AS  Vol.8 No.7 , July 2017
Utilization of Pyrosequencing to Monitor the Microbiome Dynamics of Probiotic Treated Poultry (Gallus gallus domesticus) during Downstream Poultry Processing
Abstract: Antibiotic growth promoters that have been historically employed to control pathogens and increase the rate of animal development for human consumption are currently banned in many countries. Probiotics have been proposed as an alternative to control pathogenic bacteria. Traditional culture methods typically used to monitor probiotic effects on pathogens possess significant limitations such as a lack in sensitivity to detect fastidious and non-culturable bacteria, and are both time consuming and costly. Here, we tested next generation pyrosequencing technology as a streamline and economical method to monitor the effects of a probiotic on microbial communities in juvenile poultry (Gallus gallus domesticus) after exposure to several microbiological challenges and litter conditions. Seven days and repeated again at 39 days following hatching, chicks were challenged with either Salmonella enterica serovar Enteritidis, Campylobacter jejuni, or no bacteria in the presence of, or without a probiotic (i.e., Bacillus subtilis) added to the feed. Three days following each of two challenges (i.e., days 10 and 42, respectively) the microbiome distributions of the poultry caecum were characterized based on 16S rDNA analysis. Generated PCR products were analyzed by automated identification of the samples after pooling, multiplexing and sequencing. A bioinformatics pipeline was then employed to identify microbial distributions at the phylum and genus level for the treatments. In conclusion, our results demonstrated that pyrosequencing technology is a rapid, efficient and cost-effective method to monitor the effects of probiotics on the microbiome of poultry propagated in an agricultural setting.
Cite this paper: Priya Guttala, V. , Medrano, E. , Bray, J. and Clack, B. (2017) Utilization of Pyrosequencing to Monitor the Microbiome Dynamics of Probiotic Treated Poultry (Gallus gallus domesticus) during Downstream Poultry Processing. Agricultural Sciences, 8, 675-691. doi: 10.4236/as.2017.87051.

[1]   Henry, R. and Rothwell, G. (1995) The World Poultry Industry, IFC Global Agribusiness Series, World Bank, Washington DC.

[2]   United States Department of Agriculture, Economic Research Service (2005) US and State Farm Cash Receipts 1924-2004. United States Department of Agriculture, Economic Research Service, Washington DC.

[3]   Wolfe, N.D., Dunavan, C.P. and Diamond, J. (2007) Origins of Major Human Infectious Diseases. Nature, 447, 279-283.

[4]   Tauxe, R.V. (1992) Epidemiology of Campylobacter jejuni Infection in the United States and Other Industrialized Nations. In: Nachamkin, I., Blaser, M.J. and Tompkins, L.S., Eds., Campylobacter jejuni: Current Status and Future Trends, American Association of Microbiologists, Washington DC, 9-19.

[5]   Skirrow, M.B. (1998) Campylobacteriosis. In: Palmer, S.R., Soulsby, S.R.L. and Simpson, D.I.H., Eds., Zoonoses, Oxford University Press, New York, 37-46.

[6]   Corry, J.E.L. and Atabay, H.I. (2001) Poultry as a Source of Campylobacter and Related Organisms. Journal of Applied Microbiology, 90, 96S-114S.

[7]   Majowicz, S.E., Musto, J., Scallan, E., Angulo, F.J., Kirk, M., O’Brien, S.J., Jones, T.F., Fazil, A. and Hoekstra, R.M. (2010) The Global Burden of Nontyphoidal Salmonella gastroenteritis. Clinical Infectious Diseases, 50, 882-889.

[8]   Silva, J., Leite, D., Fernandes, M., Mena, C., Gibbs, P.A. and Teixeira, P. (2011) Campylobacter Spp. As a Foodborne Pathogen: A Review. Frontiers in Microbiology, 2, 200.

[9]   Wegener, H.C., Aarestrup, F.M., Jensen, L.B., Hammerum, A.M. and Bager, F. (1999) Use of Antimicrobial Growth Promoters in Food Animals and Enterococcus faecium Resistance to Therapeutic Antimicrobial Drugs in Europe. Emerging Infectious Diseases, 5, 329-335.

[10]   Hakanen, A.J., Lehtopolku, M., Siitonen, A., Huovinen, P. and Kotilainen, P. (2003) Multidrug Resistance in Campylobacter jejuni Strains Collected from Finnish Patients During 1995-2000. Journal of Antimicrobial Chemotherapy, 52, 1035-1039.

[11]   Engberg, J., Neimann, J., Nielsen, E.M., Aarestrup, F.M. and Fussing, V. (2004) Quinolone-Resistant Campylobacter Infections in Denmark: Risk Factors and Clinical Consequences. Emerging Infectious Diseases, 10, 1056-1063.

[12]   Diarra, M.S., Delaquis, P., Rempel, H., Bach, S., Harlton, C. and Aslam, M. (2014) Antibiotic Resistance and Diversity of Salmonella enterica Serovars Associated with Broiler Chickens. Journal of Food Protection, 77, 40-49.

[13]   O’Toole, P.W. and Cooney, J.C. (2008) Probiotic Bacteria Influence the Composition and Function of the Intestinal Microbiota. Interdisciplinary Perspectives on Infectious Diseases, 2008, Article ID: 175285.

[14]   Kabir, S.M.L. (2009) The Role of Probiotics in the Poultry Industry. International Journal of Molecular Sciences, 10, 3531-3546.

[15]   Islam, M.W., Rahman, M.M., Kabir, S.M.L., Kamruzzaman, S.M. and Islam, M.N. (2004) Effects of Probiotics Supplementation on Growth Performance and Certain Haematobiochemical Parameters in Broiler Chickens. Bangladesh Journal of Veterinary Medicine, 2, 39-43.

[16]   Willis, W.L., Isikhuemhen, O.S. and Ibrahim, S.A. (2007) Performance Assessment of Broiler Chickens Given Mushroom Extract Alone or in Combination with Probiotics. Poultry Science, 86, 1856-1860.

[17]   Apata, D.F. (2008) Growth Performance, Nutrient Digestibility and Immune Response of Broiler Chicks Fed Diets Supplemented with a Culture of Lactobacillus bulgaricus. Journal of the Science of Food and Agriculture, 88, 1253-1258.

[18]   Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S Ribosomal DNA Amplification for Phylogenetic Study. Journal of Bacteriology, 173, 697-703.

[19]   Zhu, X.Y., Zhong, T., Pandya, Y. and Joerger, R.D. (2002) 16S rRNA-Based Analysis of Microbiota from the Cecum of Broiler Chickens. Applied and Environmental Microbiology, 68, 124-137.

[20]   Metzker, M.L. (2010) Sequencing Technologies—The Next Generation. Nature Reviews Genetics, 11, 31-46.

[21]   Rahaus, M., Augustinski, K., Castells, M. and Desloges, N. (2013) Application of a New Bivalent Marek’s Disease Vaccine Does Not Interfere with Infectious Bronchitis or Newcastle Disease Vaccinations and Proves Efficacious. Avian Diseases, 57, 498-502.

[22]   NRC (1994) Nutrient Requirements of Poultry. 9th Revised Edition, National Academy Press, Washington DC.

[23]   Barnes, E.M., Mead, G.C., Barnum, D.A. and Harry, E.G. (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.

[24]   Wei, S., Morrison, M. and Yu, Z. (2013) Bacterial Census of Poultry Intestinal Microbiome. Poultry Science, 92, 671-683.

[25]   Lane, D.J. (1991) 16S/23S rRNA Sequencing. In: Stackebrandt, E. and Goodfellow, M., Eds., Nucleic Acid Techniques in Bacterial Systematics, John Wiley & Sons, New York, 115-175.

[26]   Turner, S., Pryer, K.M., Miao, V.P.W. and Palmer, J.D. (1999) Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis. Journal of Eukaryotic Microbiology, 46, 327-338.

[27]   Sun, Q., Liu, L., Wu, L., Li, W., Liu, Q., Zhang, J. and Ma, J. (2015) Web Resources for Microbial Data. Genomics, Proteomics & Bioinformatics, 13, 69-72.

[28]   Dudhagara, P., Bhavsar, S., Bhagat, C., Ghelani, A., Bhatt, S. and Patel, R. (2015) Web Resources for Metagenomics Studies. Genomics, Proteomics & Bioinformatics, 13, 296-303.

[29]   JMP Version 5.1. (2011) SAS Institute Inc., Cary, NC, USA.

[30]   Lu, J., Idris, U, Harmon, B., Hofacre, C. and Maurer, J.J. (2003) Diversity and Succession of the Intestinal Bacterial Community of the Maturing Broiler Chicken. Applied and Environmental Microbiology, 69, 6816-6824.

[31]   Jozefiak, D., Rutkowski, A., Kaczmarek, S., Jensen, B.B. and Engberg, R.M. (2010) Effect of Beta-Glucanase and Xylanase Supplementation of Barley and Rye-Based Diets on Caecal Microbiota of Broiler Chickens. British Poultry Science, 51, 546-557.

[32]   Danzeisen, J.L., Kim, H.B., Isaacson, R.E., Tu, Z.J. and Johnson, T.J. (2011) Modulations of the Chicken Cecal Microbiome and Metagenome in Response to Anticoccidial and Growth Promoter Treatment. PLoS ONE, 6, e27949.

[33]   Kim, M., Morrison, M. and Yu, Z. (2011) Status of the Phylogenetic Diversity Census of Ruminal Microbiomes. FEMS Microbiology Ecology, 76, 49-63.

[34]   Kaiser, P., Howell, J., Fife, M., Sadeyen, J.R., Salmon, N. and Rothwell, L. (2009) Towards the Selection of Chickens Resistant to Salmonella and Campylobacter Infections. Bulletin et Mémoires de l’Académie Royale de Médecine de Belgique, 164, 17-25.

[35]   Wisner, A.L., Desin, T.S., Koch, B., Lam, P.K., Berberov, E.M., Mickael, C.S., Potter, A.A. and Koster, W. (2010) Salmonella enterica Subspecies enterica serovar Enteritidis Salmonella Pathogenicity Island 2 Type III Secretion System: Role in Intestinal Colonization of Chickens and Systemic Spread. Microbiology, 156, 2770-2781.

[36]   Cressman, M.D., Yu, Z., Nelson, M.C., Moeller, S.J., Lilburn, M.S. and Zerby, H.N. (2010) Interrelations between the Microbiotas in the Litter and in the Intestines of Commercial Broiler Chickens. Applied and Environmental Microbiology, 76, 6572-6582.