Intestinal Microbiome Confers Strong Colonization Resistance Against Necrotic Enteritis

Intestinal Microbiome Confers Strong Colonization Resistance Against Necrotic Enteritis | 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 Article Intestinal Microbiome Confers Strong Colonization Resistance Against Necrotic Enteritis Jing Liu, Jiaqing Guo, Isabel Tobin, Melanie A. Whitmore, Dohyung M. Kim, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8349290/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Necrotic enteritis (NE), caused by Clostridium perfringens , is a major enteric disease in poultry that leads to severe dysbiosis, morbidity, and mortality. Modulating the intestinal microbiota holds promise for enhancing animal health and disease resistance; however, specific commensal bacteria associated with NE protection remain elusive. Chicken breeds differ markedly in disease susceptibility, with Fayoumi chickens exhibiting greater resistance than Leghorn and Cobb chickens. We hypothesized that Fayoumi chickens harbor unique commensal bacteria that confer robust colonization resistance against NE. To test this, we challenged two inbred lines, Fayoumi M5.1 and Leghorn Ghs6, alongside commercial Cobb broilers with NE. Among these, M5.1 chickens demonstrated the highest resistance to NE. Cecal microbiota transplantation from the three breeds into newly hatched Cobb chicks revealed that M5.1-derived microbiota provided completion protection against NE. Comparative microbiome analysis demonstrated significant differences among breeds under both healthy and NE-challenged conditions. Notably, Bifidobacterium , largely absent in healthy chickens of all three breeds, was highly enriched in both the ileum and cecum of M5.1 chickens following NE challenge. Furthermore, oral administration of Bifidobacterium pseudolongum significantly reduced NE mortality in Cobb chickens. Collectively, these findings highlight the protective role of commensal bacteria from NE-resistant Fayoumi chickens and suggest their potential for microbiota-based strategies to mitigate NE in poultry. Biological sciences/Microbiology Biological sciences/Zoology Fayoumi chickens poultry cecal microbiota transplantation microbiota colonization resistance necrotic enteritis Clostridium perfringens Bifidobacterium Lactobacillus Ligilactobacillus salivarius Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Necrotic enteritis (NE) is an enteric disease caused by Clostridium perfringens , a Gram-positive, anaerobic, spore-forming, rod-shaped bacterium. NE remains one of the most economically devastating diseases in the global poultry industry, costing over $ 6 billion annually in production losses 1 . Various predisposing factors are known to promote NE development, such as exposure to apicomplexan protozoa, Eimeria , the etiologic agent of avian coccidiosis 2 . Traditionally, prophylactic in-feed antibiotics have proven effective for the control of clostridial infections in chickens 3 . However, with recent withdrawal of in-feed antibiotics from livestock production in a growing number of countries and increasing consumer demand for antibiotic-free animal products, NE has become more prevalent, necessitating effective antibiotic alternatives 4 . Probiotics such as Lactobacillus , Enterococcus , and Bacillus and prebiotics such as mannan-oligosaccharides have proved beneficial in NE alleviation 3 , 4 . However, no commensal bacteria have been directly linked to NE resistance, and NE-resistant commensals could potentially offer prophylactic and/or therapeutic utility against NE. Two highly inbred chicken lines, Fayoumi M5.1 and Leghorn Ghs6, are drastically different in their ability to resist Newcastle disease, with M5.1 being highly resistant and Ghs6 highly susceptible 5 , 6 . Additionally, Fayoumi chickens are known to be more resistant to coccidiosis, avian influenza, and salmonellosis than Leghorns or commercial broiler chickens 7 – 10 . The intestinal microbiota plays a significant role in host homeostasis and health, and there is mounting evidence indicating that the microbiota is shaped by host genetics 11 – 13 . Thus, we hypothesize that the intestinal microbiota from disease-resistant chicken breeds harbors a unique microbiota that offers strong colonization resistance (CR) to diseases such as NE. To test our hypothesis, we assessed the efficacy of the microbiota from M5.1, Ghs6, and Cobb chickens in protecting newly-hatched Cobb chickens from NE through cecal microbiota transplantation (CMT) and revealed that M5.1 microbiota is the most protective against NE. We further compared the intestinal microbiota profiles among the three chicken breeds under both healthy and NE conditions and identified a large number of differentially enriched bacteria. Additionally, we demonstrated that oral administration of Bifidobacterium pseudolongum , which was specifically enriched in NE-challenged M5.1 chickens, confers strong protection to Cobb chickens against NE challenge. These findings provide direct evidence that the intestinal microbiota, particularly from disease-resistant chickens, offers robust CR against NE and can be leveraged for NE mitigation. RESULTS Differential susceptibility of three chicken breeds to NE To investigate the difference in NE susceptibility, we challenged M5.1, Ghs6, and Cobb chickens to induce NE (Fig. 1 A). While approximately 80% of Cobb chickens and 91% Ghs6 chickens survived at 3 days post-infection (dpi), 100% M5.1 chickens exhibited no mortality (Fig. 1 B ) . Consistently, among surviving chickens, 75% Cobb chickens showed extensive, score-6 lesions in the small intestinal tract, and 9% Ghs6 chickens exhibited severe score-6 lesions, while none of M5.1 chickens presented any abnormalities in the small intestine at 3 dpi (Fig. 1 C ) . Additionally, NE significantly reduced weight gain in Cobb and Ghs6 chickens but had no impact on M5.1 chickens (Fig. 1 D ) . These results clearly indicated that, among three breeds of chickens, M5.1 chickens are the most resistant to NE and Cobb chickens are the most susceptible, with Ghs6 being intermediate. Differences in the ileal microbiota among three chicken breeds under healthy and NE conditions To investigate the microbiota differences among M5.1, Ghs6, and Cobb chickens and their microbiota shifts in response to NE, bacterial DNA was isolated from the ileal and cecal digesta of mock- and NE-infected chickens and subjected to 16S rRNA gene sequencing. After quality control, 7,750,326 high-quality sequencing reads were obtained, averaging 61,510 ± 7,021 sequences per sample. Following denoising and removal of amplicon sequence variants (ASVs) present in less than 5% of samples, 399 and 421 ASVs were identified in the ileum and cecum, respectively. Analysis of α-diversity of the ileal microbiota, as measured by Shannon Index, revealed a progressive decline across the three chicken breeds in relation to increasing resistance to NE (Fig. 2 A). This trend was further accentuated following NE challenge, with the NE-resistant M5.1 chickens exhibiting the least perturbation. Although β-diversity showed significant differences among the three breeds based on weighted UniFrac distance, M5.1 chickens were the least affected by NE (Fig. 2 B). The ileal microbiota profiles were drastically different among the breeds (Fig. 2 C). In healthy Cobb chickens, three dominant taxa included group A Lactobacillus F1 (25.5%), Ligilactobacillus salivarius F3 (14.0%), and Corynebacterium F45 (10.3%). In contrast, Staphylococcus gallinarum (F8) was highly abundant in healthy Ghs6 (43.9%) and M5.1 chickens (36.9%) but was largely absent (0.8%) in Cobb chickens ( Fig. S1A ). Corynebacterium was almost undetectable in both M5.1 and Ghs6 chickens. Notably, Lactobacillus johnsonii (F4) was minimal in Ghs6 chickens (0.3%), and group A Lactobacillus accounted for only 3.9% of the total ileum bacteria. In M5.1 chickens, L. johnsonii represented 34.7%, while group A Lactobacillus comprised just 2.2% in the ileum. Among the 40 most abundant ileal ASVs, the majority were significantly different among the three chicken breeds under healthy and NE-challenged conditions. The ileal microbiota profiles of M5.1 and Ghs6 chickens resembled each other more than that of Cobb chickens (Fig. 2 D). Among three major pathobionts, C. perfringens increased drastically in NE-susceptible Cobb chickens from 0.03% to 34.1% but remained minimal in NE-challenged Ghs6 (1.5%) and M5.1 (0.08%) chickens (Fig. 2 D and Fig. S1 ). Escherichia (F7) also significantly bloomed in NE-infected Cobb chickens but not in M5.1 or Ghs6 chickens. Enterococcus cecorum (F30) showed a significant increase in all three breeds following NE challenge. Major lactic acid bacteria (LAB) such as group A Lactobacillus and L. salivarius were lowly abundant in healthy M5.1 and Ghs6 chickens compared to Cobb chickens but were markedly enriched in both breeds following NE challenge ( Fig. S1 ). In response to NE, L. johnsonii was significantly reduced in Cobb chickens but increased substantially in both M5.1 and Ghs6 chickens. Differences in the cecal microbiota among three chickens breeds under healthy and NE conditions Consistent with observations in the ileum, α-diversity of the cecal microbiota progressively declined with increasing NE resistance across the three chicken breeds, and NE infection further reduced Shannon Index in Cobb but not Ghs6 or M5.1 chickens (Fig. 3 A). Moreover, β-diversity analysis using weighted UniFrac distance revealed distinct microbiota structures among the three breeds with and without NE challenge (Fig. 3 B). The taxonomic composition of the cecal microbiota also varied markedly among breeds (Fig. 3 C). Notably, Bacteroides fragilis (F2) was the dominant taxon in healthy Ghs6 (26.4%) and M5.1 (35.0%) chickens, but accounted for only 6.2% in healthy Cobb chickens (Fig. 3 C and Fig. S2 ). Following NE challenge, B. fragilis members (F2 and F16) were enriched in Cobb chickens but showed a slight reduction in Ghs6 and M5.1 chickens (Fig. 3 D). Among major LAB species including Group A Lactobacillus (F1), L. salivarius (F3), and L. johnsonii (F4) were less abundant in M5.1 chickens but increased substantially following NE infection. In contrast, L. johnsonii was significantly suppressed in NE-challenged Cobb chickens. Short-chain fatty acid (SCFA)-producing bacteria such as two Faecalibacterium members (F14 and F19) were more prevalent in Cobb chickens under healthy conditions but diminished across all breeds after NE infection. Conversely, several other SCFA-producing members of the Oscillospiraceae family (e.g., F24, F50, and F59) were more abundant in M5.1 and Ghs6 chickens and declined following NE challenge, while showing enrichment in NE-infected Cobb chickens. Regarding the three major pathobionts, C. perfringens remained at low abundance in the cecum across all breeds under both healthy and NE challenge conditions, although a statistically significant increase was observed in NE-infected Cobb and M5.1 chickens (Fig. 3 D and Fig. S2 ). Escherichia was enriched in NE-infected Cobb chickens but remained largely unchanged in Ghs6 and M5.1 chickens. E. cecorum tended to increase in Cobb and M5.1 chickens following NE challenge, with no notable changes in Ghs6 chickens. Interestingly, two other Clostridium species (F51 and F82) showed no major alterations in response to NE across all three breeds. Differential protection of Cobb chickens from NE by the cecal microbiota of three chicken breeds To directly evaluate the efficacy of intestinal microbiota in alleviating NE, cecal microbiota was prepared from all three chicken breeds and transplanted to naïve Cobb chickens, followed by NE challenge (Fig. 4 A). Our results showed that the M5.1 microbiota provided the best protection against NE, with 100% survival in the transplanted group, while approximately 40% of the chickens in the mock-transplanted control group died from severe intestinal lesions at 3 dpi (Fig. 4 B). Interestingly, the cecal microbiota from the two susceptible breeds, Ghs.6 and Cobb, also conferred significant protection to naïve Cobb chickens, although with reduced efficacy compared to the M5.1 microbiota (Fig. 4 B and 4 C). Consistently, the cecal microbiota of all three chicken breeds partially reversed NE-induced weight loss in recipient Cobb chickens (Fig. 4 D). Further examination revealed obvious changes in the ileal microbiota of recipient chickens following cecal microbiota transplantation (CMT). Except for microbiota richness ( Fig. S3A ), CMT of the M5.1 microbiota, but not other microbiota, significantly increased the Shannon Index of the ileal microbiota of recipient chickens ( Fig. S3B ) and caused significant shifts in microbiota structure based on weighted UniFrac distance ( Fig. S3C ). The ileal microbiota composition also experienced notable alterations ( Fig. S3D ). CMT of all three breeds significantly enriched two Gemmiger species (F22 and F31) with a tendency to increase many but not all LAB species. In contrast, Rombousia (F23) was significantly reduced following CMT of all three breeds. Among three major pathobionts, C. perfringens and E. cecorum were unaltered, but Escherichia was significantly diminished following CMT. In response to NE, C. perfringens and Escherichia drastically bloomed in Cobb chickens mock-transplanted or transplanted with the Ghs6 or Cobb microbiota. In contrast, CMT with the M5.1 microbiota caused no blooming of C. perfringens and a significant reduction in Escherichia and E. cecorum in the ileum of recipient chickens. CMT also caused significant changes to the cecum of recipient Cobb chickens under healthy and NE conditions ( Fig. S4A-S4D ). Similar to the ileum, two Gemmiger species (F22 and F31) were significantly enriched by all three transplanted microbiotas in both healthy and NE-infected recipients ( Fig. S4E ). Many LAB species were largely unaffected by CMT, except that group A lactobacillus and Limosilactobacillus oris (F17) were significantly enriched in recipient chickens in response to NE, with M5.1 microbiota-transplanted chickens showing the largest increase. Neither C. perfringens nor Escherichia bloomed in Cobb chickens receiving the M5.1 microbiota. Surprisingly, Bacteroides , the dominant species in the cecum of both M5.1 and Ghs6 chickens, failed to be detected in the cecum of recipient Cobb chickens following CMT, with or without NE challenge. Protection of chickens from NE by B. pseudolongum Both the ileal and cecal microbiotas of M5.1 or Ghs6 chickens resembled each other more than Cobb chickens under both mock- and NE-infected conditions. To explain the obvious difference in NE resistance between M5.1 and Ghs6 chickens, we detected a notable difference in the differential abundance of two Bifidobacterium species, namely B. anseris (F84) and B. pseudolongum (F201) in both the ileum and the cecum (Fig. 5 A- 5 D) among the three chicken breeds. Both Bifidobacterium species were largely absent in both mock- and NE-infected Ghs6 and Cobb chickens as well as in mock-infected M5.1 chickens, but showed a significant enrichment in both the ileum and cecum of M5.1 chickens following NE challenge. To directly verify if Bifidobacterium plays a role in NE resistance, we plated the cecal bacteria of M5.1 chickens on MRS plates and identified an isolate to be B. pseudolongum through Sanger sequencing of its full-length 16S rRNA gene. It showed a potent activity in inhibiting C. perfringens growth with a 5.6-fold reduction when incubated 1:1 for 24 h in a coculture assay (Fig. 5 E). To further evaluate its efficacy against NE, we orally inoculated approximately 1 × 10 7 CFU of B. pseudolongum to each Cobb chicken on days 9, 11, 13, and 15, and challenged them with E. maxima and C. perfringens to induce NE on days 10 and 14, respectively (Fig. 5 F). Only 42.2% of NE-challenged chickens survived at 3 dpi without intervention, compared to 83.3% survival in those that received B. pseudolongum (Fig. 5 G), which also significantly alleviated intestinal lesions (Fig. 5 H), but with no obvious impact on growth (Fig. 5 I). Overall, these results suggested a protective role of B. pseudolongum against NE. DISCUSSION Fayoumi chickens, originating from Egypt, have been found to be highly resistant to multiple diseases 5 – 10 . We hypothesized that, shaped by its unique genetics, this chicken breed may harbor distinct intestinal microbiota to confer enhanced disease resistance. Our results demonstrated that, among three chicken breeds studied including Fayoumi, Leghorn, and Cobb, Fayoumi chickens exhibit the highest resistance to NE, whereas Cobb chickens are the most susceptible. We further demonstrated that cecal microbiota transplantation from Fayoumi chickens offers superior protection of NE-susceptible Cobb chickens from NE. Additionally, oral administration of Bifidobacterium , a uniquely enriched commensal in NE-infected Fayoumi chickens provided significant protection of Cobb chickens against NE, suggesting the utility of intestinal bacteria and Bifidobacterium in particular in mitigating NE and perhaps other diseases. Differential intestinal microbiota responses to NE among Cobb, Ghs6, and M5.1 chickens The intestinal microbiome significantly influences health and disease, with its composition shaped by various factors, including host genetics 11 – 13 . In this study, we observed significant differences in the intestinal microbiota among three chicken breeds. For example, Bacteroides was the most abundant in the cecum of M5.1 and Ghs6 chickens but was minimally present in Cobb chickens, which is consistent with several recent analyses showing the prevalence of Bacteroides in many indigenous breeds but not in Cobb chickens 13 – 16 . Bacteroides is a genus of non-spore-forming, Gram-negative bacteria that degrade nondigestible carbohydrates to produce SCFAs, offering various host benefits and providing CR against pathogens like Clostridioides difficile 17 , 18 . However, certain Bacteroides species are opportunistic pathogens that can promote chronic inflammation 19 . While Bacteroides may aid in NE resistance, its exact role in CR against NE requires further investigation. It is noteworthy that Bacteroides seems dispensable for NE resistance, as Cobb chickens become highly resistant to NE infection despite having undetectable levels of Bacteroides following M5.1 microbiota transplantation. To our surprise, Staphylococcus , mainly S. gallinarum , was most prevalent in M5.1 and Ghs6 chickens, accounting for 35–45% of the total ileal bacteria, but was largely absent in Cobb chickens. S. gallinarum is a non-pathogenic, coagulase-negative bacterium commonly found in healthy chickens, pheasants, and humans 20 – 22 . It has probiotic properties, showing activity against pathogenic Escherichia coli and Klebsiella pneumoniae in vitro 23 . Additionally, it produces Staphyloferrin A, a siderophore that suppresses pathogenic bacteria growth by chelating iron, essential for virulence and bacterial interactions 23 . The role of Staphylococcus in NE resistance warrants further investigation. Notably, NE-resistant M5.1 chickens had a significantly higher abundance of Weissella in the ileum compared to susceptible Ghs6 and Cobb chickens. Weissella , part of the Leuconostocaceae family, is known for its probiotic and anti-inflammatory potential 24 . For example, W. cibaria can inhibit pathogenic microorganisms through metabolites like exopolysaccharides 25 . Weissella species produce bacteriocins, such as Weissellicins 25 – 27 . The role of Weissella in poultry health and disease remains underexplored, necessitating further studies. Additionally, our results clearly demonstrated varying abundances of LAB species among the three chicken breeds and their distinct responses to NE. Group A Lactobacillus , including highly related species like L. crispatus , L. acidophilus , and L. gallinarum 28 that cannot be distinguished by the V3–V4 region of the bacterial 16S rRNA gene, were more abundant in the ileum and cecum of Cobb chickens than in M5.1 and Ghs6 chickens. However, Group A Lactobacillus remained largely unchanged by NE in Cobb chickens but was significantly enriched in M5.1 and Ghs6 chickens. Conversely, L. johnsonii was significantly reduced in Cobb chickens but enriched in M5.1 and Ghs6 chickens in response to NE. The differential response of the same LAB species in different breeds suggests the possible presence of different LAB strains, explaining the variation in the NE resistance pattern. It is important to confirm and isolate LAB strains preferentially growing in Fayoumi chickens and investigate their efficacy in disease resistance. Additionally, relative contributions of different LAB species to NE resistance require further investigation, although many LAB species have shown benefits against NE 3,29 . Protection of naive Cobb chickens from NE through CMT Given recent successes of transplanting fecal or cecal microbiota in conferring CR in chickens against pathogens such as Salmonella 30 , 31 , Campylobacter jejuni 32 – 34 , and C. perfringens infections 35 , we compared the efficacy of the cecal microbiota from three chicken breeds in providing CR against NE. We observed a drastic improvement in NE resistance among naive Cobb chickens receiving the Fayoumi microbiota. Additionally, the cecal microbiota of NE-susceptible Cobb and Ghs6 chickens also provided notable, albeit less pronounced, protection against NE. These findings align with a previous report showing reduced chicken intestinal lesions in a subclinical NE model following transplantation of bioreactor-propagated cecal microbiota of adult chickens 35 . However, our results contrast with an earlier study demonstrating that CMT from a resistant chicken line (ADOL Leghorn Line 6 1 ) to a susceptible line (ADOL Leghorn Line N) failed to confer CR against C. jejuni infection 32 . The discrepancy among these studies may be attributed to differences in the CMT preparation method. In our study and that of Zaytsoff, et al. 35 , the microbiota transplants were prepared under anaerobic conditions, whereas Chintoan-Uta et al. 32 prepared CMT aerobically. It is plausible that anaerobic commensal bacteria, which are crucial for disease resistance, may not survive well during aerobic microbiota preparation. Supporting this hypothesis, transplantation of anaerobic cecal microbiota was shown to provide CR against C. jejuni 34 . However, transplantation of both aerobically and anaerobically cultured mouse fecal microbiota offered CR against C. jejuni in chickens 33 . Further research is warranted to elucidate the specific microbial communities and mechanisms underlying the protection against different pathogens. We observed enrichment of Gemmiger in both the ileum and cecum of recipient chickens following CMT from all three chicken breeds, whereas Megamonas and Bacteroides were enriched following transplantation of bioreactor-propagated cecal microbiota 35 . The differences in outcomes between the two studies likely stem from variations in the transplanted microbiota and genetic differences in recipient chickens. Gemmiger , a genus of bacteria in the family Oscillospiraceae , is closely related to Subdoligranulum and Faecalibacterium 36 , 37 , both of which produce SCFAs with anti-inflammatory properties. Gemmiger was reported to be depleted in multiple cohorts of inflammatory bowel disease patients, alongside other butyrate producers such as Faecalibacterium 38 . Administering Gemmiger or its related Faecalibacterium or Subdoligranulum may prove beneficial against NE. Bifidobacterium -mediated protection of chickens from NE Despite the similarity in intestinal microbiota between Ghs6 and M5.1 chickens, both of which were hatched in the same location, Ghs6 chickens are evidently more susceptible to NE than M5.1 chickens. A notable observation is the marked increase in Bifidobacterium in NE-challenged M5.1 chickens, a response not observed in Ghs6 or Cobb chickens. Oral administration of Bifidobacterium pseudolongum conferred substantial protection against NE, underscoring its protective role. This is consistent with the well-documented antibacterial and immunomodulatory properties of Bifidobacterium species 39 , 40 . Additionally, Bifidobacteria synthesize essential vitamins such as riboflavin, thiamine, vitamin B6, and vitamin K, along with bioactive molecules like folic acid, niacin, and pyridoxine 41 . Unlike Lactobacillus species that produce both D(-)-lactic acid and L(+)-lactic acid, Bifidobacteria predominantly produce L(+)-lactic acid, which is more readily metabolized by humans and animals 41 . Additionally, Bifidobacterium has demonstrated efficacy against subclinical NE 42 and C. perfringens in co-culture studies 43 , consistent with our in vitro and in vivo observations. However, it is noted that, although Bifidobaceterium is enriched NE-challenged Fayoumi chickens and beneficial against NE, it is unlikely to be solely responsible for NE resistance in Fayoumi chickens. This is evidenced by the absence of Bifidobacterium in recipient Cobb chickens following CMT from any chicken breed, despite the robust protection observed. Therefore, it is plausible that multiple bacterial species in the intestinal microbiota act synergistically to provide CR against NE. CONCLUSIONS Our study demonstrates that Fayoumi chickens exhibit greater resistance to NE compared to Leghorn layers and Cobb broilers. Additionally, we provide compelling evidence that the intestinal microbiota from Fayoumi chickens confers significant protection against NE in newly-hatched Cobb chickens, with B. pseudolongum playing a crucial role in this protective effect. These findings underscore the potential of leveraging disease-resistant chicken microbiota for NE mitigation. Future research should focus on identifying the specific bacterial consortia responsible for CR and exploring their application in developing probiotic treatments to enhance poultry health and productivity. METHODS Ethics statement All animal experiments described in this study were conducted according to the recommendations in the Guide for the Care and Use of Agricultural Animals in Research and Teaching, 4th edition (2020) and approved by the Institutional Animal Care and Use Committee of Oklahoma State University under protocol number AG-23-35. NE challenge of inbred chickens To investigate the difference of intestinal microbiota in response to NE, 48 day-of-hatch M5.1 and Ghs6 chicks, with 24 birds/breed, were obtained from Iowa State University (Ames, Iowa), while 24 day-of-hatch Cobb-500 chicks were obtained from Cobb-Vantress (Siloam Springs, Arkansas). Chickens were housed in floor pens (3' × 3') with 12 birds/pen and fresh wood shavings in an environmentally controlled room under standard management. Chickens had free access to tap water and an unmedicated mash corn-soybean starter diet containing 21.5% crude protein that meets or exceeds the nutrient requirements of the NRC recommendations 44 throughout the study. Within each breed, animals were weighed individually on day 16 and assigned randomly to either the mock or NE group. Each animal in the NE group was orally inoculated with 1 × 10 4 sporulated oocysts of the E. maxima M6 strain (kindly provided by Dr. John R. Barta, University of Guelph, Canada) in 1 mL PBS on day 16, followed by four sequential inoculations with approximately 5 × 10 8 CFU of netB - and tpeL -positive C. perfringens Brenda B strain (kindly provided by Dr. Lisa Bielke, North Carolina State University, Raleigh, North Carolina) in 2 mL fluid thioglycollate (FTG) broth (Thermo Fisher Scientific) twice daily on days 20 and 21, respectively, as previously described 45-47 . The mock-infected group received 1 mL PBS or 2 mL FTG each time. To minimize cross-contamination, floor pens were separated from each other with plastic sheets. Animals were observed twice daily for survival and behavior till day 23. Chickens reluctant to move were euthanized to minimize undue suffering. On day 23, all surviving chickens were weighed individually and sacrificed via CO 2 asphyxiation. Lesions in the small intestine were scored on a scale of 0-6 as described 48 . Additionally, the digesta in the proximal ileum (approximately 0.5 g) and cecum (approximately 0.2 g) were separately collected and stored at −80°C for microbial genomic DNA extraction. Preparation of the cecal microbiota Cecal microbiota was collected from 35-day-old, healthy M5.1, Ghs6, and Cobb chickens with three birds/breed. After euthanasia via CO 2 asphyxiation, the cecum was ligated at the ileal-cecal junction, excised, and transferred into BACTRON300 TM Anaerobic Chamber (Sheldon Manufacturing, Cornelius, Oregon) within 1 h. The cecal digesta was collected, combined within each breed, weighed, and diluted with five volumes (w/v) of reduced PBS. After filtration through a 70-μM cell strainer, each cecal microbiota suspension was further diluted 10-fold in PBS containing 10% glycerol and stored at −80°C until further use. On the day of CMT, frozen microbiota suspensions were thawed at 37°C and dispensed anaerobically into 1-mL syringes attached to a feeding needle and transferred to the animal facility in resealable Ziploc ® plastic bags. Cecal microbiota transplantation A total of 120 day-of-hatch Cobb chickens were obtained from Cobb-Vantress and randomly assigned to one of eight groups with 15 birds/group. Each animal received 0.2 mL PBS or 0.2 mL diluted cecal microbiota from healthy M5.1, Ghs6, or Cobb chickens on days 0, 1, 9, and 13. On days 10 and 14, four groups of animals were challenged with 1 × 10 4 sporulated oocysts of E. maxima B6 and approximately 5 × 10 8 CFU of C. perfringens Branda B to induce NE, while the other four groups were mock-infected. Animals were observed twice daily for mortalities till day 17. Chickens were weighed individually on days 0, 10, and 17. On day 17, all surviving animals were sacrificed and examined for small intestinal lesion scores. Additionally, the digesta in the proximal ileum and cecum were collected and stored at −80°C for microbial genomic DNA extraction. Isolation, culture, and oral administration of B. pseudolongum against NE The ileal and cecal digesta of three 35-day-old M5.1 chickens were collected, diluted 10-fold in in reduced PBS, and filtered through a 70-μM cell strainer in an anaerobic chamber. After 10-fold serial dilutions in reduced PBS, 100 μL of each dilution was plated on de Man, Rogosa, and Sharpe (MRS) and reinforced clostridial medium (RCM) agar plates, respectively. After 24-h anaerobic culture, colony-PCR was performed with well-isolated colonies using primers (27F: AGA GTT TGA TCC TGG CTC AG and 1492R: GGT TAC CTT GTT ACG ACT T) to amplify the entire 16S rRNA gene, followed by Sanger sequencing. An isolate was identified to share 99.5% identity to B. pseudolongum and restreaked on MRS plates three times, followed by anaerobic propagation in MRS or RCM. To evaluate the protective efficacy of B. pseudolongum against NE, 90 day-of-hatch Cobb chickens were randomly assigned to one of three treatments with 15 birds/pen and two pens/treatment. Each animal received approximately 1 × 10 7 CFU of B. pseudolongum in 1 mL reduced PBS on days 9, 11, 13, and 15. On days 10 and 14, two groups of animals were challenged with 5 × 10 3 sporulated oocysts of E. maxima B6 and approximately 5 × 10 8 CFU of C. perfringens Branda B to induce NE, while the third group was mock-infected with PBS and FTG on respective days. Animals were observed twice daily for mortalities till day 17. Chickens were weighed individually on days 0, 10, and 17. On day 17, all surviving animals were sacrificed and examined for small intestinal lesion scores. Bacterial coculture assay To directly evaluate the anti- C. perfringens activity of B. pseudolongum , both bacteria were grown anaerobically in Brain Heart Infusion (BHI) broth overnight and diluted to 2 × 10 7 CFU/mL in BHI, mixed 1:1, and incubated anaerobically for 24 h at 37°C. The survival of C. perfringens was assessed through serial plating on perfringens- selective tryptose sulfite cycloserine (TSC) agar plates (Sigma Aldrich, St. Louis, MO). Bacterial DNA isolation and 16S rRNA sequencing Fecal DNA MicroPrep and MiniPrep Kits (Zymo Research Irvine, CA) were used for isolation of DNA from the ileal and cecal digesta in animal trials, respectively. The concentration and quality of DNA was measured by Nanodrop One Spectrophotometer (Thermo Fisher Scientific). High-quality DNA samples were shipped on dry ice to Novogene (Beijing, China) for PE250 deep sequencing of the V3-V4 region of bacterial 16S rRNA gene using primers (341F: CCT AYG GGR BGC ASC AG and 806R: GGA CTA CNN GGG TAT CTA AT) on the Illumina NovaSeq 6000 system. PCR amplification and library preparation were performed by Novogene (Beijing, China) using NEBNext® Ultra™ Library Prep Kit (New England Biolabs, Ipswich, MA, USA), generating a minimum of 30,000 raw sequencing reads per sample. Bioinformatics and statistical analysis Bioinformatic analysis was conducted as we previously described 10,49-51 . Briefly, raw sequencing reads were analyzed using QIIME 2 v2023.7 52 . After filtration of low-quality reads, clean sequencing reads were trimmed to 402 nucleotides and denoised using Deblur 53 . The resulting sequences were then classified into bacterial ASVs using the RDP 16S rRNA training set (v. 18) and Bayesian classifier. A bootstrap confidence of 80% was used for taxonomic classification. ASVs with a classification confidence below 80% were assigned to the last confidently classified taxonomic level, followed by “_unclassified”. ASVs present in fewer than 5% of samples were removed from downstream analysis. The top 100 ASVs, along with all differentially enriched taxa, were further validated and reclassified, if necessary, using an updated EzBioCloud 16S database (v2023.08.23) 54 . Species-level classification was assigned to sequences sharing greater than 97% identity. Analysis and visualization of α- and β-diversities of the microbiota composition were analyzed using the ‘phyloseq’ R package v1.46.0 55 . To visualize the overall biodiversity and complexity within samples, the number of ASVs, Pielou’s evenness index, and Shannon index were used to calculate and display the richness, evenness, and overall diversity. The β-diversity was determined using weighted and unweighted UniFrac distances. Statistical significance in α-diversity and relative abundance for each sampling day was determined using non-parametric Mann-Whitney U test. Significance in β-diversity was determined using non-parametric permutational multivariate analysis of variance (PERMANOVA) with 999 permutations using the vegan package v. 2.6.4 56 . P < 0.05 was considered statistically significant. Differential abundance of bacteria among different groups of chickens was determined using ANCOM-BC2 57 . Abbreviations ASV Amplicon sequence variant BHI Brain heart infusion broth CMT Cecal microbiota transplantation CR Colonization resistance dpi Days post-infection LAB Lactic acid bacteria MRS de Man, Rogosa, and Sharpe broth NE Necrotic enteritis RCM Reinforced clostridial medium SCFA Short-chain fatty acid Declarations Data availability Raw sequencing reads of this study was deposited in the NCBI GenBank SRA database under the accession number PRJNA1132701. Acknowledgments We would like to thank Dr. John R. Barta at the University of Guelph, Canada for kindly providing E. maxima strain M6. We are grateful to Dr. Lisa Bielke at North Carolina State University for providing the C. perfringens strain Brenda B. We also thank Ms. Zijun Zhao for helping with animal handling. This research was funded by the USDA National Institute of Food and Agriculture grants (2022-67016-37208 and 2024-67016-42415), the Ralph F. and Leila W. Boulware Endowment Fund, Oklahoma Agricultural Experiment Station Project H-3268, and Iowa Agriculture and Home Economics Experiment Station Project IOW05620. I.T. and M.W. were supported by two separate USDA National Institute of Food and Agriculture Predoctoral Fellowship grants (2021-67034-35184 and 2024-67011-42944). The funders played no role in study design, data collection, analysis, and interpretation, or the writing of this manuscript. Author Contributions JL, JG, IT, MAW, PP, AS, and GZ conducted animal trials; JL and JG processed the samples; JL, JG, IT, and GZ analyzed the data; JL drafted the manuscript; GZ, MGK, and SJL revised the manuscript; GZ conceived and supervised the study. All authors reviewed the manuscript and agreed to the published version of the manuscript. Competing interests The authors declare no competing interests. References Wade, B. & Keyburn, A. The true cost of necrotic enteritis. World Poult. 31 , 16-17 (2015). Emami, N. K. & Dalloul, R. A. Centennial Review: Recent developments in host-pathogen interactions during necrotic enteritis in poultry. Poult. Sci. 100 , 101330 (2021). https://doi.org/10.1016/j.psj.2021.101330 Kulkarni, R. R., Gaghan, C., Gorrell, K., Sharif, S. & Taha-Abdelaziz, K. Probiotics as Alternatives to antibiotics for the prevention and control of necrotic enteritis in chickens. Pathogens 11 , 692 (2022). https://doi.org/10.3390/pathogens11060692 Alizadeh, M. et al. Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination. Anim. Health Res. Rev. 22 , 147-162 (2021). https://doi.org/10.1017/S146625232100013X Deist, M. S. et al. Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome. BMC Genomics 18 , 989 (2017). https://doi.org/10.1186/s12864-017-4380-4 Schilling, M. A. et al. Conserved, breed-dependent, and subline-dependent innate immune responses of Fayoumi and Leghorn chicken embryos to Newcastle disease virus infection. Sci Rep 9 , 7209 (2019). https://doi.org/10.1038/s41598-019-43483-1 Pinard-Van Der Laan, M. H., Monvoisin, J. L., Pery, P., Hamet, N. & Thomas, M. Comparison of outbred lines of chickens for resistance to experimental infection with coccidiosis ( Eimeria tenella ). Poult. Sci. 77 , 185-191 (1998). https://doi.org/10.1093/ps/77.2.185 Wang, Y., Lupiani, B., Reddy, S. M., Lamont, S. J. & Zhou, H. RNA-seq analysis revealed novel genes and signaling pathway associated with disease resistance to avian influenza virus infection in chickens. Poult. Sci. 93 , 485-493 (2014). https://doi.org/10.3382/ps.2013-03557 Cheeseman, J. H., Kaiser, M. G., Ciraci, C., Kaiser, P. & Lamont, S. J. Breed effect on early cytokine mRNA expression in spleen and cecum of chickens with and without Salmonella enteritidis infection. Dev. Comp. Immunol. 31 , 52-60 (2007). https://doi.org/10.1016/j.dci.2006.04.001 Broadwater, C. et al. Breed-specific responses to coccidiosis in chickens: identification of intestinal bacteria linked to disease resistance. J. Anim. Sci. Biotechnol. 16 , 65 (2025). https://doi.org/10.1186/s40104-025-01202-z Wilde, J., Slack, E. & Foster, K. R. Host control of the microbiome: Mechanisms, evolution, and disease. Science 385 , eadi3338 (2024). https://doi.org/10.1126/science.adi3338 Morris, A. H. & Bohannan, B. J. M. Estimates of microbiome heritability across hosts. Nat. Microbiol. 9 , 3110-3119 (2024). https://doi.org/10.1038/s41564-024-01865-w Burrows, P. B. et al. Decoding the chicken gastrointestinal microbiome. BMC Microbiol. 25 , 35 (2025). https://doi.org/10.1186/s12866-024-03690-x Xu, Y. et al. Metagenomic analysis reveals the microbiome and antibiotic resistance genes in indigenous Chinese yellow-feathered chickens. Front. Microbiol. 13 , 930289 (2022). https://doi.org/10.3389/fmicb.2022.930289 Pandit, R. J. et al. Microbial diversity and community composition of caecal microbiota in commercial and indigenous Indian chickens determined using 16S rDNA amplicon sequencing. Microbiome 6 , 115 (2018). https://doi.org/10.1186/s40168-018-0501-9 Shen, H. et al. Metagenome-assembled genome reveals species and functional composition of Jianghan chicken gut microbiota and isolation of Pediococcus acidilactic with probiotic properties. Microbiome 12 , 25 (2024). https://doi.org/10.1186/s40168-023-01745-1 Zafar, H. & Saier, M. H., Jr. Gut Bacteroides species in health and disease. Gut Microbes 13 , 1-20 (2021). https://doi.org/10.1080/19490976.2020.1848158 Shin, J. H. et al. Bacteroides and related species: The keystone taxa of the human gut microbiota. Anaerobe 85 , 102819 (2024). https://doi.org/10.1016/j.anaerobe.2024.102819 Lopez, L. R., Bleich, R. M. & Arthur, J. C. Microbiota Effects on carcinogenesis: initiation, promotion, and progression. Annu. Rev. Med. 72 , 243-261 (2021). https://doi.org/10.1146/annurev-med-080719-091604 Syed, M. A. et al. Staphylococci in poultry intestines: a comparison between farmed and household chickens. Poult. Sci. 99 , 4549-4557 (2020). https://doi.org/10.1016/j.psj.2020.05.051 Devriese*, L. A., Poutrel, B., Kilpper-BäLz, R. & Schleifer, K. H. Staphylococcus gallinarum and Staphylococcus caprae , two new species from animals. Int. J. Syst. Evol. Microbiol. 33 , 480-486 (1983). https://doi.org/https://doi.org/10.1099/00207713-33-3-480 Ohara-Nemoto, Y., Haraga, H., Kimura, S. & Nemoto, T. K. Occurrence of staphylococci in the oral cavities of healthy adults and nasal–oral trafficking of the bacteria. J. Med. Microbiol. 57 , 95-99 (2008). https://doi.org/https://doi.org/10.1099/jmm.0.47561-0 Dhanya Raj, C. T. et al. Genomic and metabolic properties of Staphylococcus gallinarum FCW1 MCC4687 isolated from naturally fermented coconut water towards GRAS assessment. Gene 867 , 147356 (2023). https://doi.org/https://doi.org/10.1016/j.gene.2023.147356 Ahmed, S. et al. The Weissella genus: clinically treatable bacteria with antimicrobial/ probiotic effects on inflammation and cancer. Microorganisms 10 , 2427 (2022). https://doi.org/10.3390/microorganisms10122427. Yeu, J.-E., Lee, H.-G., Park, G.-Y., Lee, J. & Kang, M.-S. Antimicrobial and antibiofilm activities of Weissella cibaria against pathogens of upper respiratory tract infections. Microorganisms 9 , 1181 (2021). https://doi.org/10.3390/microorganisms9061181. Teixeira, C. G. et al. Genomic Analyses of Weissella cibaria W25, a potential bacteriocin-producing strain isolated from pasture in Campos das Vertentes, Minas Gerais, Brazil. Microorganisms 10 , 314 (2022). https://doi.org/10.1016/j.fbio.2023.102421. Papagianni, M. & Papamichael, E. M. Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX. Bioresour. Technol. 102 , 6730-6734 (2011). https://doi.org/10.1016/j.biortech.2011.03.106. Fujisawa, T., Benno, Y., Yaeshima, T. & Mitsuoka, T. Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981). Int. J. Syst. Bacteriol. 42 , 487-491 (1992). https://doi.org/10.1099/00207713-42-3-487 Deng, Z., Hou, K., Zhao, J. & Wang, H. The probiotic properties of lactic acid bacteria and their applications in animal husbandry. Curr. Microbiol. 79 , 22 (2021). https://doi.org/10.1007/s00284-021-02722-3 Pottenger, S. et al. Timing and delivery route effects of cecal microbiome transplants on Salmonella Typhimurium infections in chickens: potential for in-hatchery delivery of microbial interventions. Anim. Microbiome 5 , 11 (2023). https://doi.org/10.1186/s42523-023-00232-0 Wang, X. et al. The functional role of fecal microbiota transplantation on Salmonella Enteritidis infection in chicks. Vet. Microbiol. 269 , 109449 (2022). https://doi.org/10.1016/j.vetmic.2022.109449 Chintoan-Uta, C. et al. Role of cecal microbiota in the differential resistance of inbred chicken lines to colonization by Campylobacter jejuni . Appl Environ Microbiol 86 , e02607-02619 (2020). https://doi.org/10.1128/AEM.02607-19 Almansour, A. et al. Microbiota from specific pathogen-free mice reduces Campylobacter jejuni chicken colonization. Pathogens 10 , 1387 (2021). https://doi.org/10.3390/pathogens10111387 Pang, J., Beyi, A. F., Looft, T., Zhang, Q. & Sahin, O. Fecal microbiota transplantation reduces Campylobacter jejuni colonization in young broiler chickens challenged by oral gavage but not by seeder birds. Antibiotics 12 , 1503 (2023). https://doi.org/10.3390/antibiotics12101503 Zaytsoff, S. J. M. et al. Microbiota transplantation in day-old broiler chickens ameliorates necrotic enteritis via modulation of the intestinal microbiota and host immune responses. Pathogens 11 , 972 (2022). https://doi.org/10.3390/pathogens11090972 Fitzgerald, C. B. et al. Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa. BMC Genomics 19 , 1-20 (2018). Lund, M., Bjerrum, L. & Pedersen, K. Quantification of Faecalibacterium prausnitzii -and Subdoligranulum variabile -like bacteria in the cecum of chickens by real-time PCR. Poult. Sci. 89 , 1217-1224 (2010). Ning, L. et al. Microbiome and metabolome features in inflammatory bowel disease via multi-omics integration analyses across cohorts. Nat. Commun. 14 , 7135 (2023). https://doi.org/10.1038/s41467-023-42788-0 Gavzy, S. J. et al. Bifidobacterium mechanisms of immune modulation and tolerance. Gut Microbes 15 , 2291164 (2023). https://doi.org/10.1080/19490976.2023.2291164 Lim, H. J. & Shin, H. S. Antimicrobial and immunomodulatory effects of Bifidobacterium Strains: A Review. J. Microbiol. Biotechnol. 30 , 1793-1800 (2020). https://doi.org/10.4014/jmb.2007.07046 Sharma, M., Wasan, A. & Sharma, R. K. Recent developments in probiotics: An emphasis on Bifidobacterium . Food Biosci. 41 , 100993 (2021). https://doi.org/https://doi.org/10.1016/j.fbio.2021.100993 Khalique, A. et al. Probiotics mitigating subclinical necrotic enteritis (SNE) as potential alternatives to antibiotics in poultry. AMB Express 10 , 50 (2020). https://doi.org/10.1186/s13568-020-00989-6 Schoster, A. et al. In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains. Anaerobe 20 , 36-41 (2013). https://doi.org/https://doi.org/10.1016/j.anaerobe.2013.02.006 National Research Council. Nutrient Requirements of Poultry: Ninth Revised Edition, 1994 . (The National Academies Press, 1994). Kim, D. M. et al. Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis. J. Anim. Sci. Biotechnol. 15 , 29 (2024). https://doi.org/10.1186/s40104-024-00995-9 Yang, Q. et al. Butyrate in combination with forskolin alleviates necrotic enteritis, increases feed efficiency, and improves carcass composition of broilers. J. Anim. Sci. Biotechnol. 13 , 3 (2022). https://doi.org/10.1186/s40104-021-00663-2 Yang, Q., Whitmore, M. A., Robinson, K., Lyu, W. & Zhang, G. Butyrate, forskolin, and lactose synergistically enhance disease resistance by inducing the expression of the genes involved in innate host defense and barrier function. Antibiotics 10 , 1175 (2021). https://doi.org/10.3390/antibiotics10101175 Shojadoost, B., Vince, A. R. & Prescott, J. F. The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens : a critical review. Vet. Res. 43 , 74 (2012). https://doi.org/10.1186/1297-9716-43-74 Liu, J. et al. Dynamic response of the intestinal microbiome to Eimeria maxima -induced coccidiosis in chickens. Microbiol. Spectr. 12 , e0082324 (2024). https://doi.org/10.1128/spectrum.00823-24 Guo, J. et al. Is intestinal microbiota fully restored after chickens have recovered from coccidiosis? Pathogens 14 , 81 (2025). https://doi.org/10.3390/pathogens14010081 Liu, J. et al. Anaerobutyricum and Subdoligranulum are differentially enriched in broilers with disparate weight gains. Animals 13 , 1834 (2023). https://doi.org/10.3390/ani13111834 Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37 , 852-857 (2019). https://doi.org/10.1038/s41587-019-0209-9 Amir, A. et al. Deblur rapidly resolves single-nucleotide community sequence patterns. mSystems 2 , e00191-00116 (2017). https://doi.org/10.1128/mSystems.00191-16 Yoon, S. H. et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67 , 1613-1617 (2017). https://doi.org/10.1099/ijsem.0.001755 McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8 , e61217 (2013). https://doi.org/10.1371/journal.pone.0061217 vegan: Community ecology package v. R Package version 2.5-5 (2019). Lin, H. & Peddada, S. D. Multigroup analysis of compositions of microbiomes with covariate adjustments and repeated measures. Nat. Methods 21 , 83-91 (2024). https://doi.org/10.1038/s41592-023-02092-7 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Supplementary Information Fig. S1. Relative abundances (%) of selected ileal bacteria among three chicken breeds under healthy and NE conditions. Fig. S2. Relative abundances (%) of selected cecal bacteria among three chicken breeds under healthy and NE conditions. Fig. S3. Alternations of the ileal microbiota of Cobb chickens following cecal microbiota transplantation (CMT) from three chicken breeds. Fig. S4. Alternations of the cecal microbiota of Cobb chickens following cecal microbiota transplantation (CMT) from three chicken breeds. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8349290","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":560713182,"identity":"25cd5825-7861-427a-9396-e0b2fa59e863","order_by":0,"name":"Jing Liu","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liu","suffix":""},{"id":560713183,"identity":"c5cbf3c1-a624-4951-bc21-ffb337e7626e","order_by":1,"name":"Jiaqing Guo","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqing","middleName":"","lastName":"Guo","suffix":""},{"id":560713184,"identity":"45f861c5-3358-4da2-ad01-bc3bc0e92dfd","order_by":2,"name":"Isabel Tobin","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Tobin","suffix":""},{"id":560713185,"identity":"6577948b-0062-4be0-9d13-735c37ba4a3e","order_by":3,"name":"Melanie A. Whitmore","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Melanie","middleName":"A.","lastName":"Whitmore","suffix":""},{"id":560713186,"identity":"7622c18e-97be-4f7d-8d6c-32967def25ae","order_by":4,"name":"Dohyung M. Kim","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Dohyung","middleName":"M.","lastName":"Kim","suffix":""},{"id":560713188,"identity":"6e0f1d15-e7cc-453d-844d-aa10271210f7","order_by":5,"name":"Prasiddha Paudel","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Prasiddha","middleName":"","lastName":"Paudel","suffix":""},{"id":560713189,"identity":"6fb84920-b84a-4ac2-a6fd-92c3f003edf0","order_by":6,"name":"Anisha Subedi","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Anisha","middleName":"","lastName":"Subedi","suffix":""},{"id":560713190,"identity":"77741961-fec9-4ca1-9e0f-eda45d8004ba","order_by":7,"name":"Michael G. Kaiser","email":"","orcid":"","institution":"Iowa State University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"G.","lastName":"Kaiser","suffix":""},{"id":560713191,"identity":"acb90aec-d13e-4e2c-877e-9f1e1bb727cd","order_by":8,"name":"Susan J. Lamont","email":"","orcid":"","institution":"Iowa State University","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"J.","lastName":"Lamont","suffix":""},{"id":560713192,"identity":"750e49e9-6c3c-4104-b95d-c13e74f66e94","order_by":9,"name":"Guolong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYLCCBwwMcgw8cG4CEVqAaoyBWhgbIKqJ1JLYQLQW+f41hg8SKu6k9/Mcf/7g5w8bBn72HAO8WhhnvDE2SDjzLHdmb49hY09CGoNkzxv8WpglzphJJLYdzt1wnoexgSfhMIPBDQK2sEG1pBucZ3/Y+CfhP4M9IS08/D1gLQkGZxsMm3kSDjAYSBDQIiHBVgz0y2HDmT1nDGfLpCXzSJx5VoBXi3z/4Y0PPlQclufnSX/w8Y2NnRx/e/IGvFoYJBLQXIpfOQjwHyCsZhSMglEwCkY4AAC90khvIwxAnAAAAABJRU5ErkJggg==","orcid":"","institution":"Oklahoma State University","correspondingAuthor":true,"prefix":"","firstName":"Guolong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-12-12 23:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8349290/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8349290/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98436590,"identity":"e6153322-6899-4d55-9c9b-1d83082b6c0f","added_by":"auto","created_at":"2025-12-17 16:55:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9774322,"visible":true,"origin":"","legend":"","description":"","filename":"FayoumiCRtoNEnpjMicrobiome.docx","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/48ddfe233181ca756bb83d6b.docx"},{"id":98298982,"identity":"c7c64e4d-11a7-424a-bd4e-65472cf8dda1","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10956,"visible":true,"origin":"","legend":"","description":"","filename":"f2ac5b75074942edaf63ffdcd24b8add.json","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/c5d2fafd7e6f20aa9f3e6520.json"},{"id":98435145,"identity":"a32c62c3-7ff8-4aa9-a272-8b282fe80bd6","added_by":"auto","created_at":"2025-12-17 16:53:13","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163420,"visible":true,"origin":"","legend":"","description":"","filename":"f2ac5b75074942edaf63ffdcd24b8add1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/18aae8de3aff12d87a02da80.xml"},{"id":98298997,"identity":"9fd21b9f-8592-48b5-80f4-4a4273cff7b6","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"emf","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14175568,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.emf","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/cc8c972151ad2fd164a1d5fb.emf"},{"id":98298983,"identity":"68174bf6-ce5b-4b35-8cc9-a5f9c0ad2b48","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":947376,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/8d4d67e540e005ce31039e83.jpeg"},{"id":98298999,"identity":"fe246566-ca15-4735-98f1-5b5ccf56eeef","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":988490,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/00675cf897b4ea435c20ae75.jpeg"},{"id":98298995,"identity":"9f0522f8-f090-4a03-ad7d-2c0714e72ccd","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"emf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":537756,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.emf","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/5052b486c7dfb95d026a8452.emf"},{"id":98299007,"identity":"94435de7-93e0-46f7-8077-6c48753132dc","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"emf","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32835772,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.emf","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/a2e6ed1b473f56a9ce867bad.emf"},{"id":98298984,"identity":"8f5b15bf-0249-4c7d-b826-6ea5c58c1f22","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":478020,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/85bab081a080c5f060a0ea63.jpeg"},{"id":98434579,"identity":"3773d758-6cbf-46a2-98d0-ff7b52755ac8","added_by":"auto","created_at":"2025-12-17 16:52:21","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":450156,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/6f7d14bfb12e7c3a5f644ade.jpeg"},{"id":98298990,"identity":"1c74cefb-8a16-4483-a090-c6727fa24619","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":940230,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/30c234120beef0a1f1d851c4.jpeg"},{"id":98436846,"identity":"92f3dc47-4c6d-40ce-b5db-c42e654525ab","added_by":"auto","created_at":"2025-12-17 16:56:19","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":956450,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/33cdcea834d08586a2a6c5e6.jpeg"},{"id":98435635,"identity":"84008f7b-e7c5-470c-ae97-540f8cb4ce32","added_by":"auto","created_at":"2025-12-17 16:54:09","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10766,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/70618e2fbb1c83e6d1623061.png"},{"id":98435814,"identity":"e1b88aae-9f80-4239-b0ea-430e87a5a037","added_by":"auto","created_at":"2025-12-17 16:54:27","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128520,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/9fda553358dd24040f9d9d85.png"},{"id":98435665,"identity":"6b7f7155-05e3-4e58-9f3a-f51a98ad7786","added_by":"auto","created_at":"2025-12-17 16:54:11","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133682,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/12969f6d822f2840d9cffeb4.png"},{"id":98298988,"identity":"113fd78f-703d-4352-be72-6afa475f2a33","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13654,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/773c5b821558730a5ade2959.png"},{"id":98434922,"identity":"c28a7e50-52ad-462d-9b48-e5b412a20673","added_by":"auto","created_at":"2025-12-17 16:52:46","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16115,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/eaad53192f866811a0bb8f6e.png"},{"id":98299003,"identity":"b54f93a0-40fc-4c00-8b50-9692074dfa3e","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69884,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/553d1c6ab7a0d0e98a29db95.png"},{"id":98435765,"identity":"2d4271bc-b3df-44a1-837a-2cae86e5d996","added_by":"auto","created_at":"2025-12-17 16:54:23","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66261,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/1d7dc5bb7174e0a3cc8e83a6.png"},{"id":98435741,"identity":"30bc8419-60b8-4425-9468-6de73fe5607c","added_by":"auto","created_at":"2025-12-17 16:54:20","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122261,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/3fd275fdd1ba0c547c2b77ea.png"},{"id":98435375,"identity":"291382e7-00ac-41c3-83ca-c56b868ab6c8","added_by":"auto","created_at":"2025-12-17 16:53:38","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121468,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/4206d354ca63b4c271c06151.png"},{"id":98435655,"identity":"96e5626a-703b-4e48-8f0c-12dc51b47468","added_by":"auto","created_at":"2025-12-17 16:54:10","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161130,"visible":true,"origin":"","legend":"","description":"","filename":"f2ac5b75074942edaf63ffdcd24b8add1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/f7e6684ca74c97eb0ea56831.xml"},{"id":98299000,"identity":"c8bf64dd-3e4c-4f86-8ccf-6eef86a3450e","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184096,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/b4336ed6117bae66b5aa34b9.html"},{"id":98298979,"identity":"70169723-fa01-4975-8dbc-7b95badaa8f7","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential susceptibility of three chicken breeds to necrotic enteritis (NE)\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Experimental scheme. Three chicken breeds including Fayoumi M5.1, Leghorn Ghs6, and Cobb chickens were randomly assigned to the mock or NE group with 12 birds/group. Chickens in the NE group were subjected to an initial challenge with \u003cem\u003eEimeria maxima\u003c/em\u003e on d 16 and four subsequent challenges with \u003cem\u003eClostridium perfringens\u003c/em\u003e twice daily on d 20 and 21, while the remaining chickens were mock-infected. \u003cstrong\u003e(B)\u003c/strong\u003e Animal survival rate (%) between days 20–23. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to NE-challenged Cobb chickens, based on the log-rank test. \u003cstrong\u003e(C)\u003c/strong\u003e The frequency (%) of small intestinal lesion scores (LS) of surviving chickens on d 23.\u003cstrong\u003e \u003c/strong\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 based on the Kruskal-Wallis test and post-hoc Dunn’s test.\u003cstrong\u003e (D)\u003c/strong\u003e Average individual body weight gains (g) of surviving animals between d 16–23. Data shown are mean ± SEM. Means not sharing a common superscript letter denote statistical significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) based on one-way ANOVA and post-hoc Tukey’s test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/280834c99e6ca2cc8bb0eefa.png"},{"id":98298981,"identity":"467a2a55-7ad8-4ac8-a32b-2d9aa51b9f9f","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":460638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferences in the ileal microbiota among three chicken breeds under healthy and NE conditions\u003c/strong\u003e. M5.1, Ghs.6, and Cobb chickens were either mock-infected or subjected to NE challenge (\u003cem\u003en\u003c/em\u003e = 12 per group). Ileal digesta samples were collected from surviving animals on d 23 for bacterial DNA isolation and 16S rRNA gene sequencing. (\u003cstrong\u003eA\u003c/strong\u003e) Box and whisker plot depicting Shannon Index across different treatment groups. Significance was assessed using the Kruskal–Wallis test and post-hoc Dunn’s test. Different superscript letters denote significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in pairwise comparisons. (\u003cstrong\u003eB\u003c/strong\u003e) Principal coordinates analysis (PCoA) plot of weighted UniFrac distances. Significance was determined using PERMANOVA. (\u003cstrong\u003eC\u003c/strong\u003e) Relative abundances (%) of the top 15 ASVs in the ileal microbiota. (\u003cstrong\u003eD\u003c/strong\u003e) Heatmap showing NE-induced differential enrichment of the top 40 ASVs in the ileum. The bottom panel depicts log2 fold changes (log2FC) in ileal bacterial abundance across six groups relative to mock-infected Cobb chickens. Groups not sharing a common superscript letter in a column denote statistically significant differences (P \u0026lt; 0.05) based on ANCOM-BC2 analysis \u003csup\u003e57\u003c/sup\u003e. The top panel indicates log2FC values comparing NE-infected chickens to their respective mock-infected controls. *\u003cem\u003eP\u003c/em\u003e-adjusted \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e-adjusted \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e-adjusted \u0026lt; 0.001 as determined by the Kruskal-Wallis test and post-hoc Dunn’s test with Benjamini-Hochberg correction.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/2b689140ece850d7ac87d10c.png"},{"id":98436404,"identity":"4d388434-e3dc-49d4-80c4-1f8ebbda0aeb","added_by":"auto","created_at":"2025-12-17 16:55:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":461011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferences in the cecal microbiota among three chicken breeds under healthy and NE conditions\u003c/strong\u003e. M5.1, Ghs.6, and Cobb chickens were either mock-infected or subjected to NE challenge (\u003cem\u003en\u003c/em\u003e = 12 per group). Cecal digesta samples were collected from surviving animals on d 23 for bacterial DNA isolation and 16S rRNA gene sequencing. (\u003cstrong\u003eA\u003c/strong\u003e) Box and whisker plot depicting Shannon Index across different treatment groups. Significance was assessed using the Kruskal–Wallis test and post-hoc Dunn’s test. Different superscript letters denote significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in pairwise comparisons. (\u003cstrong\u003eB\u003c/strong\u003e) Principal coordinates analysis (PCoA) plot of weighted UniFrac distances. Significance was determined using PERMANOVA. (\u003cstrong\u003eC\u003c/strong\u003e) Relative abundances (%) of the top 15 ASVs in the cecal microbiota. (\u003cstrong\u003eD\u003c/strong\u003e) Heatmap showing NE-induced differential enrichment of the top 50 ASVs in the cecum. The bottom panel depicts log2 fold changes (log2FC) in bacterial abundance across six groups relative to mock-infected Cobb chickens. Groups not sharing a common superscript letter in a column denote statistically significant differences (P \u0026lt; 0.05) based on ANCOM-BC2 analysis \u003csup\u003e57\u003c/sup\u003e. The top panel indicates log2FC values comparing NE-infected chickens to their respective mock-infected controls. *\u003cem\u003eP\u003c/em\u003e-adjusted \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e-adjusted \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e-adjusted \u0026lt; 0.001 as determined by the Kruskal-Wallis test and post-hoc Dunn’s test with Benjamini-Hochberg correction.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/e335c8bb920c3e426435c86f.png"},{"id":98298980,"identity":"e921c38d-f78c-4e20-abe0-f069e060d540","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential protection of Cobb chickens from NE by the cecal microbiota of three chicken breeds. (A) \u003c/strong\u003eExperimental scheme. A total of 120 day-of-hatch Cobb chicks were randomly divided into eight groups (\u003cem\u003en\u003c/em\u003e= 15 per group), with each receiving the cecal microbiota prepared from M5.1, Ghs6, or Cobb chickens or an equal volume of PBS via oral gavage on days 0, 1, 9, and 13. Four groups were challenged with \u003cem\u003eE. maxima\u003c/em\u003e on day 10 and \u003cem\u003eC. perfringens\u003c/em\u003e on day 14 to induce NE, while the other four groups receiving the same volume of PBS or fluid thioglycollate broth (FTG) on respective days.\u003cstrong\u003e(B)\u003c/strong\u003e Animal survival (%) between day 14–17 among four groups of NE-infected Cobb chickens that received PBS or the cecal microbiota from Cobb, Ghs6, or M5.1 chickens. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the NE group receiving only PBS, based on the log-rank test. (\u003cstrong\u003eC)\u003c/strong\u003e The frequency (%) of intestinal lesion scores (LS) among four groups of NE-infected Cobb chickens that received PBS or the cecal microbiota on day 17. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 based on the Kruskal-Wallis test and post-hoc Dunn’s test.\u003cstrong\u003e \u003c/strong\u003eNo other groups are significantly different.\u003cstrong\u003e (D)\u003c/strong\u003e Average body weight gains of surviving animals among eight groups of recipient Cobb chickens between day 10–17. Data shown are means ± SEM. Means not sharing a common superscript letter denote statistical significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) based on one-way ANOVA and post-hoc Tukey’s test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/d064349912f78c826361b313.png"},{"id":98298987,"identity":"fb642f7f-3a23-470c-9122-fdda307fa804","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":149019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtection of chickens from NE by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBifidobacterium pseudolongum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eRelative abundances (%) of \u003cem\u003eB. anseris\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e) and \u003cem\u003eB. pseudolongum\u003c/em\u003e (\u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e) in the ileum and cecum of healthy and NE-challenged M5.1, Ghs6, and Cobb chickens (\u003cem\u003en\u003c/em\u003e = 12 per group). (\u003cstrong\u003eE\u003c/strong\u003e) \u003cem\u003eC. perfringens \u003c/em\u003ecounts following 24-h incubation with or without \u003cem\u003eB. pseudolongum\u003c/em\u003e at the 1:1 ratio. (\u003cstrong\u003eF\u003c/strong\u003e) Experimental scheme assessing \u003cem\u003eB. pseudolongum\u003c/em\u003e-mediated protection against NE. A total of 90 day-of-hatch Cobb chicks were randomly divided into three groups (\u003cem\u003en\u003c/em\u003e = 30 per group), with each receiving \u003cem\u003eB. pseudolongum\u003c/em\u003e or an equal volume of PBS via oral gavage on days 9, 11, 13, and 15. Two groups were challenged with \u003cem\u003eE. maxima\u003c/em\u003eon day 10 and \u003cem\u003eC. perfringens\u003c/em\u003e on day 14 to induce NE, while the third group receiving the same volume of PBS or fluid thioglycollate broth (FTG) on respective days.\u003cstrong\u003e (G)\u003c/strong\u003e Animal survival (%) between day 14–17 among three groups. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 compared to the NE group receiving only PBS, based on the log-rank test. (\u003cstrong\u003eH)\u003c/strong\u003e The frequency (%) of intestinal lesion scores (LS) among three groups. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 based on the Kruskal-Wallis test and post-hoc Dunn’s test.\u003cstrong\u003e (I)\u003c/strong\u003e Average body weight gains of animals among three groups between day 10–17. Data shown are means ± SEM. Means not sharing a common superscript letter denote statistical significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) based on one-way ANOVA and post-hoc Tukey’s test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/da742eac01380851d9d8a866.png"},{"id":99313087,"identity":"50d0aa68-37cf-4a56-b6c4-5349a82b83ee","added_by":"auto","created_at":"2025-12-31 16:19:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2452423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/cdeaae6e-b218-4796-a8d1-f73ab1f2ec46.pdf"},{"id":98298989,"identity":"c9105429-72af-4dbd-9a1f-6ae710cbb4d3","added_by":"auto","created_at":"2025-12-16 09:47:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2843401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. S1. Relative abundances (%) of selected ileal bacteria among three chicken breeds under healthy and NE conditions. Fig. S2. Relative abundances (%) of selected cecal bacteria among three chicken breeds under healthy and NE conditions. Fig. S3. Alternations of the ileal microbiota of Cobb chickens following cecal microbiota transplantation (CMT) from three chicken breeds. Fig. S4. Alternations of the cecal microbiota of Cobb chickens following cecal microbiota transplantation (CMT) from three chicken breeds.\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8349290/v1/440ad423e80cd71381818c6c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intestinal Microbiome Confers Strong Colonization Resistance Against Necrotic Enteritis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNecrotic enteritis (NE) is an enteric disease caused by \u003cem\u003eClostridium perfringens\u003c/em\u003e, a Gram-positive, anaerobic, spore-forming, rod-shaped bacterium. NE remains one of the most economically devastating diseases in the global poultry industry, costing over \u003cspan\u003e$\u003c/span\u003e6\u0026nbsp;billion annually in production losses \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Various predisposing factors are known to promote NE development, such as exposure to apicomplexan protozoa, \u003cem\u003eEimeria\u003c/em\u003e, the etiologic agent of avian coccidiosis \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Traditionally, prophylactic in-feed antibiotics have proven effective for the control of clostridial infections in chickens \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, with recent withdrawal of in-feed antibiotics from livestock production in a growing number of countries and increasing consumer demand for antibiotic-free animal products, NE has become more prevalent, necessitating effective antibiotic alternatives \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Probiotics such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e and prebiotics such as mannan-oligosaccharides have proved beneficial in NE alleviation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, no commensal bacteria have been directly linked to NE resistance, and NE-resistant commensals could potentially offer prophylactic and/or therapeutic utility against NE.\u003c/p\u003e \u003cp\u003eTwo highly inbred chicken lines, Fayoumi M5.1 and Leghorn Ghs6, are drastically different in their ability to resist Newcastle disease, with M5.1 being highly resistant and Ghs6 highly susceptible \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Additionally, Fayoumi chickens are known to be more resistant to coccidiosis, avian influenza, and salmonellosis than Leghorns or commercial broiler chickens \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The intestinal microbiota plays a significant role in host homeostasis and health, and there is mounting evidence indicating that the microbiota is shaped by host genetics \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Thus, we hypothesize that the intestinal microbiota from disease-resistant chicken breeds harbors a unique microbiota that offers strong colonization resistance (CR) to diseases such as NE.\u003c/p\u003e \u003cp\u003eTo test our hypothesis, we assessed the efficacy of the microbiota from M5.1, Ghs6, and Cobb chickens in protecting newly-hatched Cobb chickens from NE through cecal microbiota transplantation (CMT) and revealed that M5.1 microbiota is the most protective against NE. We further compared the intestinal microbiota profiles among the three chicken breeds under both healthy and NE conditions and identified a large number of differentially enriched bacteria. Additionally, we demonstrated that oral administration of \u003cem\u003eBifidobacterium pseudolongum\u003c/em\u003e, which was specifically enriched in NE-challenged M5.1 chickens, confers strong protection to Cobb chickens against NE challenge. These findings provide direct evidence that the intestinal microbiota, particularly from disease-resistant chickens, offers robust CR against NE and can be leveraged for NE mitigation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDifferential susceptibility of three chicken breeds to NE\u003c/h2\u003e \u003cp\u003eTo investigate the difference in NE susceptibility, we challenged M5.1, Ghs6, and Cobb chickens to induce NE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). While approximately 80% of Cobb chickens and 91% Ghs6 chickens survived at 3 days post-infection (dpi), 100% M5.1 chickens exhibited no mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Consistently, among surviving chickens, 75% Cobb chickens showed extensive, score-6 lesions in the small intestinal tract, and 9% Ghs6 chickens exhibited severe score-6 lesions, while none of M5.1 chickens presented any abnormalities in the small intestine at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Additionally, NE significantly reduced weight gain in Cobb and Ghs6 chickens but had no impact on M5.1 chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. These results clearly indicated that, among three breeds of chickens, M5.1 chickens are the most resistant to NE and Cobb chickens are the most susceptible, with Ghs6 being intermediate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDifferences in the ileal microbiota among three chicken breeds under healthy and NE conditions\u003c/h3\u003e\n\u003cp\u003eTo investigate the microbiota differences among M5.1, Ghs6, and Cobb chickens and their microbiota shifts in response to NE, bacterial DNA was isolated from the ileal and cecal digesta of mock- and NE-infected chickens and subjected to 16S rRNA gene sequencing. After quality control, 7,750,326 high-quality sequencing reads were obtained, averaging 61,510\u0026thinsp;\u0026plusmn;\u0026thinsp;7,021 sequences per sample. Following denoising and removal of amplicon sequence variants (ASVs) present in less than 5% of samples, 399 and 421 ASVs were identified in the ileum and cecum, respectively.\u003c/p\u003e \u003cp\u003eAnalysis of α-diversity of the ileal microbiota, as measured by Shannon Index, revealed a progressive decline across the three chicken breeds in relation to increasing resistance to NE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This trend was further accentuated following NE challenge, with the NE-resistant M5.1 chickens exhibiting the least perturbation. Although β-diversity showed significant differences among the three breeds based on weighted UniFrac distance, M5.1 chickens were the least affected by NE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The ileal microbiota profiles were drastically different among the breeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In healthy Cobb chickens, three dominant taxa included group A \u003cem\u003eLactobacillus\u003c/em\u003e F1 (25.5%), \u003cem\u003eLigilactobacillus salivarius\u003c/em\u003e F3 (14.0%), and \u003cem\u003eCorynebacterium\u003c/em\u003e F45 (10.3%). In contrast, \u003cem\u003eStaphylococcus gallinarum\u003c/em\u003e (F8) was highly abundant in healthy Ghs6 (43.9%) and M5.1 chickens (36.9%) but was largely absent (0.8%) in Cobb chickens (\u003cb\u003eFig. S1A\u003c/b\u003e). \u003cem\u003eCorynebacterium\u003c/em\u003e was almost undetectable in both M5.1 and Ghs6 chickens. Notably, \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e (F4) was minimal in Ghs6 chickens (0.3%), and group A \u003cem\u003eLactobacillus\u003c/em\u003e accounted for only 3.9% of the total ileum bacteria. In M5.1 chickens, \u003cem\u003eL. johnsonii\u003c/em\u003e represented 34.7%, while group A \u003cem\u003eLactobacillus\u003c/em\u003e comprised just 2.2% in the ileum.\u003c/p\u003e \u003cp\u003eAmong the 40 most abundant ileal ASVs, the majority were significantly different among the three chicken breeds under healthy and NE-challenged conditions. The ileal microbiota profiles of M5.1 and Ghs6 chickens resembled each other more than that of Cobb chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Among three major pathobionts, \u003cem\u003eC. perfringens\u003c/em\u003e increased drastically in NE-susceptible Cobb chickens from 0.03% to 34.1% but remained minimal in NE-challenged Ghs6 (1.5%) and M5.1 (0.08%) chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cb\u003eFig. S1\u003c/b\u003e). \u003cem\u003eEscherichia\u003c/em\u003e (F7) also significantly bloomed in NE-infected Cobb chickens but not in M5.1 or Ghs6 chickens. \u003cem\u003eEnterococcus cecorum\u003c/em\u003e (F30) showed a significant increase in all three breeds following NE challenge. Major lactic acid bacteria (LAB) such as group A \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e were lowly abundant in healthy M5.1 and Ghs6 chickens compared to Cobb chickens but were markedly enriched in both breeds following NE challenge (\u003cb\u003eFig. S1\u003c/b\u003e). In response to NE, \u003cem\u003eL. johnsonii\u003c/em\u003e was significantly reduced in Cobb chickens but increased substantially in both M5.1 and Ghs6 chickens.\u003c/p\u003e\n\u003ch3\u003eDifferences in the cecal microbiota among three chickens breeds under healthy and NE conditions\u003c/h3\u003e\n\u003cp\u003eConsistent with observations in the ileum, α-diversity of the cecal microbiota progressively declined with increasing NE resistance across the three chicken breeds, and NE infection further reduced Shannon Index in Cobb but not Ghs6 or M5.1 chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, β-diversity analysis using weighted UniFrac distance revealed distinct microbiota structures among the three breeds with and without NE challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The taxonomic composition of the cecal microbiota also varied markedly among breeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Notably, \u003cem\u003eBacteroides fragilis\u003c/em\u003e (F2) was the dominant taxon in healthy Ghs6 (26.4%) and M5.1 (35.0%) chickens, but accounted for only 6.2% in healthy Cobb chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cb\u003eFig. S2\u003c/b\u003e). Following NE challenge, \u003cem\u003eB. fragilis\u003c/em\u003e members (F2 and F16) were enriched in Cobb chickens but showed a slight reduction in Ghs6 and M5.1 chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAmong major LAB species including Group A \u003cem\u003eLactobacillus\u003c/em\u003e (F1), \u003cem\u003eL. salivarius\u003c/em\u003e (F3), and \u003cem\u003eL. johnsonii\u003c/em\u003e (F4) were less abundant in M5.1 chickens but increased substantially following NE infection. In contrast, \u003cem\u003eL. johnsonii\u003c/em\u003e was significantly suppressed in NE-challenged Cobb chickens. Short-chain fatty acid (SCFA)-producing bacteria such as two \u003cem\u003eFaecalibacterium\u003c/em\u003e members (F14 and F19) were more prevalent in Cobb chickens under healthy conditions but diminished across all breeds after NE infection. Conversely, several other SCFA-producing members of the \u003cem\u003eOscillospiraceae\u003c/em\u003e family (e.g., F24, F50, and F59) were more abundant in M5.1 and Ghs6 chickens and declined following NE challenge, while showing enrichment in NE-infected Cobb chickens.\u003c/p\u003e \u003cp\u003eRegarding the three major pathobionts, \u003cem\u003eC. perfringens\u003c/em\u003e remained at low abundance in the cecum across all breeds under both healthy and NE challenge conditions, although a statistically significant increase was observed in NE-infected Cobb and M5.1 chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD \u003cb\u003eand Fig. S2\u003c/b\u003e). \u003cem\u003eEscherichia\u003c/em\u003e was enriched in NE-infected Cobb chickens but remained largely unchanged in Ghs6 and M5.1 chickens. \u003cem\u003eE. cecorum\u003c/em\u003e tended to increase in Cobb and M5.1 chickens following NE challenge, with no notable changes in Ghs6 chickens. Interestingly, two other \u003cem\u003eClostridium\u003c/em\u003e species (F51 and F82) showed no major alterations in response to NE across all three breeds.\u003c/p\u003e\n\u003ch3\u003eDifferential protection of Cobb chickens from NE by the cecal microbiota of three chicken breeds\u003c/h3\u003e\n\u003cp\u003e To directly evaluate the efficacy of intestinal microbiota in alleviating NE, cecal microbiota was prepared from all three chicken breeds and transplanted to na\u0026iuml;ve Cobb chickens, followed by NE challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Our results showed that the M5.1 microbiota provided the best protection against NE, with 100% survival in the transplanted group, while approximately 40% of the chickens in the mock-transplanted control group died from severe intestinal lesions at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Interestingly, the cecal microbiota from the two susceptible breeds, Ghs.6 and Cobb, also conferred significant protection to na\u0026iuml;ve Cobb chickens, although with reduced efficacy compared to the M5.1 microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Consistently, the cecal microbiota of all three chicken breeds partially reversed NE-induced weight loss in recipient Cobb chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eFurther examination revealed obvious changes in the ileal microbiota of recipient chickens following cecal microbiota transplantation (CMT). Except for microbiota richness (\u003cb\u003eFig. S3A\u003c/b\u003e), CMT of the M5.1 microbiota, but not other microbiota, significantly increased the Shannon Index of the ileal microbiota of recipient chickens (\u003cb\u003eFig. S3B\u003c/b\u003e) and caused significant shifts in microbiota structure based on weighted UniFrac distance (\u003cb\u003eFig. S3C\u003c/b\u003e). The ileal microbiota composition also experienced notable alterations (\u003cb\u003eFig. S3D\u003c/b\u003e). CMT of all three breeds significantly enriched two \u003cem\u003eGemmiger\u003c/em\u003e species (F22 and F31) with a tendency to increase many but not all LAB species. In contrast, \u003cem\u003eRombousia\u003c/em\u003e (F23) was significantly reduced following CMT of all three breeds. Among three major pathobionts, \u003cem\u003eC. perfringens\u003c/em\u003e and \u003cem\u003eE. cecorum\u003c/em\u003e were unaltered, but \u003cem\u003eEscherichia\u003c/em\u003e was significantly diminished following CMT. In response to NE, \u003cem\u003eC. perfringens\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e drastically bloomed in Cobb chickens mock-transplanted or transplanted with the Ghs6 or Cobb microbiota. In contrast, CMT with the M5.1 microbiota caused no blooming of \u003cem\u003eC. perfringens\u003c/em\u003e and a significant reduction in \u003cem\u003eEscherichia\u003c/em\u003e and \u003cem\u003eE. cecorum\u003c/em\u003e in the ileum of recipient chickens.\u003c/p\u003e \u003cp\u003eCMT also caused significant changes to the cecum of recipient Cobb chickens under healthy and NE conditions (\u003cb\u003eFig. S4A-S4D\u003c/b\u003e). Similar to the ileum, two \u003cem\u003eGemmiger\u003c/em\u003e species (F22 and F31) were significantly enriched by all three transplanted microbiotas in both healthy and NE-infected recipients (\u003cb\u003eFig. S4E\u003c/b\u003e). Many LAB species were largely unaffected by CMT, except that group A \u003cem\u003elactobacillus\u003c/em\u003e and \u003cem\u003eLimosilactobacillus oris\u003c/em\u003e (F17) were significantly enriched in recipient chickens in response to NE, with M5.1 microbiota-transplanted chickens showing the largest increase. Neither \u003cem\u003eC. perfringens\u003c/em\u003e nor \u003cem\u003eEscherichia\u003c/em\u003e bloomed in Cobb chickens receiving the M5.1 microbiota. Surprisingly, \u003cem\u003eBacteroides\u003c/em\u003e, the dominant species in the cecum of both M5.1 and Ghs6 chickens, failed to be detected in the cecum of recipient Cobb chickens following CMT, with or without NE challenge.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtection of chickens from NE by\u003c/b\u003e \u003cb\u003eB. pseudolongum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBoth the ileal and cecal microbiotas of M5.1 or Ghs6 chickens resembled each other more than Cobb chickens under both mock- and NE-infected conditions. To explain the obvious difference in NE resistance between M5.1 and Ghs6 chickens, we detected a notable difference in the differential abundance of two \u003cem\u003eBifidobacterium\u003c/em\u003e species, namely \u003cem\u003eB. anseris\u003c/em\u003e (F84) and \u003cem\u003eB. pseudolongum\u003c/em\u003e (F201) in both the ileum and the cecum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) among the three chicken breeds. Both \u003cem\u003eBifidobacterium\u003c/em\u003e species were largely absent in both mock- and NE-infected Ghs6 and Cobb chickens as well as in mock-infected M5.1 chickens, but showed a significant enrichment in both the ileum and cecum of M5.1 chickens following NE challenge.\u003c/p\u003e \u003cp\u003eTo directly verify if \u003cem\u003eBifidobacterium\u003c/em\u003e plays a role in NE resistance, we plated the cecal bacteria of M5.1 chickens on MRS plates and identified an isolate to be \u003cem\u003eB. pseudolongum\u003c/em\u003e through Sanger sequencing of its full-length 16S rRNA gene. It showed a potent activity in inhibiting \u003cem\u003eC. perfringens\u003c/em\u003e growth with a 5.6-fold reduction when incubated 1:1 for 24 h in a coculture assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). To further evaluate its efficacy against NE, we orally inoculated approximately 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU of \u003cem\u003eB. pseudolongum\u003c/em\u003e to each Cobb chicken on days 9, 11, 13, and 15, and challenged them with \u003cem\u003eE. maxima\u003c/em\u003e and \u003cem\u003eC. perfringens\u003c/em\u003e to induce NE on days 10 and 14, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Only 42.2% of NE-challenged chickens survived at 3 dpi without intervention, compared to 83.3% survival in those that received \u003cem\u003eB. pseudolongum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), which also significantly alleviated intestinal lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), but with no obvious impact on growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Overall, these results suggested a protective role of \u003cem\u003eB. pseudolongum\u003c/em\u003e against NE.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eFayoumi chickens, originating from Egypt, have been found to be highly resistant to multiple diseases \u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We hypothesized that, shaped by its unique genetics, this chicken breed may harbor distinct intestinal microbiota to confer enhanced disease resistance. Our results demonstrated that, among three chicken breeds studied including Fayoumi, Leghorn, and Cobb, Fayoumi chickens exhibit the highest resistance to NE, whereas Cobb chickens are the most susceptible. We further demonstrated that cecal microbiota transplantation from Fayoumi chickens offers superior protection of NE-susceptible Cobb chickens from NE. Additionally, oral administration of \u003cem\u003eBifidobacterium\u003c/em\u003e, a uniquely enriched commensal in NE-infected Fayoumi chickens provided significant protection of Cobb chickens against NE, suggesting the utility of intestinal bacteria and \u003cem\u003eBifidobacterium\u003c/em\u003e in particular in mitigating NE and perhaps other diseases.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDifferential intestinal microbiota responses to NE among Cobb, Ghs6, and M5.1 chickens\u003c/h2\u003e \u003cp\u003eThe intestinal microbiome significantly influences health and disease, with its composition shaped by various factors, including host genetics \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this study, we observed significant differences in the intestinal microbiota among three chicken breeds. For example, \u003cem\u003eBacteroides\u003c/em\u003e was the most abundant in the cecum of M5.1 and Ghs6 chickens but was minimally present in Cobb chickens, which is consistent with several recent analyses showing the prevalence of \u003cem\u003eBacteroides\u003c/em\u003e in many indigenous breeds but not in Cobb chickens \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eBacteroides\u003c/em\u003e is a genus of non-spore-forming, Gram-negative bacteria that degrade nondigestible carbohydrates to produce SCFAs, offering various host benefits and providing CR against pathogens like \u003cem\u003eClostridioides difficile\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, certain \u003cem\u003eBacteroides\u003c/em\u003e species are opportunistic pathogens that can promote chronic inflammation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003eBacteroides\u003c/em\u003e may aid in NE resistance, its exact role in CR against NE requires further investigation. It is noteworthy that \u003cem\u003eBacteroides\u003c/em\u003e seems dispensable for NE resistance, as Cobb chickens become highly resistant to NE infection despite having undetectable levels of \u003cem\u003eBacteroides\u003c/em\u003e following M5.1 microbiota transplantation.\u003c/p\u003e \u003cp\u003eTo our surprise, \u003cem\u003eStaphylococcus\u003c/em\u003e, mainly \u003cem\u003eS. gallinarum\u003c/em\u003e, was most prevalent in M5.1 and Ghs6 chickens, accounting for 35\u0026ndash;45% of the total ileal bacteria, but was largely absent in Cobb chickens. \u003cem\u003eS. gallinarum\u003c/em\u003e is a non-pathogenic, coagulase-negative bacterium commonly found in healthy chickens, pheasants, and humans \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It has probiotic properties, showing activity against pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in vitro \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Additionally, it produces Staphyloferrin A, a siderophore that suppresses pathogenic bacteria growth by chelating iron, essential for virulence and bacterial interactions \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The role of \u003cem\u003eStaphylococcus\u003c/em\u003e in NE resistance warrants further investigation.\u003c/p\u003e \u003cp\u003eNotably, NE-resistant M5.1 chickens had a significantly higher abundance of \u003cem\u003eWeissella\u003c/em\u003e in the ileum compared to susceptible Ghs6 and Cobb chickens. \u003cem\u003eWeissella\u003c/em\u003e, part of the \u003cem\u003eLeuconostocaceae\u003c/em\u003e family, is known for its probiotic and anti-inflammatory potential \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For example, \u003cem\u003eW. cibaria\u003c/em\u003e can inhibit pathogenic microorganisms through metabolites like exopolysaccharides \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eWeissella\u003c/em\u003e species produce bacteriocins, such as Weissellicins \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The role of \u003cem\u003eWeissella\u003c/em\u003e in poultry health and disease remains underexplored, necessitating further studies.\u003c/p\u003e \u003cp\u003eAdditionally, our results clearly demonstrated varying abundances of LAB species among the three chicken breeds and their distinct responses to NE. Group A \u003cem\u003eLactobacillus\u003c/em\u003e, including highly related species like \u003cem\u003eL. crispatus\u003c/em\u003e, \u003cem\u003eL. acidophilus\u003c/em\u003e, and \u003cem\u003eL. gallinarum\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e that cannot be distinguished by the V3\u0026ndash;V4 region of the bacterial 16S rRNA gene, were more abundant in the ileum and cecum of Cobb chickens than in M5.1 and Ghs6 chickens. However, Group A \u003cem\u003eLactobacillus\u003c/em\u003e remained largely unchanged by NE in Cobb chickens but was significantly enriched in M5.1 and Ghs6 chickens. Conversely, \u003cem\u003eL. johnsonii\u003c/em\u003e was significantly reduced in Cobb chickens but enriched in M5.1 and Ghs6 chickens in response to NE. The differential response of the same LAB species in different breeds suggests the possible presence of different LAB strains, explaining the variation in the NE resistance pattern. It is important to confirm and isolate LAB strains preferentially growing in Fayoumi chickens and investigate their efficacy in disease resistance. Additionally, relative contributions of different LAB species to NE resistance require further investigation, although many LAB species have shown benefits against NE \u003csup\u003e3,29\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtection of naive Cobb chickens from NE through CMT\u003c/h3\u003e\n\u003cp\u003eGiven recent successes of transplanting fecal or cecal microbiota in conferring CR in chickens against pathogens such as \u003cem\u003eSalmonella\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eCampylobacter jejuni\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eC. perfringens\u003c/em\u003e infections \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, we compared the efficacy of the cecal microbiota from three chicken breeds in providing CR against NE. We observed a drastic improvement in NE resistance among naive Cobb chickens receiving the Fayoumi microbiota. Additionally, the cecal microbiota of NE-susceptible Cobb and Ghs6 chickens also provided notable, albeit less pronounced, protection against NE. These findings align with a previous report showing reduced chicken intestinal lesions in a subclinical NE model following transplantation of bioreactor-propagated cecal microbiota of adult chickens \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, our results contrast with an earlier study demonstrating that CMT from a resistant chicken line (ADOL Leghorn Line 6\u003csub\u003e1\u003c/sub\u003e) to a susceptible line (ADOL Leghorn Line N) failed to confer CR against \u003cem\u003eC. jejuni\u003c/em\u003e infection \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe discrepancy among these studies may be attributed to differences in the CMT preparation method. In our study and that of Zaytsoff, et al. \u003csup\u003e35\u003c/sup\u003e, the microbiota transplants were prepared under anaerobic conditions, whereas Chintoan-Uta et al. \u003csup\u003e32\u003c/sup\u003e prepared CMT aerobically. It is plausible that anaerobic commensal bacteria, which are crucial for disease resistance, may not survive well during aerobic microbiota preparation. Supporting this hypothesis, transplantation of anaerobic cecal microbiota was shown to provide CR against \u003cem\u003eC. jejuni\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, transplantation of both aerobically and anaerobically cultured mouse fecal microbiota offered CR against \u003cem\u003eC. jejuni\u003c/em\u003e in chickens \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Further research is warranted to elucidate the specific microbial communities and mechanisms underlying the protection against different pathogens.\u003c/p\u003e \u003cp\u003eWe observed enrichment of \u003cem\u003eGemmiger\u003c/em\u003e in both the ileum and cecum of recipient chickens following CMT from all three chicken breeds, whereas \u003cem\u003eMegamonas\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e were enriched following transplantation of bioreactor-propagated cecal microbiota \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The differences in outcomes between the two studies likely stem from variations in the transplanted microbiota and genetic differences in recipient chickens. \u003cem\u003eGemmiger\u003c/em\u003e, a genus of bacteria in the family \u003cem\u003eOscillospiraceae\u003c/em\u003e, is closely related to \u003cem\u003eSubdoligranulum\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, both of which produce SCFAs with anti-inflammatory properties. \u003cem\u003eGemmiger\u003c/em\u003e was reported to be depleted in multiple cohorts of inflammatory bowel disease patients, alongside other butyrate producers such as \u003cem\u003eFaecalibacterium\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Administering \u003cem\u003eGemmiger\u003c/em\u003e or its related \u003cem\u003eFaecalibacterium\u003c/em\u003e or \u003cem\u003eSubdoligranulum\u003c/em\u003e may prove beneficial against NE.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBifidobacterium\u003c/b\u003e \u003cb\u003e-mediated protection of chickens from NE\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDespite the similarity in intestinal microbiota between Ghs6 and M5.1 chickens, both of which were hatched in the same location, Ghs6 chickens are evidently more susceptible to NE than M5.1 chickens. A notable observation is the marked increase in \u003cem\u003eBifidobacterium\u003c/em\u003e in NE-challenged M5.1 chickens, a response not observed in Ghs6 or Cobb chickens. Oral administration of \u003cem\u003eBifidobacterium pseudolongum\u003c/em\u003e conferred substantial protection against NE, underscoring its protective role. This is consistent with the well-documented antibacterial and immunomodulatory properties of \u003cem\u003eBifidobacterium\u003c/em\u003e species \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Additionally, Bifidobacteria synthesize essential vitamins such as riboflavin, thiamine, vitamin B6, and vitamin K, along with bioactive molecules like folic acid, niacin, and pyridoxine \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Unlike \u003cem\u003eLactobacillus\u003c/em\u003e species that produce both D(-)-lactic acid and L(+)-lactic acid, Bifidobacteria predominantly produce L(+)-lactic acid, which is more readily metabolized by humans and animals \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, \u003cem\u003eBifidobacterium\u003c/em\u003e has demonstrated efficacy against subclinical NE \u003csup\u003e42\u003c/sup\u003e and \u003cem\u003eC. perfringens\u003c/em\u003e in co-culture studies \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, consistent with our in vitro and in vivo observations. However, it is noted that, although \u003cem\u003eBifidobaceterium\u003c/em\u003e is enriched NE-challenged Fayoumi chickens and beneficial against NE, it is unlikely to be solely responsible for NE resistance in Fayoumi chickens. This is evidenced by the absence of \u003cem\u003eBifidobacterium\u003c/em\u003e in recipient Cobb chickens following CMT from any chicken breed, despite the robust protection observed. Therefore, it is plausible that multiple bacterial species in the intestinal microbiota act synergistically to provide CR against NE.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eOur study demonstrates that Fayoumi chickens exhibit greater resistance to NE compared to Leghorn layers and Cobb broilers. Additionally, we provide compelling evidence that the intestinal microbiota from Fayoumi chickens confers significant protection against NE in newly-hatched Cobb chickens, with \u003cem\u003eB. pseudolongum\u003c/em\u003e playing a crucial role in this protective effect. These findings underscore the potential of leveraging disease-resistant chicken microbiota for NE mitigation. Future research should focus on identifying the specific bacterial consortia responsible for CR and exploring their application in developing probiotic treatments to enhance poultry health and productivity.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments described in this study were conducted according to the recommendations in the Guide for the Care and Use of Agricultural Animals in Research and Teaching, 4th edition (2020) and approved by the Institutional Animal Care and Use Committee of Oklahoma State University under protocol number AG-23-35.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNE challenge of inbred chickens\u003c/p\u003e\n\u003cp\u003eTo investigate the difference of intestinal microbiota in response to NE, 48 day-of-hatch M5.1 and Ghs6 chicks, with 24 birds/breed, were obtained from Iowa State University (Ames, Iowa), while 24 day-of-hatch Cobb-500 chicks were obtained from Cobb-Vantress (Siloam Springs, Arkansas). Chickens were housed in floor pens (3' × 3') with 12 birds/pen and fresh wood shavings in an environmentally controlled room under standard management. Chickens had free access to tap water and an unmedicated mash corn-soybean starter diet containing 21.5% crude protein that meets or exceeds the nutrient requirements of the NRC recommendations \u003csup\u003e44\u003c/sup\u003e throughout the study. Within each breed, animals were weighed individually on day 16 and assigned randomly to either the mock or NE group. Each animal in the NE group was orally inoculated with 1 × 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003esporulated oocysts of the \u003cem\u003eE. maxima\u003c/em\u003e M6 strain (kindly provided by Dr. John R. Barta, University of Guelph, Canada) in 1 mL PBS on day 16, followed by four sequential inoculations with approximately 5 × 10\u003csup\u003e8\u003c/sup\u003e CFU of \u003cem\u003enetB\u003c/em\u003e- and \u003cem\u003etpeL\u003c/em\u003e-positive \u003cem\u003eC. perfringens\u003c/em\u003e Brenda B strain (kindly provided by \u0026nbsp;Dr. Lisa Bielke, North Carolina State University, Raleigh, North Carolina) in 2 mL fluid thioglycollate (FTG) broth (Thermo Fisher Scientific) twice daily on days 20 and 21, respectively, as previously described \u003csup\u003e45-47\u003c/sup\u003e. The mock-infected group received 1 mL PBS or 2 mL FTG each time.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To minimize cross-contamination, floor pens were separated from each other with plastic sheets. Animals were observed twice daily for survival and behavior till day 23. Chickens reluctant to move were euthanized to minimize undue suffering. On day 23, all surviving chickens were weighed individually and sacrificed via CO\u003csub\u003e2\u003c/sub\u003e asphyxiation. Lesions in the small intestine were scored on a scale of 0-6 as described\u0026nbsp;\u003csup\u003e48\u003c/sup\u003e. Additionally, the digesta in the proximal ileum (approximately 0.5 g) and cecum (approximately 0.2 g) were separately collected and stored at −80°C for microbial genomic DNA extraction.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc171328384\"\u003e\u003cstrong\u003ePreparation of the cecal microbiota\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCecal microbiota was collected from 35-day-old, healthy M5.1, Ghs6, and Cobb chickens with three birds/breed. After euthanasia via CO\u003csub\u003e2\u003c/sub\u003e asphyxiation, the cecum was ligated at the ileal-cecal junction, excised, and transferred into BACTRON300\u003csup\u003eTM\u003c/sup\u003e Anaerobic Chamber (Sheldon Manufacturing, Cornelius, Oregon) within 1 h. The cecal digesta was collected, combined within each breed, weighed, and diluted with five volumes (w/v) of reduced PBS. After filtration through a 70-μM cell strainer, each cecal microbiota suspension was further diluted 10-fold in PBS containing 10% glycerol and stored at −80°C until further use. On the day of CMT, frozen microbiota suspensions were thawed at 37°C and dispensed anaerobically into 1-mL syringes attached to a feeding needle and transferred to the animal facility in resealable Ziploc\u003csup\u003e®\u003c/sup\u003e plastic bags. \u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc171328385\"\u003e\u003cstrong\u003eCecal microbiota transplantation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 120 day-of-hatch Cobb chickens were obtained from Cobb-Vantress and randomly assigned to one of eight groups with 15 birds/group. Each animal received 0.2 mL PBS or 0.2 mL diluted cecal microbiota from healthy M5.1, Ghs6, or Cobb chickens on days 0, 1, 9, and 13. On days 10 and 14, four groups of animals were challenged with 1 × 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003esporulated oocysts of \u003cem\u003eE. maxima\u003c/em\u003e B6 and approximately 5 × 10\u003csup\u003e8\u003c/sup\u003e CFU of \u003cem\u003eC. perfringens\u003c/em\u003e Branda B to induce NE, while the other four groups were mock-infected. Animals were observed twice daily for mortalities till day 17. Chickens were weighed individually on days 0, 10, and 17. On day 17, all surviving animals were sacrificed and examined for small intestinal lesion scores. Additionally, the digesta in the proximal ileum and cecum were collected and stored at −80°C for microbial genomic DNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, culture, and oral administration of \u003cem\u003eB. pseudolongum\u003c/em\u003e against NE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ileal and cecal digesta of three 35-day-old M5.1 chickens were collected, diluted 10-fold in in reduced PBS, and filtered through a 70-μM cell strainer in an anaerobic chamber. After 10-fold serial dilutions in reduced PBS, 100 μL of each dilution was plated on de Man, Rogosa, and Sharpe (MRS) and reinforced clostridial medium (RCM) agar plates, respectively. After 24-h anaerobic culture, colony-PCR was performed with well-isolated colonies using primers (27F: AGA GTT TGA TCC TGG CTC AG and 1492R: GGT TAC CTT GTT ACG ACT T) to amplify the entire 16S rRNA gene, followed by Sanger sequencing. An isolate was identified to share 99.5% identity to \u003cem\u003eB. pseudolongum\u003c/em\u003e and restreaked on MRS plates three times, followed by anaerobic propagation in MRS or RCM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the protective efficacy of \u003cem\u003eB. pseudolongum\u003c/em\u003e against NE, 90 day-of-hatch Cobb chickens were randomly assigned to one of three treatments with 15 birds/pen and two pens/treatment. Each animal received approximately 1 × 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003eCFU of \u003cem\u003eB. pseudolongum\u003c/em\u003e in 1 mL reduced PBS on days 9, 11, 13, and 15. On days 10 and 14, two groups of animals were challenged with 5 × 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003esporulated oocysts of \u003cem\u003eE. maxima\u003c/em\u003e B6 and approximately 5 × 10\u003csup\u003e8\u003c/sup\u003e CFU of \u003cem\u003eC. perfringens\u003c/em\u003e Branda B to induce NE, while the third group was mock-infected with PBS and FTG on respective days. Animals were observed twice daily for mortalities till day 17. Chickens were weighed individually on days 0, 10, and 17. On day 17, all surviving animals were sacrificed and examined for small intestinal lesion scores.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial coculture assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo directly evaluate the anti-\u003cem\u003eC. perfringens\u003c/em\u003e activity of \u003cem\u003eB. pseudolongum\u003c/em\u003e, both bacteria were grown anaerobically in Brain Heart Infusion (BHI) broth overnight and diluted to 2 × 10\u003csup\u003e7\u003c/sup\u003e CFU/mL in BHI, mixed 1:1, and incubated anaerobically for 24 h at 37°C. The survival of \u003cem\u003eC. perfringens\u003c/em\u003e was assessed through serial plating on \u003cem\u003eperfringens-\u003c/em\u003eselective tryptose sulfite cycloserine (TSC) agar plates (Sigma Aldrich, St. Louis, MO).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial DNA isolation and 16S rRNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFecal DNA MicroPrep and MiniPrep Kits (Zymo Research Irvine, CA) were used for isolation of DNA from the ileal and cecal digesta in animal trials, respectively. The concentration and quality of DNA was measured by Nanodrop One Spectrophotometer (Thermo Fisher Scientific). High-quality DNA samples were shipped on dry ice to Novogene (Beijing, China) for PE250 deep sequencing of the V3-V4 region of bacterial 16S rRNA gene using primers (341F: CCT AYG GGR BGC ASC AG and 806R: GGA CTA CNN GGG TAT CTA AT) on the Illumina NovaSeq 6000 system. PCR amplification and library preparation were performed by Novogene (Beijing, China) using NEBNext® Ultra™ Library Prep Kit (New England Biolabs, Ipswich, MA, USA), generating a minimum of 30,000 raw sequencing reads per sample.\u003c/p\u003e\n\u003cp id=\"_Toc171328387\"\u003e\u003cstrong\u003eBioinformatics and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioinformatic analysis was conducted as we previously described \u003csup\u003e10,49-51\u003c/sup\u003e. Briefly, raw sequencing reads were analyzed using QIIME 2 v2023.7 \u003csup\u003e52\u003c/sup\u003e. After filtration of low-quality reads, clean sequencing reads were trimmed to 402 nucleotides and denoised using Deblur \u003csup\u003e53\u003c/sup\u003e. The resulting sequences were then classified into bacterial ASVs using the RDP 16S rRNA training set (v. 18) and Bayesian classifier. A bootstrap confidence of 80% was used for taxonomic classification. ASVs with a classification confidence below 80% were assigned to the last confidently classified taxonomic level, followed by “_unclassified”. ASVs present in fewer than 5% of samples were removed from downstream analysis. The top 100 ASVs, along with all differentially enriched taxa, were further validated and reclassified, if necessary, using an updated EzBioCloud 16S database (v2023.08.23) \u003csup\u003e54\u003c/sup\u003e. Species-level classification was assigned to sequences sharing greater than 97% identity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Analysis and visualization of α- and β-diversities of the microbiota composition were analyzed using the ‘phyloseq’ R package v1.46.0 \u003csup\u003e55\u003c/sup\u003e. To visualize the overall biodiversity and complexity within samples, the number of ASVs, Pielou’s evenness index, and Shannon index were used to calculate and display the richness, evenness, and overall diversity. The β-diversity was determined using weighted and unweighted UniFrac distances. Statistical significance in α-diversity and relative abundance for each sampling day was determined using non-parametric Mann-Whitney U test. Significance in β-diversity was determined using non-parametric permutational multivariate analysis of variance (PERMANOVA) with 999 permutations using the vegan package v. 2.6.4 \u003csup\u003e56\u003c/sup\u003e. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant. Differential abundance of bacteria among different groups of chickens was determined using ANCOM-BC2 \u003csup\u003e57\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ch2\u003eASV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Amplicon sequence variant\u003c/h2\u003e\n\u003ch2\u003eBHI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Brain heart infusion broth\u003c/h2\u003e\n\u003ch2\u003eCMT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cecal microbiota transplantation\u003c/h2\u003e\n\u003ch2\u003eCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Colonization resistance\u003c/h2\u003e\n\u003ch2\u003edpi \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Days post-infection\u003c/h2\u003e\n\u003ch2\u003eLAB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lactic acid bacteria\u0026nbsp;\u003c/h2\u003e\n\u003ch2\u003eMRS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;de Man, Rogosa, and Sharpe broth\u003c/h2\u003e\n\u003ch2\u003eNE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Necrotic enteritis\u003c/h2\u003e\n\u003ch2\u003eRCM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reinforced clostridial medium\u003c/h2\u003e\n\u003cp\u003eSCFA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Short-chain fatty acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequencing reads of this study was deposited in the NCBI GenBank SRA database under the accession number PRJNA1132701.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. John R. Barta at the University of Guelph, Canada for kindly providing \u003cem\u003eE. maxima\u003c/em\u003e strain M6. We are grateful to Dr. Lisa Bielke at North Carolina State University for providing the \u003cem\u003eC. perfringens\u003c/em\u003e strain Brenda B. We also thank Ms. Zijun Zhao for helping with animal handling.\u003c/p\u003e\n\u003cp id=\"_Toc171328402\"\u003eThis research was funded by the USDA National Institute of Food and Agriculture grants (2022-67016-37208 and 2024-67016-42415), the Ralph F. and Leila W. Boulware Endowment Fund, Oklahoma Agricultural Experiment Station Project H-3268, and Iowa Agriculture and Home Economics Experiment Station Project IOW05620. I.T. and M.W. were supported by two separate USDA National Institute of Food and Agriculture Predoctoral Fellowship grants (2021-67034-35184 and 2024-67011-42944). The funders played no role in study design, data collection, analysis, and interpretation, or the writing of this manuscript.\u003c/p\u003e\n\u003cp id=\"_Toc171328388\"\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJL, JG, IT, MAW, PP, AS, and GZ conducted animal trials; JL and JG processed the samples; JL, JG, IT, and GZ analyzed the data; JL drafted the manuscript; GZ, MGK, and SJL revised the manuscript; GZ conceived and supervised the study. All authors reviewed the manuscript and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWade, B. \u0026amp; Keyburn, A. The true cost of necrotic enteritis. \u003cem\u003eWorld Poult.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 16-17 (2015). \u003c/li\u003e\n\u003cli\u003eEmami, N. K. \u0026amp; Dalloul, R. A. Centennial Review: Recent developments in host-pathogen interactions during necrotic enteritis in poultry. \u003cem\u003ePoult. Sci.\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 101330 (2021). https://doi.org/10.1016/j.psj.2021.101330\u003c/li\u003e\n\u003cli\u003eKulkarni, R. R., Gaghan, C., Gorrell, K., Sharif, S. \u0026amp; Taha-Abdelaziz, K. Probiotics as Alternatives to antibiotics for the prevention and control of necrotic enteritis in chickens. \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 692 (2022). https://doi.org/10.3390/pathogens11060692\u003c/li\u003e\n\u003cli\u003eAlizadeh, M.\u003cem\u003e et al.\u003c/em\u003e Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination. \u003cem\u003eAnim. Health Res. Rev.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 147-162 (2021). https://doi.org/10.1017/S146625232100013X\u003c/li\u003e\n\u003cli\u003eDeist, M. S.\u003cem\u003e et al.\u003c/em\u003e Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 989 (2017). https://doi.org/10.1186/s12864-017-4380-4\u003c/li\u003e\n\u003cli\u003eSchilling, M. A.\u003cem\u003e et al.\u003c/em\u003e Conserved, breed-dependent, and subline-dependent innate immune responses of Fayoumi and Leghorn chicken embryos to Newcastle disease virus infection. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 7209 (2019). https://doi.org/10.1038/s41598-019-43483-1\u003c/li\u003e\n\u003cli\u003ePinard-Van Der Laan, M. H., Monvoisin, J. L., Pery, P., Hamet, N. \u0026amp; Thomas, M. Comparison of outbred lines of chickens for resistance to experimental infection with coccidiosis (\u003cem\u003eEimeria tenella\u003c/em\u003e). \u003cem\u003ePoult. Sci.\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 185-191 (1998). https://doi.org/10.1093/ps/77.2.185\u003c/li\u003e\n\u003cli\u003eWang, Y., Lupiani, B., Reddy, S. M., Lamont, S. J. \u0026amp; Zhou, H. RNA-seq analysis revealed novel genes and signaling pathway associated with disease resistance to avian influenza virus infection in chickens. \u003cem\u003ePoult. Sci.\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 485-493 (2014). https://doi.org/10.3382/ps.2013-03557\u003c/li\u003e\n\u003cli\u003eCheeseman, J. H., Kaiser, M. G., Ciraci, C., Kaiser, P. \u0026amp; Lamont, S. J. Breed effect on early cytokine mRNA expression in spleen and cecum of chickens with and without Salmonella enteritidis infection. \u003cem\u003eDev. Comp. Immunol.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 52-60 (2007). https://doi.org/10.1016/j.dci.2006.04.001\u003c/li\u003e\n\u003cli\u003eBroadwater, C.\u003cem\u003e et al.\u003c/em\u003e Breed-specific responses to coccidiosis in chickens: identification of intestinal bacteria linked to disease resistance. \u003cem\u003eJ. Anim. Sci. Biotechnol.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 65 (2025). https://doi.org/10.1186/s40104-025-01202-z\u003c/li\u003e\n\u003cli\u003eWilde, J., Slack, E. \u0026amp; Foster, K. R. Host control of the microbiome: Mechanisms, evolution, and disease. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e385\u003c/strong\u003e, eadi3338 (2024). https://doi.org/10.1126/science.adi3338\u003c/li\u003e\n\u003cli\u003eMorris, A. H. \u0026amp; Bohannan, B. J. M. Estimates of microbiome heritability across hosts. \u003cem\u003eNat. Microbiol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 3110-3119 (2024). https://doi.org/10.1038/s41564-024-01865-w\u003c/li\u003e\n\u003cli\u003eBurrows, P. B.\u003cem\u003e et al.\u003c/em\u003e Decoding the chicken gastrointestinal microbiome. \u003cem\u003eBMC Microbiol.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 35 (2025). https://doi.org/10.1186/s12866-024-03690-x\u003c/li\u003e\n\u003cli\u003eXu, Y.\u003cem\u003e et al.\u003c/em\u003e Metagenomic analysis reveals the microbiome and antibiotic resistance genes in indigenous Chinese yellow-feathered chickens. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 930289 (2022). https://doi.org/10.3389/fmicb.2022.930289\u003c/li\u003e\n\u003cli\u003ePandit, R. J.\u003cem\u003e et al.\u003c/em\u003e Microbial diversity and community composition of caecal microbiota in commercial and indigenous Indian chickens determined using 16S rDNA amplicon sequencing. \u003cem\u003eMicrobiome\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 115 (2018). https://doi.org/10.1186/s40168-018-0501-9\u003c/li\u003e\n\u003cli\u003eShen, H.\u003cem\u003e et al.\u003c/em\u003e Metagenome-assembled genome reveals species and functional composition of Jianghan chicken gut microbiota and isolation of \u003cem\u003ePediococcus acidilactic\u003c/em\u003e with probiotic properties. \u003cem\u003eMicrobiome\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 25 (2024). https://doi.org/10.1186/s40168-023-01745-1\u003c/li\u003e\n\u003cli\u003eZafar, H. \u0026amp; Saier, M. H., Jr. Gut \u003cem\u003eBacteroides\u003c/em\u003e species in health and disease. \u003cem\u003eGut Microbes\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1-20 (2021). https://doi.org/10.1080/19490976.2020.1848158\u003c/li\u003e\n\u003cli\u003eShin, J. H.\u003cem\u003e et al.\u003c/em\u003e \u003cem\u003eBacteroides\u003c/em\u003e and related species: The keystone taxa of the human gut microbiota. \u003cem\u003eAnaerobe\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 102819 (2024). https://doi.org/10.1016/j.anaerobe.2024.102819\u003c/li\u003e\n\u003cli\u003eLopez, L. R., Bleich, R. M. \u0026amp; Arthur, J. C. Microbiota Effects on carcinogenesis: initiation, promotion, and progression. \u003cem\u003eAnnu. Rev. Med.\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 243-261 (2021). https://doi.org/10.1146/annurev-med-080719-091604\u003c/li\u003e\n\u003cli\u003eSyed, M. A.\u003cem\u003e et al.\u003c/em\u003e Staphylococci in poultry intestines: a comparison between farmed and household chickens. \u003cem\u003ePoult. Sci.\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 4549-4557 (2020). https://doi.org/10.1016/j.psj.2020.05.051\u003c/li\u003e\n\u003cli\u003eDevriese*, L. A., Poutrel, B., Kilpper-B\u0026auml;Lz, R. \u0026amp; Schleifer, K. H. \u003cem\u003eStaphylococcus gallinarum\u003c/em\u003e and \u003cem\u003eStaphylococcus caprae\u003c/em\u003e, two new species from animals. \u003cem\u003eInt. J. Syst. Evol. Microbiol.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 480-486 (1983). https://doi.org/https://doi.org/10.1099/00207713-33-3-480\u003c/li\u003e\n\u003cli\u003eOhara-Nemoto, Y., Haraga, H., Kimura, S. \u0026amp; Nemoto, T. K. Occurrence of staphylococci in the oral cavities of healthy adults and nasal\u0026ndash;oral trafficking of the bacteria. \u003cem\u003eJ. Med. Microbiol.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 95-99 (2008). https://doi.org/https://doi.org/10.1099/jmm.0.47561-0\u003c/li\u003e\n\u003cli\u003eDhanya Raj, C. T.\u003cem\u003e et al.\u003c/em\u003e Genomic and metabolic properties of \u003cem\u003eStaphylococcus gallinarum \u003c/em\u003eFCW1 MCC4687 isolated from naturally fermented coconut water towards GRAS assessment. \u003cem\u003eGene\u003c/em\u003e \u003cstrong\u003e867\u003c/strong\u003e, 147356 (2023). https://doi.org/https://doi.org/10.1016/j.gene.2023.147356\u003c/li\u003e\n\u003cli\u003eAhmed, S.\u003cem\u003e et al.\u003c/em\u003e The \u003cem\u003eWeissella\u003c/em\u003e genus: clinically treatable bacteria with antimicrobial/ probiotic effects on inflammation and cancer. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2427 (2022). https://doi.org/10.3390/microorganisms10122427.\u003c/li\u003e\n\u003cli\u003eYeu, J.-E., Lee, H.-G., Park, G.-Y., Lee, J. \u0026amp; Kang, M.-S. Antimicrobial and antibiofilm activities of \u003cem\u003eWeissella cibaria \u003c/em\u003eagainst pathogens of upper respiratory tract infections. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1181 (2021). https://doi.org/10.3390/microorganisms9061181.\u003c/li\u003e\n\u003cli\u003eTeixeira, C. G.\u003cem\u003e et al.\u003c/em\u003e Genomic Analyses of \u003cem\u003eWeissella cibaria\u003c/em\u003e W25, a potential bacteriocin-producing strain isolated from pasture in Campos das Vertentes, Minas Gerais, Brazil. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 314 (2022). https://doi.org/10.1016/j.fbio.2023.102421.\u003c/li\u003e\n\u003cli\u003ePapagianni, M. \u0026amp; Papamichael, E. M. Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by \u003cem\u003eWeissella paramesenteroides\u003c/em\u003e DX. \u003cem\u003eBioresour. Technol.\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 6730-6734 (2011). https://doi.org/10.1016/j.biortech.2011.03.106. \u003c/li\u003e\n\u003cli\u003eFujisawa, T., Benno, Y., Yaeshima, T. \u0026amp; Mitsuoka, T. Taxonomic study of the \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e group, with recognition of \u003cem\u003eLactobacillus gallinarum\u003c/em\u003e sp. nov. and \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e sp. nov. and synonymy of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e group A3 (Johnson et al. 1980) with the type strain of \u003cem\u003eLactobacillus amylovorus\u003c/em\u003e (Nakamura 1981). \u003cem\u003eInt. J. Syst. Bacteriol.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 487-491 (1992). https://doi.org/10.1099/00207713-42-3-487\u003c/li\u003e\n\u003cli\u003eDeng, Z., Hou, K., Zhao, J. \u0026amp; Wang, H. The probiotic properties of lactic acid bacteria and their applications in animal husbandry. \u003cem\u003eCurr. Microbiol.\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 22 (2021). https://doi.org/10.1007/s00284-021-02722-3\u003c/li\u003e\n\u003cli\u003ePottenger, S.\u003cem\u003e et al.\u003c/em\u003e Timing and delivery route effects of cecal microbiome transplants on \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium infections in chickens: potential for in-hatchery delivery of microbial interventions. \u003cem\u003eAnim. Microbiome\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 11 (2023). https://doi.org/10.1186/s42523-023-00232-0\u003c/li\u003e\n\u003cli\u003eWang, X.\u003cem\u003e et al.\u003c/em\u003e The functional role of fecal microbiota transplantation on \u003cem\u003eSalmonella \u003c/em\u003eEnteritidis infection in chicks. \u003cem\u003eVet. Microbiol.\u003c/em\u003e \u003cstrong\u003e269\u003c/strong\u003e, 109449 (2022). https://doi.org/10.1016/j.vetmic.2022.109449\u003c/li\u003e\n\u003cli\u003eChintoan-Uta, C.\u003cem\u003e et al.\u003c/em\u003e Role of cecal microbiota in the differential resistance of inbred chicken lines to colonization by \u003cem\u003eCampylobacter jejuni\u003c/em\u003e. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e \u003cstrong\u003e86\u003c/strong\u003e, e02607-02619 (2020). https://doi.org/10.1128/AEM.02607-19\u003c/li\u003e\n\u003cli\u003eAlmansour, A.\u003cem\u003e et al.\u003c/em\u003e Microbiota from specific pathogen-free mice reduces \u003cem\u003eCampylobacter jejuni\u003c/em\u003e chicken colonization. \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1387 (2021). https://doi.org/10.3390/pathogens10111387\u003c/li\u003e\n\u003cli\u003ePang, J., Beyi, A. F., Looft, T., Zhang, Q. \u0026amp; Sahin, O. Fecal microbiota transplantation reduces \u003cem\u003eCampylobacter jejuni\u003c/em\u003e colonization in young broiler chickens challenged by oral gavage but not by seeder birds. \u003cem\u003eAntibiotics\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1503 (2023). https://doi.org/10.3390/antibiotics12101503\u003c/li\u003e\n\u003cli\u003eZaytsoff, S. J. M.\u003cem\u003e et al.\u003c/em\u003e Microbiota transplantation in day-old broiler chickens ameliorates necrotic enteritis via modulation of the intestinal microbiota and host immune responses. \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 972 (2022). https://doi.org/10.3390/pathogens11090972\u003c/li\u003e\n\u003cli\u003eFitzgerald, C. B.\u003cem\u003e et al.\u003c/em\u003e Comparative analysis of \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e genomes shows a high level of genome plasticity and warrants separation into new species-level taxa. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 1-20 (2018). \u003c/li\u003e\n\u003cli\u003eLund, M., Bjerrum, L. \u0026amp; Pedersen, K. Quantification of \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e-and \u003cem\u003eSubdoligranulum variabile\u003c/em\u003e-like bacteria in the cecum of chickens by real-time PCR. \u003cem\u003ePoult. Sci.\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e, 1217-1224 (2010). \u003c/li\u003e\n\u003cli\u003eNing, L.\u003cem\u003e et al.\u003c/em\u003e Microbiome and metabolome features in inflammatory bowel disease via multi-omics integration analyses across cohorts. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 7135 (2023). https://doi.org/10.1038/s41467-023-42788-0\u003c/li\u003e\n\u003cli\u003eGavzy, S. J.\u003cem\u003e et al.\u003c/em\u003e \u003cem\u003eBifidobacterium\u003c/em\u003e mechanisms of immune modulation and tolerance. \u003cem\u003eGut Microbes\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 2291164 (2023). https://doi.org/10.1080/19490976.2023.2291164\u003c/li\u003e\n\u003cli\u003eLim, H. J. \u0026amp; Shin, H. S. Antimicrobial and immunomodulatory effects of \u003cem\u003eBifidobacterium\u003c/em\u003e Strains: A Review. \u003cem\u003eJ. Microbiol. Biotechnol.\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1793-1800 (2020). https://doi.org/10.4014/jmb.2007.07046\u003c/li\u003e\n\u003cli\u003eSharma, M., Wasan, A. \u0026amp; Sharma, R. K. Recent developments in probiotics: An emphasis on \u003cem\u003eBifidobacterium\u003c/em\u003e. \u003cem\u003eFood Biosci.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 100993 (2021). https://doi.org/https://doi.org/10.1016/j.fbio.2021.100993\u003c/li\u003e\n\u003cli\u003eKhalique, A.\u003cem\u003e et al.\u003c/em\u003e Probiotics mitigating subclinical necrotic enteritis (SNE) as potential alternatives to antibiotics in poultry. \u003cem\u003eAMB Express\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 50 (2020). https://doi.org/10.1186/s13568-020-00989-6\u003c/li\u003e\n\u003cli\u003eSchoster, A.\u003cem\u003e et al.\u003c/em\u003e In vitro inhibition of \u003cem\u003eClostridium difficile\u003c/em\u003e and \u003cem\u003eClostridium perfringens\u003c/em\u003e by commercial probiotic strains. \u003cem\u003eAnaerobe\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 36-41 (2013). https://doi.org/https://doi.org/10.1016/j.anaerobe.2013.02.006\u003c/li\u003e\n\u003cli\u003eNational Research Council. \u003cem\u003eNutrient Requirements of Poultry: Ninth Revised Edition, 1994\u003c/em\u003e. (The National Academies Press, 1994).\u003c/li\u003e\n\u003cli\u003eKim, D. M.\u003cem\u003e et al.\u003c/em\u003e Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis. \u003cem\u003eJ. Anim. Sci. Biotechnol.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 29 (2024). https://doi.org/10.1186/s40104-024-00995-9\u003c/li\u003e\n\u003cli\u003eYang, Q.\u003cem\u003e et al.\u003c/em\u003e Butyrate in combination with forskolin alleviates necrotic enteritis, increases feed efficiency, and improves carcass composition of broilers. \u003cem\u003eJ. Anim. Sci. Biotechnol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 3 (2022). https://doi.org/10.1186/s40104-021-00663-2\u003c/li\u003e\n\u003cli\u003eYang, Q., Whitmore, M. A., Robinson, K., Lyu, W. \u0026amp; Zhang, G. Butyrate, forskolin, and lactose synergistically enhance disease resistance by inducing the expression of the genes involved in innate host defense and barrier function. \u003cem\u003eAntibiotics\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1175 (2021). https://doi.org/10.3390/antibiotics10101175\u003c/li\u003e\n\u003cli\u003eShojadoost, B., Vince, A. R. \u0026amp; Prescott, J. F. The successful experimental induction of necrotic enteritis in chickens by \u003cem\u003eClostridium perfringens\u003c/em\u003e: a critical review. \u003cem\u003eVet. Res.\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 74 (2012). https://doi.org/10.1186/1297-9716-43-74\u003c/li\u003e\n\u003cli\u003eLiu, J.\u003cem\u003e et al.\u003c/em\u003e Dynamic response of the intestinal microbiome to \u003cem\u003eEimeria maxima\u003c/em\u003e-induced coccidiosis in chickens. \u003cem\u003eMicrobiol. Spectr.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0082324 (2024). https://doi.org/10.1128/spectrum.00823-24\u003c/li\u003e\n\u003cli\u003eGuo, J.\u003cem\u003e et al.\u003c/em\u003e Is intestinal microbiota fully restored after chickens have recovered from coccidiosis? \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 81 (2025). https://doi.org/10.3390/pathogens14010081\u003c/li\u003e\n\u003cli\u003eLiu, J.\u003cem\u003e et al.\u003c/em\u003e \u003cem\u003eAnaerobutyricum\u003c/em\u003e and \u003cem\u003eSubdoligranulum \u003c/em\u003eare differentially enriched in broilers with disparate weight gains. \u003cem\u003eAnimals \u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 1834 (2023). https://doi.org/10.3390/ani13111834\u003c/li\u003e\n\u003cli\u003eBolyen, E.\u003cem\u003e et al.\u003c/em\u003e Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 852-857 (2019). https://doi.org/10.1038/s41587-019-0209-9\u003c/li\u003e\n\u003cli\u003eAmir, A.\u003cem\u003e et al.\u003c/em\u003e Deblur rapidly resolves single-nucleotide community sequence patterns. \u003cem\u003emSystems\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, e00191-00116 (2017). https://doi.org/10.1128/mSystems.00191-16\u003c/li\u003e\n\u003cli\u003eYoon, S. H.\u003cem\u003e et al.\u003c/em\u003e Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. \u003cem\u003eInt. J. Syst. Evol. Microbiol.\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 1613-1617 (2017). https://doi.org/10.1099/ijsem.0.001755\u003c/li\u003e\n\u003cli\u003eMcMurdie, P. J. \u0026amp; Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e61217 (2013). https://doi.org/10.1371/journal.pone.0061217\u003c/li\u003e\n\u003cli\u003evegan: Community ecology package v. R Package version 2.5-5 (2019).\u003c/li\u003e\n\u003cli\u003eLin, H. \u0026amp; Peddada, S. D. Multigroup analysis of compositions of microbiomes with covariate adjustments and repeated measures. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 83-91 (2024). https://doi.org/10.1038/s41592-023-02092-7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fayoumi chickens, poultry, cecal microbiota transplantation, microbiota, colonization resistance, necrotic enteritis, Clostridium perfringens, Bifidobacterium, Lactobacillus, Ligilactobacillus salivarius ","lastPublishedDoi":"10.21203/rs.3.rs-8349290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8349290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNecrotic enteritis (NE), caused by \u003cem\u003eClostridium perfringens\u003c/em\u003e, is a major enteric disease in poultry that leads to severe dysbiosis, morbidity, and mortality. Modulating the intestinal microbiota holds promise for enhancing animal health and disease resistance; however, specific commensal bacteria associated with NE protection remain elusive. Chicken breeds differ markedly in disease susceptibility, with Fayoumi chickens exhibiting greater resistance than Leghorn and Cobb chickens. We hypothesized that Fayoumi chickens harbor unique commensal bacteria that confer robust colonization resistance against NE. To test this, we challenged two inbred lines, Fayoumi M5.1 and Leghorn Ghs6, alongside commercial Cobb broilers with NE. Among these, M5.1 chickens demonstrated the highest resistance to NE. Cecal microbiota transplantation from the three breeds into newly hatched Cobb chicks revealed that M5.1-derived microbiota provided completion protection against NE. Comparative microbiome analysis demonstrated significant differences among breeds under both healthy and NE-challenged conditions. Notably, \u003cem\u003eBifidobacterium\u003c/em\u003e, largely absent in healthy chickens of all three breeds, was highly enriched in both the ileum and cecum of M5.1 chickens following NE challenge. Furthermore, oral administration of \u003cem\u003eBifidobacterium pseudolongum\u003c/em\u003e significantly reduced NE mortality in Cobb chickens. Collectively, these findings highlight the protective role of commensal bacteria from NE-resistant Fayoumi chickens and suggest their potential for microbiota-based strategies to mitigate NE in poultry.\u003c/p\u003e","manuscriptTitle":"Intestinal Microbiome Confers Strong Colonization Resistance Against Necrotic Enteritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 09:47:00","doi":"10.21203/rs.3.rs-8349290/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0670504-97d4-425d-8e31-d00fd2031904","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59695097,"name":"Biological sciences/Microbiology"},{"id":59695098,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-12-26T09:39:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 09:47:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8349290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8349290","identity":"rs-8349290","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}