Effect of Broiler Age and Breeding Region on Bacterial Population Changes of Ileum

Document Type : Original Paper


1 Department of Anaerobic Bacterial Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

2 Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran


Chicken gut microbiota is affected by factors such as diets, environmental ,and bird age. In the current study, the effects of age and region on the ileum bacterial population of broiler chickens were investigated. A total of 2679 chickens in four geographical regions of Iran were sacrificed in the first to eighth week of age. Stool samples were collected and DNA was extracted and analyzed for the detection of Lactobacillus, Enterococcaceae, Clostridiaceae, Streptococcaceae, and Actinobacteria, using specific primers and probes. Purified amplicons were quantified by QuantiFluor® and pooled for sequencing. Findings showed that L. acidophilus was the dominant bacterium during the first four weeks, and was substituted with L. crispatus and L. salivarius in the next four weeks. So, the Lactobacillus family was the most dominant bacteria at all ages showing its essential role in chicken physiology. The age of chickens significantly affected the percentage of L. crispatus, L. acidophilus, L. salivarius, Clostridiaceae, Enterococcaceae, Streptococcaceae, and Actinobacteria. The breeding region influenced Streptococcaceae, with the highest percentage in the hot region. Chicken weight had a significant effect on Enterococcaceae. Broiler breeder age and distance to the nearest farm had no effect on ileum bacterial populations. This study showed there are several factors during the broiler breeding period that have an impact on microbial population changes at different ages.


Ballou AL, Ali RA, Mendoza MA, Ellis JC, Hassan HM, Croom WJ & Koci MD. 2016. Development of the chick microbiome: How early exposure influences future microbial diversity. Frontiers in Veterinary Science, 3: 2. DOI: 10.3389/fvets.2016.00002
Barnes EM. 1979. The intestinal microflora of poultry and game birds during life and after storage. Journal of Applied Bacteriology, 46: 407-419. DOI: 10.1111/j.1365-2672.1979.tb00838.x
Barnes EM, Mead GC, Barnuml DA & Harry EG. 1972. The intestinal flora of the chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria. British Poultry Science, 13: 311-326. DOI: 10.1080/00071667208415953
Barrow PA. 1992. Probiotics for chickens. In: Hill M. (ed.) Probiotics: The scientific basis. Springer Netherlands. Dordrecht. Pages, 225-257. DOI: 10.1007/978-94-011-2364-8_10
Brisbin JT, Parvizi P & Sharif S. 2012. Differential cytokine expression in T-cell subsets of chicken caecal tonsils co-cultured with three species of Lactobacillus. Beneficial Microbes, 3(3): 205-210. DOI: 10.3920/BM2012.0014
Brooks JP, Edwards DJ, Harwich MD, Rivera MC, Fettweis JM, Serrano MG, Reris RA, Sheth NU, Huang B, Girerd P, Strauss JF, Jefferson KK & Buck GA. 2015. The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiology, 15: 66. DOI: 10.1186/s12866-015-0351-6
Brolazo EM, Leite DS, Tiba MR, Villarroel M, Marconi C & Simoes JA. 2011. Correlation between API 50 CH and multiplex polymerase chain reaction for the identification of vaginal lactobacilli in isolates. Brazilian Journal of Microbiology, 42(1): 225-232. DOI: 10.1590/S1517-83822011000100028
Burkholder KM, Thompson KL, Einstein ME, Applegate TJ & Patterson JA. 2008. Influence of stressors on normal intestinal microbiota, intestinal morphology, and susceptibility to salmonella enteritidis colonization in broilers. Poultry Science, 87: 1734-1741. DOI: 10.3382/ps.2008-00107
Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA & Hunter N. 2004. Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. Journal of Clinical Microbiology, 42(7): 3128-3136. DOI: 10.1128/JCM.42.7.3128-3136.2004
Danzeisen JL, Calvert AJ, Noll SL, McComb B, Sherwood JS, Logue CM & Johnson TJ. 2013. Succession of the turkey gastrointestinal bacterial microbiome related to weight gain. PeerJ, 1: e237-e237. DOI: 10.7717/peerj.237
Dibner JJ & Richards JD. 2005. Antibiotic growth promoters in agriculture: history and mode of action. Poultry Science, 84: 634-643. DOI: 10.1093/ps/84.4.634
Droleskey RE, Oyofo BA, Hargis BM, Corrier DE & DeLoach JR. 1994. Effect of mannose on salmonella typhimurium-mediated loss of mucosal epithelial integrity in cultured chick intestinal segments. Avian Diseases, 38: 275-281. DOI: 10.2307/1591949
Gong J, Forster RJ, Yu H, Chambers JR, Wheatcroft R, Sabour PM & Chen S. 2002. Molecular analysis of bacterial populations in the ileum of broiler chickens and comparison with bacteria in the cecum. FEMS Microbiology Ecology, 41: 171-179. DOI: 10.1111/j.1574-6941.2002.tb00978.x
Han GG, Kim EB, Lee J, Lee JY, Jin G, Park J, Huh CS, Kwon IK, Kil DY, Choi YJ & Kong C. 2016. Relationship between the microbiota in different sections of the gastrointestinal tract, and the body weight of broiler chickens. SpringerPlus, 5: 911. DOI: 10.1186/s40064-016-2604-8
Hildebrandt MA Hoffmann C, Sherrill–Mix SA, Keilbaugh SA, Hamady M, Chen YY, Knight R, Ahima RS, Bushman F & Wu GD. 2009. High-fat diet determines the composition of the murine gut microbiome independently of obesity. Gastroenterology, 137: 1716-1724.e1712. DOI: 10.1053/j.gastro.2009.08.042
Haesler F. 2008. Microbial biocontrol of the pathogen Phytophthora citricola in the rhizosphere of European beech (Fagus sylvatica L.): Impact of elevated O3 and CO2 on the antagonistic community structure and function. Technische Universitat Munchen, Munich, Germany. 136 pages.
Kers JG, Velkers FC, Fischer EAJ, Hermes GDA, Stegeman JA & Smidt H. 2018. Host and environmental factors affecting the intestinal microbiota in chickens. Frontiers in Microbiology, 9: 235. DOI: 10.3389/fmicb.2018.00235
Klasing KC, Johnston BK & Benson BN. 1999. Implications of an immune response on growth and nutrition requirements of chicks. In: Wiseman J & Garnsworthy PC (Ed.). Recent developments in poultry nutrition. Nottingham University Press, Pages, 35–47. DOI: 10.1016/B978-0-7506-1397-2.50012-5
Kikuchi E, Miyamoto Y, Narushima S & Itoh K. 2002. Design of species-specific primers to identify 13 species of Clostridium harbored in human intestinal tracts. Microbiology and Immunology,46: 353–358. DOI: 10.1111/j.1348-0421.2002.tb02706.x
Lan PTN, Sakamoto M & Benno Y. 2004. Effects of two probiotic lactobacillus strains on jejunal and cecal microbiota of broiler chicken under acute heat stress condition as revealed by molecular analysis of 16s rRNA genes. Microbiology and Immunology, 48: 917-929. DOI: 10.1111/j.1348-0421.2004.tb03620.x
Laudadio V, Dambrosio A, Normanno G, Khan RU, Naz S, Rowghani E & Tufarelli V. 2012. Effect of reducing dietary protein level on performance responses and some microbiological aspects of broiler chickens under summer environmental conditions. Avian Biology Research, 5: 88-92. DOI: 10.3184/175815512X13350180713553
Lee S, La TM, Lee HJ, Choi IS, Song CS. Park SY, Lee JB & Lee SW. 2019. Characterization of microbial communities in the chicken oviduct and the origin of chicken embryo gut microbiota. Scientific Reports, 9: 6838. DOI: 10.1038/s41598-019-43280-w
Ley RE, Bäckhed F, Turnbaugh PJ, Lozupone CA, Knight RD & Gordon JI. 2005. Obesity alters gut microbial ecology. Proceeding of National Academy of Sciences United  States of America, 102: 11070–11075. DOI: 10.1073/pnas.0504978102
Ley RE, Turnbaugh PJ, Klein S & Gordon JI. 2006. Human gut microbes associated with obesity. Nature, 444: 1022-1023. DOI: 10.1038/4441022a
Lozupone CA, Stombaugh J, Gonzalez A, Ackermann G, Wendel D, Vázquez-Baeza Y, Jansson JK, Gordon JI & Knight R. 2013. Meta-analyses of studies of the human microbiota. Genome Research, 23: 1704-1714. DOI: 10.1101/gr.151803.112
Lu J, Idris U, Harmon B, Hofacre C, Maurer JJ & Lee MD. 2003a. Diversity and succession of the intestinal bacterial community of the maturing broiler chicken. Applied and Environmental Microbiology, 69: 6816-6824. DOI: 10.1128/AEM.69.11.6816-6824.2003
Lu J, Sanchez S, Hofacre C, Maurer JJ, Harmon BG & Lee MD. 2003b. Evaluation of broiler litter with reference to the microbial composition as assessed by using 16s rRNA and functional gene markers. Applied and Environmental Microbiology, 69: 901-908. DOI: 10.1128/AEM.69.2.901-908.2003
Luo J, Zheng A, Meng K, Chang W, Bai Y, Li K, Cai H, Liu G & Yao B. 2013. Proteome changes in the intestinal mucosa of broiler (Gallus gallus) activated by probiotic Enterococcus faecium. Journal of Proteomics, 91: 226-241. DOI: 10.1016/j.jprot.2013.07.017
McVeigh HP, Munro J & Embley TM. 1996. Molecular evidence for the presence of novel actinomycete lineages in a temperate forest soil. Journal of Industrial Microbiology, 17: 197-204. DOI: 10.1007/BF01574693
Mead GC. 1989. Microbes of the avian cecum: Types present and substrates utilized. Journal of Experimental Zoology, 252: 48-54. DOI: 10.1002/jez.1402520508
Nathiya S, Dhinakar RG, Rajasekar A, Vijayalakshmi D & Devasena T. 2012. Identification of microbial diversity in caecal content of broiler chicken. African Journal of Microbiology Research, 6: 4897-4902. DOI: 10.5897/AJMR11.1146
Oakley BB & Kogut MH. 2016. Spatial and temporal changes in the broiler chicken cecal and fecal microbiomes and correlations of bacterial taxa with cytokine gene expression. Frontiers in Veterinary Science, 3: 11. DOI: 10.3389/fvets.2016.00011
Pan D & Yu Z. 2014. Intestinal microbiome of poultry and its interaction with host and diet. Gut Microbes, 5: 108-119. DOI: 10.4161/gmic.26945
Pedroso AA, Menten JF, Lambais MR, Racanicci, AMC, Longo FA & Sorbara JOB. 2006. Intestinal bacterial community and growth performance of chickens fed diets containing antibiotics. Poultry Science, 85: 747-752. DOI: 10.1093/ps/85.4.747
Ranjitkar S, Lawley B, Tannock G & Engberg RM. 2016. Bacterial succession in the broiler gastrointestinal tract. Applied and Environmental Microbiology, 82: 2399-2410. DOI: 10.1128/AEM.02549-15
Ricke SC & Pillai SD. 1999. Conventional and molecular methods for understanding probiotic bacteria functionality in gastrointestinal tracts. Critical Reviews in Microbiology, 25: 19-38. DOI: 10.1080/10408419991299176
Salanitro JP, Fairchilds IG & Zgornicki YD. 1974. Isolation, culture characteristics, and identification of anaerobic bacteria from the chicken cecum. Applied Microbiology, 27: 678-687. DOI: 10.1128/am.27.4.678-687.1974
Seppälä H, Varkila JV, Österblad M, Jahkola M, Rummukainen M, Holm SE & Huovinen P. 1994. Evaluation of methods for epidemiologic typing of group A streptococci. The Journal of Infectious Diseases, 169: 519-525. DOI: 10.1093/infdis/169.3.519
Smits CHM, Veldman A, Verstegen MWA & Beynen AC. 1997. Dietary carboxymethylcellulose with high instead of low viscosity reduces macronutrient digestion in broiler chickens. The Journal of Nutrition, 127: 483-487. DOI: 10.1093/jn/127.3.483
Sohail MU, Hume ME, Byrd JA, Nisbet DJ, Shabbir MZ, Ijaz A & Rehman H. 2015. Molecular analysis of the caecal and tracheal microbiome of heat-stressed broilers supplemented with prebiotic and probiotic. Avian Pathology, 44: 67-74. DOI: 10.1080/03079457.2015.1004622
Soliman E, Taha E, Infante K, Laboy K, Sobieh M & Reddy P. 2009. Stressors influence on Salmonella enterica serovar enteritidis colonization in broilers. American Journal of Animal and Veterinary Sciences, 4: 42-48. DOI: 10.3844/ajavsp.2009.42.48
Stanley D, Hughes RJ & Moore RJ. 2014. Microbiota of the chicken gastrointestinal tract: influence on health, productivity and disease. Applied Microbiology and Biotechnology, 98: 4301-4310. DOI: 10.1007/s00253-014-5646-2
Tannock GW. 1999. Analysis of the intestinal microflora: a renaissance, Antonie van Leeuwenhoek, 76: 265-278. DOI: 10.1007/978-94-017-2027-4_13
Tellez G, Higgins SE, Donoghue AM & Hargis BM. 2006. Digestive physiology and the role of microorganisms. Journal of Applied Poultry Research, 15: 136-144. DOI: 10.1093/japr/15.1.136
Theron J & Cloete TE. 2000. Molecular techniques for determining microbial diversity and community structure in natural environments. Critical Reviews in Microbiology, 26: 37-57. DOI: 10.1080/10408410091154174
Van Leeuwen P, Mouwen JMVM, Van Der Klis JD & Verstegen MWA. 2004. Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance. British Poultry Science, 45: 41-48. DOI: 10.1080/00071660410001668842
Videnska P, Rahman MM, Faldynova M, Babak V, Matulova ME, Prukner-Radovcic E, Krizek I, Smole-Mozina S, Kovac J, Szmolka A, Nagy B, Sedlar K, Cejkova D & Rychlik I. 2014. Characterization of egg laying hen and broiler fecal microbiota in poultry farms in Croatia, Czech Republic, Hungary and Slovenia. PLoS One, 9: e110076. DOI: 10.1371/journal.pone.0110076
Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K & Alatossava T. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Applied and Environmental Microbiology, 66: 297–303. DOI: 10.1128/AEM.66.1.297-303.2000
Yegani M & Korver DR. 2008. Factors affecting intestinal health in poultry. Poultry Science, 87: 2052-2063. DOI: 10.3382/ps.2008-00091
Yeoman CJ, Chia N, Jeraldo P, Sipos M, Goldenfeld ND & White BA. 2012. The microbiome of the chicken gastrointestinal tract. Animal Health Research Reviews, 13: 89-99. DOI: 10.1017/S1466252312000138
Zhang X, Akhtar M, Chen Y, Ma Z, Liang Y, Shi D, Cheng R, Cui L, Hu Y,  Nafady AA,  Ansari AR, Abdel-Kafy ESM & Liu H. 2022. Chicken jejunal microbiota improves growth performance by mitigating intestinal inflammation. Microbiome, 10: 107. DOI: 10.1186/s40168-022-01299-8
Zhao L, Wang G, Siegel P, He C, Wang H, Zhao W, Zhai Z, Tian F, Zhao J, Zhang H, Sun Z, Chen W, Zhang Y & Meng H. 2013. Quantitative genetic background of the host influences gut microbiomes in chickens. Scientific Reports, 3: 1163. DOI: 10.1038/srep01163
Zheng A, Luo J, Meng K, Li J, Zhang S, Li K, Liu G, Cai H, Bryden WL & Yao B. 2014. Proteome changes underpin improved meat quality and yield of chickens (Gallus gallus) fed the probiotic Enterococcus faecium. BMC Genomics, 15: 1167. DOI: 10.1186/1471-2164-15-1167
Zhou X, Jiang X, Yang C, Ma B, Lei C, Xu C, Zhang A, Yang X, Xiong Q, Zhang P, Men S, Xiang R & Wang H. 2016. Cecal microbiota of Tibetan chickens from five geographic regions were determined by 16S rRNA sequencing. Microbiology Open, 5: 753-762. DOI: 10.1002/mbo3.367
Zhu Y, Li T, Din AU, Hassan A, Wang Y & Wang G. 2019. Beneficial effects of Enterococcus faecalis in hypercholesterolemic mice on cholesterol transportation and gut microbiota. Applied Microbiology and Biotechnology, 103(7): 3181-3191. DOI: 10.1007/s00253-019-09681-7