Study of Bacterial Diversity in Poultry Gut Using Denaturing Gradient Gel Electrophoresis

Subject Areas : Camel

V.B. Ahir ¹ , K.M. Singh ² , A.K. Tripathi ³ , R.A. Mathakiya ⁴ , S.J. Jakhesara ⁵ , P.G. Koringa ⁶ , D.N. Rank ⁷ , M.K. Jhala ⁸ , C.G. Joshi ⁹

1 - Department of Animal Biotechnology, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
2 - Department of Animal Biotechnology, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
3 - Department of Animal Biotechnology, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
4 - Department of Veterinary Microbiology. Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
5 - Department of Animal Biotechnology, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
6 - Department of Animal Biotechnology, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
7 - Department of Animal Genetics and Breeding, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
8 - Department of Veterinary Microbiology. Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India
9 - Department of Animal Biotechnology, Collegeof VeterinaryScience and Animal Science, Anand Agricultural University, 388001, Anand, India

Received: 2011-08-22 Accepted : 2011-10-21 Published : 2012-09-01

Keywords: Bacterial diversity, electrophoresis, poultry gut,

Abstract :

Bacteria play an important role in the initiation and progression of diseases (not only in disease but affect health status of individual). Populations of digestive microflora in chicken gut change with age and also affected by diet, stressors, and performance enhancers as well as in disease conditions. The aim of the pre-sent study was to know bacterial makeup in chicken gut, using denaturing gradient gel electrophore-sis (DGGE). DGGE results showed 11 different band patterns from the six birds (P1-P6). A total of 11 bands were cloned and sequenced and similarities to known 16S rDNA sequences were examined. All the sequences had 93 to 100% similarity to 16S rDNA database sequences. In P1 birds, total 8 bands were ob-served; which had varying degree of homology with Proteus mirabilis, Gallibacterium anatis, uncultured bacterium, bacteroidetes bacterium, rumen bacterium and Bacillus subtilis. Similarly in second bird (P2), one clone was showing 98% similarities with uncultured bacterium. Further one clone from P3 was similar to uncultured prokaryote clone (93%). Likewise in P4, one clone was similar (100%) to Escherichia spe-cies. Moreover, in P5 and P6 birds, DGGE bands were having some common migration pattern to those that were observed in other four birds (P1-P4). These results may help poultry producers to develop more cost-effective health management strategies as well as differences in the composition of the gut microbial com-munity with improved performance, which implies that the presence of specific beneficial and / or absence of specific detrimental bacterial species may contribute to the improved performance of the birds.

References:

Abrams G.D., Bauer H. and Sprinz H. (1963). Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. A comparison of germfree and conventional mice. Lab. Invest. 12, 355-364.
Amit-Romach E., Sklan D. and Uni Z. (2004). Microflora ecology of the chicken intestine using 16S ribosomal DNA primers. Poult. Sci. 83, 1093-1098.
Apajalahti J., Kettunen A. and Graham H. (2004). Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World’s Poult. Sci. J.60, 223-232.
Apajalahti J.H.A., Sarkilahti L.K., Maki B.R.E., Heikkinen J.P., Nurminen P.H. and Holben W.E. (1998). Effective recovery of bacterial DNA and percent-guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens. Appl. Environ. Microbiol. 64, 4084-4088.
Barnes E.M. (1979) The intestinal microflora of poultry and game birds during life and after storage. J. Appl. Bacteriol. 46, 407-419.
De Angelis M., Siragusa S., Berloco M., Caputo L., Settanni L., Alfonsi G., Amerio M., Grandi A., Ragni A. and Gobbetti M. (2006). Selection of potential probiotic lactobacilli from pig faeces to be used as additives in pelleted feeding. Res. Microbiol. 157, 792-801.
Fischer S.G. and Lerman L.S. (1979). Length-independent separation of DNA restriction fragments in two dimensional gel electrophoresis. Cell. 16, 191-200.
Fischer S.G. and Lerman L.S. (1983). DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc. Natl. Acad. Sci. 80, 1579-1583.
Gong J., Forster R.J., Yu H., Chambers J.R., Sabour P.M., Wheatcroft R. and Chen S. (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.
Gong J., Yu H., Liu T., Gill J.J., Chambers J.R. and Wheatcroft R. (2008). Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens. J. Appl. Microbiol. 104, 1372-1382.
Gordon H.A. and Pesti L. (1971). The gnotobiotic animal as a tool in the study of host-microbial relationships. Bacteriol. Rev. 35, 390-429.
Hong T.T.T., Passoth V. and Lindberg J.E. (2011). Bacterial diversity at different sites of the digestive tract of weaned piglets fed liquid diets. Asian-Aust. J. Anim. Sci.24, 834-843.
HumeM.E., Kubena L.F., Edrington T.S., Donskey C.J., MooreR.W., RickeS.C.and Nisbet D.J. (2003). Poultry digestive microflora biodiversity as indicated by denaturing gradient gel electrophoresis.Poult. Sci. 82, 1100-1107.
Kelly D., Conway S. and Aminov R. (2005). Commensal gut bacteria: mechanisms of immune modulation.Trends. Immunol. 26, 326-333.
Kisand V. and Wikner J. (2003). Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences. J. Microbiol. Methods. 54,183-191.
Klasing K.C., Johnston B.K. and Benson B.N. (1999). Implications of an immune response on growth and nutrition requirements of chicks, Pp. 35-47 inRecent Developments in Poultry Nutrition, P.C. Garnsworthy and J. Wiseman Ed., vol. 2. NottinghamUniversityPress, Nottingham, United Kingdom.
Lan P.T.N., Hayashi H., Sakamoto M. and Benno Y. (2002). Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries. Microbiol. Immunol. 46, 371-382.
Lerman L.S., Fischer S.G., Hurley I., Silverstein K. and Lumelsky N. (1984). Sequence-determined DNA separations. Annu. Rev. Biophys. Bioeng. 13, 399-423.
Liu B.Y., Wang Z.Y., Wang H.R., Hu P., Xu D. and Wang Q. (2011). Molecular profiling of bacterial species in the caecum of geese. Czech J. Anim. Sci.56, 192-203.
Lu J., Idris U., Harmon B., Hofacre C., Maurer J.J. and Lee M.D. (2003). Diversity and succession of the intestinal bacterial communities of the maturing boiler chicken. Appl. Environ. Microbiol. 69, 6816-6824.
Matsui H., Kato Y., Chikaraishi T., Moritani M., Ban- Tokuda T. and Wakita M. (2010). Microbial diversity in ostrich ceca as revealed by 16S ribosomal RNA gene clone library and detection of novel Fibrobacterspecies. Anaerobe. 16, 83-93.
Mead G.C. (1989). Microbes of the avian cecum: types present and substrates utilized. J. Exp. Zool. 3, 48-54.
Mead G.C. (2000). Prospects for competitive exclusion treatment to control salmonellas and other foodborne pathogens in poultry. Vet. J. 159, 111-123.
Muyzer G., De Waal E.C. and Uitterlinden A.G. (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.
Muyzer G. and Smalla K. (1998). Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Leeuwenhoek. 73, 127-141.
Muyzer G., Brinkhoff T., Nu¨bel U., Santegoeds C., Scha¨fer H. and Wawer C. (1998). Denaturing gradient gel electrophoresis (DGGE) in microbial ecology, Pp. 3441-3442 inMolecular microbial ecology manual, A.D.L. Akkermans, J.D. Van Elsas and F.J. De Bruijn Ed. 3^rd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Myers R.M., Lumelsky N., Lerman L.S. and Maniatis T. (1985). Detection of single base substitutions in total genomic DNA. Nature. 313, 495-498.
Noll W.W. and Collins M. (1987). Detection of human DNA polymorphism with a simplified denaturing gradient gel electrophoresis technique. Proc. Natl. Acad. Sci. 84, 3339-3343.
Ovreas L., Forney L., Daae F.L. and Torsvik V. (1997). Distribution of bacterioplankton in meromictic LakeSaelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.Appl. Environ. Microbiol. 63, 3367-3373.
Reeson A.F., Jankovic T., Kasper M.L., Rogers S. and Austin A.D. (2003). Application of 16S rDNA-DGGE to examine the microbial ecology associated with a social wasp Vespula germanica. Insect. Mol. Biol. 12, 85-91.
Salanitro J.P., Fairchild I.G. and Zgornicki Y.D. (1974). Isolation, culture characteristics and identification of anaerobic bacteria from the chicken caecum. Appl. Microbiol. 27, 678-687.
Simpson J.M., McCracken V.J., Gaskins H.R., White B.A. and MackieR.I.(1999). Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. J. Microbiol. Methods. 36, 167-179.
Smits C.H., Veldman A., Verstegen M.W. and Beynen A.C. (1997). Dietary carboxymethyl cellulose with high instead of low viscosity reduces macronutrient digestion in broiler chickens. J. Nutr. 127, 483-487.
Steenfeldt S., Bach Knudsen K.E., Børsting C.F. and Eggum B.O. (1995). The nutritive value of decorticated mill fractions of wheat. 2. Evaluation with raw and enzyme treated fractions using adult cockerels. Anim. Feed Sci. Technol. 54, 249-265.
Swift S., Vaughan E.E. and De Vos W.M. (2000). Quorum sensing within the gut ecosystem. Microb. Ecol. Health Dis. 2, 81-92.
Tellez G., Higgins S.E., Donoghue A.M. and Hargin B.M. (2006). Digestive physiology and the role of microorganisms. J. Appl. Poult. Res. 15, 136-144.
Thi Ngoc Lan P., Hayashi H., Sakamoto M. and Benno Y. (2002). Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries. Microbiol. Immunol. 46, 371-382.
Torok V.A., Ophel-Keller K., Loo M. and Hughes R.J. (2008). Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl. Environ. Microbiol.74, 783-791.
Van Leeuwen P., Mouwen J.M., Van Der Klis J.D. and Verstegen M.W. (2004). Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance. Br. Poult. Sci. 45, 41-48.
Wang Z.Y., Shi S.R., Xu M.J. and Yang H.M. (2009). 16S rRNA-based analysis of bacterial diversity in the microbial flora of the goose intestinal tract. Anim. Feed Sci. Technol.18, 531-540.
Wintzingerode F.V., Gobel U.B. and Stackebrandt E. (1997). Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analyses. FEMS Microbiol. Rev. 21, 213-229.
Wright A.D.G., Dehority B.A. and Lynn D.H. (1997). Phylogeny of the rumen ciliates Entodinium, Epidinium and Polyplastron (Litostomatea: Entodiniomorphida) inferred from small subunit ribosomal RNA sequences. J. Eukaryot. Microbiol. 44, 61-67.
Xiao W., Peng Q., Liu H.W., Wen M.L. and Cui X.L. (2007). Prokaryotic microbial diversity of the ancient salt deposits in the Kunming Salt Mine, P.R. China. Acta. Microbiol. Sinica. 47, 295-300.
Yu Z.T. and Morrison M. (2004). Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 70, 4800-4806.
Zhou H., Gong J., Brisbin J.T., Yu H., Sanei B., Sabour P. and Sharif S. (2007). Appropriate chicken sample size for identifying the composition of broiler intestinal microbiota affected by dietary antibiotics, using the polymerase chain reaction-denaturing gradient gel electrophoresis technique. Poult. Sci. 86, 2541-2549.
Zhu X.Y. and Joerger R.D. (2003). Composition of microbiota in content and mucus from caeca of broiler chickens as measured by fluorescent in situ hybridization with group-specific, 16S rRNA-targeted oligonucleotide probes. Poult. Sci. 82, 1242-1249.
Zhu X.Y., Zhong T.Z., Pandya Y. and Joerger R.D. (2002). 16S rRNAbased analysis of microbiota from the cecum of boiler chickens. Appl. Environ. Microbiol. 68, 124-137.