level Among Groups\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/8db1d7602e7897c6745cf8bb.png"},{"id":79802021,"identity":"68398884-1edf-44c4-88a0-39d848ac543f","added_by":"auto","created_at":"2025-04-03 04:14:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1182660,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the Taxonomic Relative Abundances of Bacterial Communities at the Genus Level Among Groups\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/bb75f3f141bfa228e70b626e.png"},{"id":79802195,"identity":"22621de7-e0ed-4a2c-a1ed-86f2f68f6026","added_by":"auto","created_at":"2025-04-03 04:22:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":704140,"visible":true,"origin":"","legend":"\u003cp\u003eIntergroup Taxon Counts by Taxonomic Levels A) Trial Start Control Group (DB) B) Post-Trial Control Group (DS) C) \u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e Administered Group (NE) D) \u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e + \u003cem\u003e\u003cstrong\u003eB. velezensis\u003c/strong\u003e\u003c/em\u003e Administered Group (BV) E) \u003cem\u003e\u003cstrong\u003eC. perfringens\u003c/strong\u003e\u003c/em\u003e + Amoxicillin Administered Group (AB)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/1e738d2d456918c4cc40a3e5.png"},{"id":79801631,"identity":"0403a334-1e41-4923-b438-183bc7e48876","added_by":"auto","created_at":"2025-04-03 04:06:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":295036,"visible":true,"origin":"","legend":"\u003cp\u003eVenn Diagram at the Family Level\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/264cd7a8ad6b9fff58b4417b.png"},{"id":79801634,"identity":"6932904d-0c92-463c-9a78-83dab72c2fe7","added_by":"auto","created_at":"2025-04-03 04:06:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":291140,"visible":true,"origin":"","legend":"\u003cp\u003eVenn Diagram at the Genus Level\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/cf951e713df487ef093da034.png"},{"id":79801641,"identity":"ed144b21-7554-4d2c-b565-168c92cf52fd","added_by":"auto","created_at":"2025-04-03 04:06:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":297352,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha-Diversity Scores of the Groups\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/7eac3eef99aa2e90a835911d.png"},{"id":79802023,"identity":"9bd00f24-caac-4315-8c02-64154b723f0e","added_by":"auto","created_at":"2025-04-03 04:14:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":308939,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Coordinate Analysis (PCA-plot) of the Similarities in Microbiome Analysis Results from the Groups Based on the Bray-Curtis Index\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/38c8a8c8e84494e66686f5ac.png"},{"id":79802614,"identity":"4a75d6d6-079b-49f9-8c35-b0118b79284f","added_by":"auto","created_at":"2025-04-03 04:30:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5155338,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6190428/v1/3a35ebda-f66f-442b-b9b9-26f45b8bb7ec.pdf"}],"financialInterests":"","formattedTitle":"Intestinal Microbiom in Necrotic Enteritis Infection of Broiler and Comparison of Treatment Alternatives","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNecrotic enteritis (NE), a gastrointestinal disease that results in major financial losses for poultry, is caused by \u003cem\u003eC. perfringens\u003c/em\u003e. This disease, particularly affecting broiler chickens, results in reduced feed conversion rates, high mortality rates, and severe deterioration of animal welfare (M\u0026rsquo;sadeq et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Opengart \u0026amp; Songer, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). NE, forecasted to impose an annual financial burden of \u003cspan\u003e$\u003c/span\u003e6\u0026nbsp;billion on the global poultry industry, has become a widespread and challenging problem to control (Wade \u0026amp; Keyburn, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). With the ban on antimicrobial feed additives, the incidence of this disease has increased, further emphasizing the need for alternative control strategies (Van Immerseel et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe spore-forming, anaerobic, Gram-positive bacterium \u003cem\u003eC. perfringens\u003c/em\u003e is frequently found in the environment. It proliferates primarily in the small intestine, causing necrotic enteritis through toxin production and mechanical mucosal degradation in the intestines. While healthy chickens have \u003cem\u003eC. perfringens\u003c/em\u003e concentrations of 10\u0026sup2;\u0026ndash;10⁴ CFU/g in their intestines, concentrations of 10⁷\u0026ndash;10⁹ CFU/g can cause disease (Shojadoost et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The bacterium produces several virulence factors, such as alpha-toxin and NetB toxin, which cause severe damage to the intestinal epithelium (Keyburn et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The NetB toxin is particularly recognized as an essential virulence factor in the NE pathogenesis (Keyburn et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Prescott et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The gene encoding NetB toxin is carried on a conjugative plasmid, enabling the bacterium to easily spread its pathogenic genetic traits (Bannam et al., 2011; Parreira et al., 2012). Additionally, dietary components, physical damage to the intestinal mucosa, and environmental factors are predispositional contributors to the disease (Moorea, 2016).\u003c/p\u003e \u003cp\u003eProlonged antibiotic use for managing and efforts to treat NE have facilitated the rise of antibiotic resistance in \u003cem\u003eC. perfringens\u003c/em\u003e (Gillings et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This situation has increased interest in alternative therapeutic methods, Including probiotics, prebiotics, organic acids, and plant-derived compounds (Ramlucken et al., 2020c). Probiotics have been shown to regulate intestinal microbiota, inhibit toxin production, and support the host immune system by competing with pathogenic bacteria (Pan \u0026amp; Ye, 2014; Amara \u0026amp; Shibl, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among probiotics, \u003cem\u003eB. velezensis\u003c/em\u003e stands out for its ability to colonize the gut, produce antimicrobial metabolites, and support gut health (Ye et al., 2018; Grady et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies have proven that Bacillus species improve villus morphology and feed conversion rates by colonizing the intestinal surface (Ramlucken et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA healthy gut microbiome is vital for both host well-being and immune responses, and metabolic processes. It supports diverse functions, involving nutrient digestion and absorption, exclusion of pathogens, and regulation of immune responses (Kogut et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Maintaining a balanced gut microbiota contributes to preventing diseases like NE by restricting the growth of harmful bacteria (Wilson et al., 2018). However, factors such as antibiotic use, poor nutrition, and stress can disrupt the gut microbiome, creating a predisposition to disease (Pan \u0026amp; Yu, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Regulating the microbiome is essential to enhancing animal welfare and preventing diseases (Kogut et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis research focuses on evaluating the effects of \u003cem\u003eB. velezensis\u003c/em\u003e in regulating necrotic enteritis triggered by \u003cem\u003eC. perfringens.\u003c/em\u003e The study investigates the probiotic's impact on the gut microbiome, its potential to inhibit toxin production, and its role in promoting resistance to the disease. The findings are expected to contribute to sustainable and effective control strategies that reduce the need for antibiotics.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eDaily chicks with similar body weights (~\u0026thinsp;45 g) were used in the study. Eighty chicks in total were allocated into five distinct groups: Trial Baseline Group (DB), Post-Trial Control Group (DS), \u003cem\u003eC. perfringens\u003c/em\u003e-exposed Group (NE), \u003cem\u003eC. perfringens\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. velezensis\u003c/em\u003e Group (BV), and \u003cem\u003eC. perfringens\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Amoxicillin Group (AB).\u003c/p\u003e \u003cp\u003eFor the isolation and culture of C. perfringens, Tryptose Sulfite Cyclocerine (TSC) Agar, blood agar, Fluid Thioglycollate (FTG) Medium, and Cooked Meat Medium (CMM) were used.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. perfringens\u003c/em\u003e NCTC 8239 strain was used for the challenge application. The \u003cem\u003eB. velezensis\u003c/em\u003e strain was obtained from the culture collection of Aydın Adnan Menderes University Department of Microbiology.\u003c/p\u003e \u003cp\u003eThe chicks received a corn-soy-based diet during the first 13 days. As of day 14, all groups other than the control group received specific experimental treatments were fed a diet containing 30% fish meal.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. perfringens\u003c/em\u003e strain was incubated under anaerobic conditions on blood agar for 18 hours. The resulting colonies were sequentially transferred to minced meat broth and FTG media, and their culture concentration was increased with 12\u0026ndash;15 hour incubations. As a result, a bacterial culture with a density of 2\u0026ndash;5\u0026times;10⁸ CFU/ml was prepared for use in the challenge.\u003c/p\u003e\n\u003ch3\u003eChallenge\u003c/h3\u003e\n\u003cp\u003eThe chicks were fed a corn-soy-based diet from day 1 to day 13. Starting on day 14, all groups except the control group were switched to a diet containing 30% fish meal. Throughout the trial, water was made available to the chicks ad libitum, and sawdust was used as bedding material. From days 21 to 25, \u003cem\u003eC. perfringens\u003c/em\u003e culture (1.25 FTG\u0026ndash;1.5 feed [v/w]), previously prepared, was administered twice daily along with the feed to the NE, BV, and AB groups. From days 26 to 30, the AB group was given 20 mg/kg of amoxicillin diluted in drinking water once daily orally. In the BV group, the lyophilized form of \u003cem\u003eB. velezensis\u003c/em\u003e (10\u0026sup1;\u0026sup1; spores/g sucrose-filled) was used. The lyophilized powder was freshly diluted in distilled water to a dose of 10⁸ spores and administered orally to each chick twice daily. After the \u003cem\u003eB. velezensis\u003c/em\u003e and antibiotic applications, euthanasia was performed on day 32 via cervical dislocation for all groups, including the control group. Cecal contents were collected from the chickens for molecular analysis and stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eMicrobiome Analysis\u003c/h3\u003e\n\u003cp\u003eMetagenomic analysis targeting the 16S rRNA gene was performed using previously defined workflows (Cusco et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The primer pair used for amplicon library preparation targeted approximately 1400 bp covering the V1\u0026ndash;V9 regions of the 16S rRNA gene (Zeng et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Klindworth et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Oxford Nanopore Technologies\u0026rsquo; Nanopore barcode DNA sequences were added to the 5\u0026rsquo; ends of the target-specific primer pairs. The target-specific primer-connector sequences for 16S rRNA were as follows: forward primer 5\u0026rsquo;-TTTCTGTTGGTGCTGATATTGC-AGRGTTTGATYHTGGCTCAG-3\u0026rsquo; and reverse primer 5\u0026rsquo;-ACTTGCCTGTCGCTCTATCTTC-TACCTTGTTAYGACTT-3\u0026rsquo;. The initial PCR was performed using a Proof Reading DNA Polymerase 2x Reaction Mix with 200 nm of each primer. The thermal cycling program was as follows: 95\u0026deg;C for 3 minutes; 25 cycles of 95\u0026deg;C for 30 seconds, 55\u0026deg;C for 30 seconds, and 72\u0026deg;C for 90 seconds; and a final extension at 72\u0026deg;C for 5 minutes. The PCR product was electrophoresed on an agarose gel to confirm its size (~\u0026thinsp;1450 bp) and purified using a PCR Product Purification Kit.\u003c/p\u003e \u003cp\u003eFor amplicon library preparation, the Ligase Sequencing Kit 1D (SQK-LSK108; Oxford Nanopore Technologies) was used, and the library was loaded onto the MinION\u0026trade; (Oxford Nanopore Technologies) device. A mixture containing 1\u0026ndash;1.5 \u0026micro;g DNA with 45 \u0026micro;l of barcoded DNA and 5 \u0026micro;l of lambda phage DNA as a positive control was loaded onto the device. The NEBNext End Repair/dA-tailing Module (New England Biolabs) kit was used for DNA end repair and dA-tailing. Purification was performed using the Agencourt AMPure XP beads (Beckman Coulter) kit.\u003c/p\u003e \u003cp\u003eFor adapter ligation, 0.2 pmol of DNA ends were mixed with 50 \u0026micro;l of Blunt/TA Ligase Master Mix (New England Biolabs) and incubated at room temperature for 10 minutes with the addition of 20 \u0026micro;l of adapter mix. The final purification of the DNA library was completed using the Adapter Bead Binding Buffer (provided in the SQK-LSK108 kit) and 0.5X Agencourt AMPure XP beads (Beckman Coulter).\u003c/p\u003e \u003cp\u003eThe sequencing mix (14 \u0026micro;l DNA library), loading beads (25.5 \u0026micro;l), and running buffer mixture (35.5 \u0026micro;l) were prepared and loaded onto the primed R9.4 flow cell. A 48-hour sequencing protocol was performed using MinION\u0026trade; control software, MinKNOW\u0026trade; version 0.46.1.9 (R9.4). Read data were generated by base-calling with the Guppy v3.1.5 software and demultiplexed. Barcode and adapter sequences were trimmed using the Porechop v0.2.3 software. Reads with lengths of 1350\u0026ndash;1550 bp were filtered, and other reads were excluded from the analysis.\u003c/p\u003e \u003cp\u003eFiltered reads were analyzed with the Mothur v.1.39.5 platform using a customized workflow. The sequences were de-chimerized, aligned, and clustered into operational taxonomic units (OTUs) based on \u0026gt;\u0026thinsp;99% similarity. OTUs were taxonomically annotated using the RDP 16S rRNA database, and statistical results were obtained by grouping similar OTUs.\u003c/p\u003e\n\u003ch3\u003eBioinformatic Analysis\u003c/h3\u003e\n\u003cp\u003eThe similarities of bacterial communities in the cecal contents collected from the groups (DB, DS, NE, BV, AB) were evaluated using Principal Coordinate Analysis (PCA) applied to the Bray-Curtis distance matrix obtained from the 'vegan' R package. The first two principal components, PC1 and PC2, were visualized in two dimensions. Visualizations were performed using the 'factoextra' and 'ggplot2' packages in R.\u003c/p\u003e \u003cp\u003eRelative abundance (%) graphs were created using Excel 2013 (Microsoft Office, USA). Statistical analyses were performed using SPSS version 22.0 (IBM Corp., USA). Differences in phylum, family, and genus levels between groups were evaluated using the Kruskal-Wallis test and one-way ANOVA for independent samples. The significance of differences was determined using the post-hoc Tukey test, with a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eClinical Observations and Pathological Changes\u003c/h2\u003e \u003cp\u003eDuring the trial, no clinical signs or mortalities were observed in any group. Post-trial necropsy revealed pathological alterations observed exclusively in the intestines of chickens from the \u003cem\u003eC. perfringens\u003c/em\u003e group (NE). The intestines were swollen, and the mucosa displayed mild hemorrhages. No macroscopic findings were observed in the Baseline group (DB), Post-trial Control group (DS), \u003cem\u003eC. perfringens\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. velezensis\u003c/em\u003e group (BV), or \u003cem\u003eC. perfringens\u003c/em\u003e\u0026thinsp;+\u0026thinsp;amoxicillin group (AB).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicrobiome Analysis\u003c/h3\u003e\n\u003cp\u003eThe read counts obtained from the V1-V9 regions of the 16S rRNA gene ranged from 33,517 to 35,437 in the DB group, 28,945 to 38,920 in the DS group, 13,998 to 15,518 in the NE group, 41,781 to 49,590 in the BV group, and 37,469 to 44,506 in the AB group.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCecal Microbiome of the DB Group\u003c/h2\u003e \u003cp\u003eIn the DB group, the most dominant phylum was Firmicutes (56.21%), followed by Bacteroidetes (27.38%), Proteobacteria (15.20%), Fusobacteria (0.9%), and Actinobacteria (0.31%). No bacteria belonging to the Synergistetes or Tenericutes phyla were detected.\u003c/p\u003e \u003cp\u003eThe number of bacterial families identified in each of the 16 chickens in the DB group is presented in Supplementary Data 1. At the family level, Oscillospiraceae (23.42%) and Enterobacteriaceae (14.11%) were the most abundant, followed by Porphyromonadaceae (11.09%), Lachnospiraceae (10.28%), Clostridiaceae (9.78%), and Rikenellaceae (8.57%). Families with a prevalence below 1% included 22 additional families. Relative changes at the family level were consistent among the 16 chickens.\u003c/p\u003e \u003cp\u003eAt the genus level, Enterobacteriaceae gen. (14%), Porphyromonas (11%), and Faecalibacterium (8.5%) were the most abundant, followed by Clostridium (6.7%), Coprococcus (5.1%), and Alistipes (4.9%). Genera with less than 1% abundance included 32 genera. The relative distribution of bacterial genera was consistent across the group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCecal Microbiome of the DS Group\u003c/h2\u003e \u003cp\u003eThe dominant phylum in the DS group was Firmicutes (72.64%), followed by Bacteroidetes (22.41%), Proteobacteria (4.04%), Actinobacteria (0.48%), Synergistetes (0.38%), and Fusobacteria (0.05%). No bacteria from the Tenericutes phylum were detected.\u003c/p\u003e \u003cp\u003eFamily-level bacterial compositions in the DS group are provided in Supplementary Data 3. The most abundant families were Oscillospiraceae (38.54%), Lachnospiraceae (14.68%), Clostridiaceae (13.57%), Rikenellaceae (9.17%), and Bacteroidaceae (11.96%). Families with less than 1% abundance totaled 30. The 16 chickens in the group exhibited a similar relative abundance of bacterial families.\u003c/p\u003e \u003cp\u003eAt the genus level, the DS group had a similar pattern, with Faecalibacterium (21.94%) being the most abundant, followed by Bacteroides (11.18%), Ruminiclostridium (9.62%), and Clostridium (7.93%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCecal Microbiome of the NE Group\u003c/h2\u003e \u003cp\u003eIn the NE group, which was exposed to \u003cem\u003eC. perfringens\u003c/em\u003e, the dominant phyla were Firmicutes (52.30%) and Proteobacteria (44.98%). These were followed by Bacteroidetes (1.13%), Fusobacteria (0.90%), Actinobacteria (0.60%), Synergistetes (0.06%), and Tenericutes (0.03%).\u003c/p\u003e \u003cp\u003eThe most abundant family was Enterobacteriaceae (38.70%), followed by Clostridiaceae (23.70%), Erysipelotrichaceae (17.80%), Oscillospiraceae (6%), and Desulfovibrionaceae (5.8%). Families with less than 1% abundance included 19 additional families. The distribution of bacterial families remained stable across the 16 chickens in the group.\u003c/p\u003e \u003cp\u003eAt the genus level, the most dominant genera in the NE group were Enterobacteriaceae gen. (38.7%), Clostridium (20.3%), and Erysipelatoclostridium (16.1%). Other significant genera included Desulfovibrio (4.3%), Clostridiaceae incertae sedis (3.4%), and Bilophila (1.5%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCecal Microbiome of the BV Group\u003c/h2\u003e \u003cp\u003eIn the BV group, which received \u003cem\u003eC. perfringens\u003c/em\u003e and \u003cem\u003eB. velezensis\u003c/em\u003e, Firmicutes (71.48%) was the dominant phylum, followed by Bacteroidetes (20.37%), Proteobacteria (5.07%), Synergistetes (1.53%), Actinobacteria (1.48%), and Fusobacteria (0.08%). No bacteria from the Tenericutes phylum were detected.\u003c/p\u003e \u003cp\u003eOscillospiraceae (33.21%) was the most abundant family, followed by Lachnospiraceae (15.82%), Clostridiaceae (15.80%), Porphyromonadaceae (8.53%), and Rikenellaceae (5.39%). Families with less than 1% abundance totaled 26. The relative abundance of bacterial families was consistent among the 16 chickens in the group.\u003c/p\u003e \u003cp\u003eAt the genus level, Faecalibacterium (20.25%) was the most abundant genus in the BV group, followed by Porphyromonas (8.54%), Clostridium (8.41%), and Clostridiaceae incertae sedis (7.19%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCecal Microbiome of the AB Group\u003c/h2\u003e \u003cp\u003eIn the AB group, which received \u003cem\u003eC. perfringens\u003c/em\u003e and amoxicillin, Firmicutes (49.95%) was the dominant phylum, followed by Bacteroidetes (43.27%), Proteobacteria (5.90%), Actinobacteria (0.52%), Synergistetes (0.19%), Tenericutes (0.16%), and Fusobacteria (0.03%).\u003c/p\u003e \u003cp\u003eOscillospiraceae (32.34%) was the most abundant family, followed by Lachnospiraceae (4.91%), Clostridiaceae (7.15%), and Rikenellaceae (19.62%). Families with less than 1% abundance totaled 23. The relative abundance of bacterial families was consistent among the 16 chickens in the group.\u003c/p\u003e \u003cp\u003eAt the genus level, Faecalibacterium (21.06%) was the most abundant genus in the AB group, followed by Bacteroides (17.72%), Rikenella (13.73%), and Ruminiclostridium (7.66%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Next-Generation Sequencing Results\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eTaxonomic Diversity/Relative Abundance\u003c/h2\u003e \u003cp\u003eA significant variation between groups at the phylum level was identified through statistical analysis (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Relative abundance diagrams were used to visualize these differences:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of Statistical Analysis Between Groups at the Phylum Level\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePhylum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ep Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eNE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eBV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eAB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFirmicutes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55,93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,38b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52,29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,95c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e71,49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,68a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e49,96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2,53d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroidetes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27,24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,79b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22,31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,37c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1,13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,06d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20,37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,88c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e43,24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2,45a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eProteobacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15,12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,05b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,49e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44,99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,91a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5,07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,57d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e5,91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,68c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eActinobacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,03b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,03b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1,47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0,52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSynergistetes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,08b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,02d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1,53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,13a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0,19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,03c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,02bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,03b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0,03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,01c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTenericutes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,03b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0,16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,04a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eFirmicutes was most prevalent in the BV group (71.48%), followed closely by the DS group (72.64%). It was significantly lower in the AB group (49.95%) and further reduced in the NE group (52.30%).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eBacteroidetes showed higher prevalence in the AB (43.27%) and BV (20.37%) groups compared to the DS group (22.41%), while its abundance was significantly lower in the NE group (1.13%).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eProteobacteria was most abundant in the NE group (44.98%) and least in the DS group (4.04%).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eOther phyla such as Actinobacteria, Synergistetes, and Fusobacteria showed varying trends across the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eA comparison of the taxonomic relative abundances of bacterial communities at the family level and genus level across groups is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomic Richness\u003c/h2\u003e \u003cp\u003eA quantitative assessment of bacterial taxonomic variations across phylum, class, order, family, and genus levels indicated that the BV group exhibited the highest similarity to the DS group in taxonomic richness. Conversely, the NE and AB groups elicited reduced taxonomic richness compared to the DS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCommon Taxa Between Groups\u003c/h2\u003e \u003cp\u003eAt the phylum level, six shared phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Synergistetes, and Fusobacteria) were identified in the DS and BV groups. The NE and AB groups contained these six phyla as well as Tenericutes. The DB group shared five phyla with the DS group, excluding Synergistetes.\u003c/p\u003e \u003cp\u003eAt the family level, the BV group shared 34 families with the DS group, the highest among the groups. The DB group shared 32 families, the AB group shared 28 families, and the NE group shared 21 families with the DS group.\u003c/p\u003e \u003cp\u003eAt the genus level, the BV group shared 72 genera with the DS group, the highest among the groups. The DB group shared 50 genera, the AB group shared 55 genera, and the NE group shared 36 genera with the DS group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eVenn Diagram Analysis\u003c/h2\u003e \u003cp\u003eVenn diagrams were utilized to visualize the overlap of bacterial taxa between groups at the family and genus levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The BV group showed the greatest similarity to the DS group in terms of both family and genus richness, followed by the AB group. The NE group was the most distinct from the DS group.\u003c/p\u003e \u003cp\u003eAt the family level, the similarity percentages between the DS group and other groups were as follows: DS vs. BV: 73.9%, DS vs. AB: 70.09%, DS vs. NE: 45.7%, DS vs. DB: 84.21%.\u003c/p\u003e \u003cp\u003eAt the genus level, the similarity percentages between the DS group and other groups were as follows: DS vs. BV: 78.3%, DS vs. AB: 60.9%, DS vs. NE: 39.2%, DS vs. DB: 63.29%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAlpha Diversity\u003c/h2\u003e \u003cp\u003eAlpha diversity analysis of cecal microbiota was performed to compare OTU (Operational Taxonomic Unit) richness across the five experimental groups. The diversity indices revealed: Lower diversity in the DB and NE groups. Significantly higher diversity in the DS, BV, and AB groups. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Statistical analysis confirmed significant differences in alpha diversity across the groups (Kruskal-Wallis chi-squared\u0026thinsp;=\u0026thinsp;74.475, df\u0026thinsp;=\u0026thinsp;4, p-value\u0026thinsp;=\u0026thinsp;2.573e-15).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eBeta Diversity\u003c/h2\u003e \u003cp\u003eBeta diversity analysis demonstrated significant compositional differences among the groups. The observed variations explained 30.4% of the total variance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Among the groups, BV was the closest to the healthy controls (DS group), with AB ranking next in similarity. The NE group showed the lowest beta diversity, suggesting the most significant microbial community disruption.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNE is a serious intestinal disorder that can profoundly impact the composition of the gut microbiota (Stanley et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Various predisposing factors contribute to NE, with fishmeal being one of the most commonly used triggers. As previously reported, fishmeal diets increase intestinal mucus secretion and viscosity, creating an environment conducive to pathogen proliferation and leading to NE infections (Shojadoost et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eC. perfringens\u003c/em\u003e exerts its effects through the production of alpha toxin, NetB toxin, and various enzymes (Takehara et al., 2016).\u003c/p\u003e \u003cp\u003eIn animal health, the host microbiota plays a key role by improving nutrient uptake, promoting growth and metabolism, safeguarding against pathogenic bacteria, and influencing immune function. This study demonstrated the beneficial impact of \u003cem\u003eB. velezensis\u003c/em\u003e on gut microbiota. The bacterium likely improves the intestinal barrier function, inhibits endotoxins and pathogens from entering the bloodstream, and produces metabolites that enhance antimicrobial activity. Furthermore, \u003cem\u003eB. velezensis\u003c/em\u003e enhances immune responses by promoting lymphocyte activation, boosting immunoglobulin levels, and improving both cellular and humoral immunity (Deng et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, the cecal microbiota of the DS group was predominantly composed of the phyla Firmicutes, Bacteroidetes, and Proteobacteria, aligning with previous findings in normal chickens (Qu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Oakley et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The BV group exhibited a microbial composition similar to that of the DS group. In contrast, the NE group showed a significant reduction in Firmicutes and Bacteroidetes, accompanied by an increase in Proteobacteria levels. Meanwhile, in the AB group, Firmicutes levels declined, whereas Bacteroidetes abundance doubled.\u003c/p\u003e \u003cp\u003eBacteria within the Firmicutes (Anaerotruncus, Anaerostipes, Faecalibacterium, Megasphaera, Oscillibacter, Subdoligranum, and Butyrivibrio) and Bacteroidetes (Alistipes, Bacteroides, Parabacteroides, Paraprevotella, Prevotella, Tannerella) phyla contribute to the synthesis of short-chain fatty acids (SCFAs) such as butyrate and propionate (Polansky et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). SCFAs are taken up through passive diffusion across the cecal epithelium and participate in various metabolic pathways. Additionally, these SCFAs influence intestinal blood circulation, promote enterocyte growth and proliferation, regulate mucin secretion, and play a role in intestinal immune responses (Tellez et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pan \u0026amp; Yu, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe reduction of Firmicutes and Bacteroidetes in the NE group likely weakened intestinal immunity, leading to the proliferation of Proteobacteria. The latter group includes pathogenic Gram-negative bacteria that produce lipopolysaccharides, triggering inflammatory responses in the host. The altered Firmicutes/Bacteroidetes ratio in the AB group appears to be due to the selective pressure of antibiotics.\u003c/p\u003e \u003cp\u003eMembers of the Lachnospiraceae family adhere to the intestinal epithelium and influence the host immune system (Thompson et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The presence of fishmeal leads to a reduction in butyrate-producing strains of Oscillospiraceae. Likewise, fishmeal decreases the abundance of butyrate-producing bacteria within the Lachnospiraceae family. These microbial alterations suggest that the gut microbiota's immunoregulatory effects are crucial in counteracting the necrotic effects of C. perfringens. The colonization of butyrate-producing bacteria is vital for reducing inflammation and preserving gut integrity.\u003c/p\u003e \u003cp\u003eIn our study, Oscillospiraceae and Lachnospiraceae levels were similar in the DS and BV groups but significantly reduced in the NE group. In the AB group, Oscillospiraceae levels were similar to the DS group, whereas Lachnospiraceae levels decreased significantly. The Enterobacteriaceae family was most abundant in the NE group, consistent with its role as an enteric pathogen that colonizes the gut and triggers disease (Mora et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genus Faecalibacterium was the most abundant in the DS group (21.94%) and was similar in the BV (20.25%) and AB (21.06%) groups. However, this genus was absent in the NE group. Faecalibacterium is known to contribute to gut health through SCFA production, immune modulation, and anti-inflammatory properties. The genus Bacteroides, another key SCFA producer, showed increased abundance in the AB group but was reduced in the BV group compared to the DS group.\u003c/p\u003e \u003cp\u003eInterestingly, Lactobacillus was entirely absent in the NE group, despite its documented ability to produce lactic acid, lower intestinal pH, and inhibit pathogenic bacterial growth (Belenguer et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sengupta et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Other studies have reported varying impacts of \u003cem\u003eC. perfringens\u003c/em\u003e on Lactobacillus populations, suggesting that these interactions may depend on host diet and immune system differences.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eProbiotics are increasingly used in poultry farming to promote growth and improve animal health by protecting against enteric pathogens, particularly as antibiotic growth promoters have been banned in many countries. Bacillus species are favored among probiotics for their resilience against environmental stressors, including heat, UV radiation, extended storage, low pH, and the harsh conditions of the gastrointestinal tract. \u003cem\u003eB. velezensis\u003c/em\u003e exhibits antimicrobial properties through the synthesis of metabolites and volatile organic compounds, including surfactin, fengycin, bacillibactin, difficidin, bacillaene, macrolactin, and acetoin (Rabbee et al., 2019).\u003c/p\u003e\n\u003cp\u003eThis study, for the first time, highlighted the significant role of \u003cem\u003eB. velezensis\u003c/em\u003e supplementation in regulating gut microbiota in chickens affected by necrotic enteritis. The microbial diversity and richness in the BV group were statistically similar to those in the DS control group, suggesting that \u003cem\u003eB. velezensis\u003c/em\u003e may act as a biological antagonist, preventing microbial dysbiosis by strengthening mucosal immune responses and enhancing epithelial barrier function.\u0026nbsp;The results indicate that \u003cem\u003eB. velezensis\u003c/em\u003e supplementation may help alleviate the detrimental effects of necrotic enteritis on gut microbiota. These findings emphasize its potential as an alternative to prophylactic antibiotics in reducing the negative impact of necrotic enteritis on the microbiome. The data from this study offer new perspectives on the prevention and management of necrotic enteritis in poultry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther research is needed to assess the long-term effects of \u003cem\u003eB. velezensis\u003c/em\u003e on gut microbiota and its possible contributions to enhancing poultry health and productivity.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and material generated and/or analysed during the current study are available from the corresponding author on a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis thesis was supported by the Aydın Adnan Menderes University Scientific Research Projects Unit under project number VTF-190002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“OYÇ and SD contributed to the study conception and design. Material preparation, data collection and analysis were performed by OYÇ, SD and SK. The first draft of the manuscript was written by OYÇ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was carried out with the authorization of the Ethics Committee of İzmir/Bornova Veterinary Control Institute, dated 25/07/2018, with decision number 71705440-170-2228.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eAmara, A. 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The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review. \u003cem\u003eAvian Pathology\u003c/em\u003e, 45(3), 288-294. doi: 10.1080/03079457.2016.1139688\u003c/li\u003e\n\u003cli\u003eQu, A., Brulc, J.M., Wilson, M.K., Law, B.F., Theoret, J.R., \u0026hellip; Joens, L.A. (2008). Comparative metagenomics reveals host-specific metavirulomes and horizontal gene transfer elements in the chicken cecum microbiome. \u003cem\u003ePLoS One\u003c/em\u003e, 3(8), e2945. https://doi.org/10.1371/journal.pone.0002945\u003c/li\u003e\n\u003cli\u003eRabbee, M. F., Ali, M., Choi, J., Hwang, B. S., Jeong, S. C., Baek, K. (2019). Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes. \u003cem\u003eMolecules\u003c/em\u003e, 24(6), 1046. doi: 10.3390/molecules24061046\u003c/li\u003e\n\u003cli\u003eRamlucken, U., Ramchuran, S. O., Moonsamy, G., Lalloo, R., Thantsha, M. S., Jansen van Rensburg, C. (2020b). 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Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms. \u003cem\u003ePLoS One\u003c/em\u003e, 10, e117732. https://doi.org/10.1371/journal.pone.0117732\u003c/li\u003e\n\u003cli\u003eYang, W.Y., Lee, Y., Lu, H., Chou, C.H., Wang, C. (2019). Analysis of gut microbiota and the effect of lauric acid against necrotic enteritis in Clostridium perfringens and Eimeria side-by-side challenge model. \u003cem\u003ePLoS ONE\u003c/em\u003e, 14(5): e0205784. doi: 10.1371/journal.pone.0205784\u003c/li\u003e\n\u003cli\u003eYe, M., Wei, C., Khalid, A., Hu, Q., Yang, R., Dai1, B., \u0026hellip; Wang, Z. (2020). Effect of Bacillus velezensis to substitute in-feed antibiotics on the production, blood biochemistry and egg quality indices of laying hens. \u003cem\u003eBMC Veterinary Research\u003c/em\u003e, 16(1), 400. doi: 10.1186/s12917-020-02570-6\u003c/li\u003e\n\u003cli\u003eZeng, H., Ishaq, S.L., Zhao, F., Wright, A.G. (2013). Comparative metagenomics of the rumen microbiome associated with different diets in dairy cattle. \u003cem\u003eMicrobial Ecology, 65\u003c/em\u003e(3), 528-538. doi: 10.1007/s00248-012-0158-9\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Data","content":"\u003cp\u003eSupplemental Data files are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"B. velezensis, C. perfringes, microbiome, challenge, broiler","lastPublishedDoi":"10.21203/rs.3.rs-6190428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6190428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eClostridium perfringens\u003c/em\u003eis the primary causative agent of necrotic enteritis (NE), a gastrointestinal disease that leads to substantial economic losses in poultry. This study aims to characterize the intestinal microbiome of chickens and assess the effects of Bacillus velezensis on gut microbiota and recovery from necrotic enteritis, comparing its efficacy to antibiotic treatment. The experiment involved five groups, each consisting of 16 chickens. The first group, the start-of-challenge (DB) group, included 1-day-old chicks. The second group, the post-challenge control (DS) group, was reared until the end of the trial. The third group was infected with \u003cem\u003eC. perfringens\u003c/em\u003e (NE group). The fourth group received both \u003cem\u003eC. perfringens\u003c/em\u003e and \u003cem\u003eB. velezensis\u003c/em\u003e (BV group), while the fifth group was treated with \u003cem\u003eC. perfringens\u003c/em\u003e and amoxicillin (AB group). All chickens were euthanized via cervical dislocation following the experimental infection. Fecal samples collected from the cecum underwent 16S rRNA gene-based metagenomic analysis, and the resulting data were statistically evaluated. Macroscopic examination after euthanasia revealed pathological changes in the intestines of chickens in the NE group, which had received only \u003cem\u003eC. perfringens\u003c/em\u003e. Their intestines appeared swollen, with slight mucosal bleeding. In contrast, no macroscopic lesions were observed in the DB, DS, BV, or AB groups. Microbiome analysis showed a decline in microbial diversity within the NE group. The BV group exhibited a microbial composition most similar to that of healthy animals, followed by the AB group. The study concludes that B. velezensis could serve as an alternative to prophylactic antibiotics in mitigating the adverse effects of necrotic enteritis on the gut microbiome.\u003c/p\u003e","manuscriptTitle":"Intestinal Microbiom in Necrotic Enteritis Infection of Broiler and Comparison of Treatment Alternatives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 04:06:01","doi":"10.21203/rs.3.rs-6190428/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-24T05:15:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-23T13:41:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T12:59:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2025-03-17T04:32:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"914607f1-5603-4242-ba79-d387c9c78ad3","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T17:00:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-03 04:06:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6190428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6190428","identity":"rs-6190428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}