Zootecnica International - World Poultry Journal

Agtech Products, Inc. Waukesha, WI, U.S.A.

Direct fed microbial products (DFM) have been historically called as “Foo Foo dust” by most nutritionists and production Vets. I am one of those Vets. This is partly due to the poor understanding of the microbial flora in the gastrointestinal track of commercial broilers and turkeys.

Based on new technologies that I will describe, it has become possible to study the diversity of microflora in the GI track of birds and how they might influence presence or absence of disease. With the application of new technologies we can understand how we can design DFM that are more ideal for better health and performance without the help of conventional antibiotics.

The gastrointestinal tract of poultry harbours a dense and metabolically active microbial community. During the production process, antibiotics are used in feed and water as an intervention tool to control gastrointestinal diseases and modify growth. However, the effects of antibiotics on the gastrointestinal microbial community are poorly understood. Traditional plating methods have been used to characterize microbial populations in the gastrointestinal tract of poultry. Unfortunately, these techniques are limited in their ability to detect only cultivable microorganisms.

New advances in molecular biology have allowed for the application of nonculture-based methods to investigate biological questions. Polymerase chain reaction (PCR) based methods bypass the need for culture steps, yielding information about both the culturable and non-culturable microflora present. The most common target for these PCR techniques is the 16S rRNA gene, which codes for the large subunit of the ribosomal complex, essential to every bacterium. The 16S gene is of particular interest because it contains both nonvariable and variable regions, making it an ideal target for phylogenetic comparisons. Terminal restriction length polymorphism (T-RFLP) is an important method for studying microbial ecology and has been used to characterize bacterial communities from soil, humans, rats, pigs, poultry and many other natural environments (1, 2, 3, 4, 5, 6, 7). T-RFLP combines PCR with restriction enzyme digests to create a “profile” of the bacterial community present. Using TRFLP, GI communities from birds can be compared to reveal bacteria which are unique to a treatment or health status.

The microbial community of the gastrointestinal (GI) tract has been the focus of many studies because of the critical role it plays in the nutritional, physiological, and immunological processes in the host animal (8). Snoeyenbos et al. (1977) found that both young poults and chicks need protection early in life against GI tract diseases. Enteric diseases concern the poultry industry because of lost productivity, increased mortality, and contamination of poultry products for human consumption (9). To combat these diseases during the production process, antibiotics and other antimicrobials are commonly used. Antibiotics are not always effective against all enteric diseases; in addition increased concern of antibiotic resistance has prompted efforts to develop alternative treatments (10).

Traditional attempts to characterize the GI microflora have relied upon microbiological culturing techniques. Unfortunately, these techniques are limited in their ability to detect only cultivable microorganisms, which are estimated to be only 10-50% of the total microbial community (11). To obtain total community information, culture independent techniques have been employed utilizing the 16S ribosomal RNA (rRNA) molecule. The 16S rRNA gene is ideal for culture independent studies because it has a conserved function and can easily be sequenced. The 16S contains both highly conserved and strain dependent variable regions, making it an ideal target for PCR and subsequent community analysis. Denaturing gradient gel electrophoresis (DGGE) was first used for bacterial community analysis in 1993 by Muyzer et al. DGGE allows for the separation of similarly sized segments of DNA (PCR products) based upon nucleotide content, creating a community profile. DGGE can be used together with 16S rDNA primers to determine the makeup of complex communities.

For example, DGGE has successfully been used to study the GI tract microflora for humans, pigs, cattle, dogs, rodents, and chickens (12,13). The objective is to study the microbial diversity and succession of the bacterial community in the gastrointestinal tract of turkey poults with and without penicillin using denaturing gradient gel electrophoresis (DGGE) and combine this information with performance data to identify candidates for a new probiotic.

References

1. Gong, J., R. J. Forster, H. Yu, J. R. Chambers, P. M. Sabour, R. Wheatcroft, and S. Chen. 2002. Diversity and phylogenetic analysis of bacteria in the mucosa of chicken ceca and comparison with bacteria in the cecal lumen. FEMS Microbiol Lett 208:1-7.
2. Hartmann, M., B. Frey, R. Kolliker, and F. Widmer. 2005. Semiautomated genetic analyses of soil microbial communities: comparison of T-RFLP and RISA based on descriptive and discriminative statistical approaches. J Microbiol Methods 61:349-60.
3. &
4. Kaplan, C. W., J. C. Astaire, M. E. Sanders, B. S. Reddy, and C. L. Kitts. 2001. 16S ribosomal DNA terminal restriction fragment pattern analysis of bacterial communities in feces of rats fed Lactobacillus acidophilus NCFM. Appl Environ Microbiol 67:1935-9.
5. Lan, P. T., M. Sakamoto, and Y. Benno. 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. Microbiol Immunol 48:917-29.
6. Leser, T. D., R. H. Lindecrona, T. K. Jensen, B. B. Jensen, and K. Moller. 2000. Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl Environ Microbiol 66:3290-6.
7. Yannarell, A. C., and E. W. Triplett. 2005. Geographic and environmental sources of variation in lake bacterial community composition. Appl Environ Microbiol 71:227-39.
8. Zoetendal, E. G., C. T. Collier, S. Koike, R. I. Mackie, and H. R. Gaskins. 2004. Molecular Ecological Analysis of the Gastrointestinal Microbiota: A Review. J. Nutr. 134:465-472.
9. Patterson, J. A., and K. M. Burkholder. 2003. Application of Prebiotics and Probiotics in Poultry Production. Poultry Science. 83:627-631.
10. Brosius, J., T. Dull, D. D. Sleeter, and H. F. Noller. 1981. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801-4805.
11. Hayashi, H., R. Takahashi, T. Nishi, M. Sakamoto, and Y. Benno. 2005. Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J Med Microbiol 54:1093-101.
12. Muyzer, G., E. C. De Waal, and A. G. Uitterlinden. 1993. Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700.
13. Wiard, T., M. King, and T. Rehberger. 2003. Application of Plating Enumerations and Denaturing Gradient Gel Electrophoresis to Study Turkey Poult Gastrointestinal Tract Bacterial Diversity. Poult. Sci. Asso. Supplement 1:58-59.

From Proceedings of “Midwest Poultry Federation Convention”, St. Paul, Minnesota, U.S.A.