K. Ophel-Keller1
R.J. Hughes2
1 Sardi,
Field Crops Pathology Unit, Plant Research Centre, Urrbrae,
Australia
2 Sardi,
Pig and Poultry Production Institute, University of Adelaide, Roseworthy
Australia
The microorganisms that colonise the gastrointestinal tract during the early post-hatch period form a synergistic relationship with their poultry host, releasing and providing essential nutrients as well as competitively excluding pathogenic species. Microorganisms can also directly interact with the lining of the gastrointestinal tract, which may alter the uptake of essential nutrients and the immunological status of the bird. Several Australian studies indicate that microbial colonisation of the gut is a variable process and that it can differ substantially between male and female chickens.
This report outlines the development and application of terminal restriction fragment length polymorphism (T-RFLP), a DNA-based analytical tool, for examining diet-induced changes in the microbial community of the chicken gut.
Introduction
Gut microbiology and its role in animal health has become increasingly important, particularly now that the prophylactic use of antibiotics in animal feeds to promote growth has been questioned (Grimes, 2000). Gastrointestinal microorganisms have a highly significant impact on uptake and utilisation of energy (Choct et al., 1996) and other nutrients (Smits et al., 1997; Steenfeldt et al., 1995), and on the response of poultry to anti-nutritional factors (such as non-starch polysaccharides), pre- and pro-biotic feed additives and feed enzymes (Bedford and Apajalahti, 2001).
Microorganisms can also directly interact with the lining of the gastrointestinal tract (Van Leeuwen et al., 2004), which may alter the physiology of the tract and immunological status of the bird (Klasing et al., 1999). Current methods for analysis of intestinal flora rely on culturing, which is not only laborious, but more importantly, misses a large part of the uncultivable microflora. Alternatively, DNA techniques have the advantages of being rapid, relatively inexpensive and capable of monitoring gene regions of complex populations.
This report is a brief summary of current work supported by the Australian Poultry Cooperative Research Centre (CRC) to develop DNA-based analytical tools for examining diet-induced changes in the microbial community of the chicken gut.
Current molecular approaches to the analysis of complex biological microbial communities
Currently the techniques of choice for microbial community analysis in many disciplines are denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) (Muyzer, 1999). These techniques amplify the 16S subunit of bacterial ribosomal DNA by PCR (polymerase chain reaction), and then separate the amplicons on a denaturing gel to visualise fragment size differences in the ribosomal DNA. However, this technique is not conducive to high throughput and there are issues with reproducibility and analysis based on presence/absence/position of bands. To this end, there has been emphasis placed on the need to standardize conditions across research groups and the need for good analytical software. Of further concern with DGGE/TGGE and other nucleic acid-based techniques involving PCR is the choice of appropriate primers in order to obtain a true representation of the actual microbial community present.
An alternate technique for bacterial community analysis is terminal restriction fragment length polymorphism (T-RFLP) (Osborn et al., 2000). This technique also amplifies the 16S subunit of the bacterial ribosomal DNA present in biological samples; however, all bacterial sequences amplified are labelled with a fluorescent dye. The amplified and labelled bacterial sequences are cut with sequence specific enzymes. The resulting fragments are separated according to size and detected by fluorescence emission from the incorporated dye by a DNA sequencing machine. Results are converted to graphical profiles where peaks can represent taxonomically related groups and/or strains of bacteria. These can be easily compared between samples to identify changes in bacterial community composition. The T-RFLP technique has great potential for large scale profiling, as 96 samples can be run at a time.
Development of a high throughput approach for chicken gut microbial analysis
The aim of this work is to establish a microbial profiling technique for chicken intestinal microflora based on high-throughput, high resolution fingerprinting of bacterial ribosomal gene regions. This will enable changes in the intestinal microbial community under different environmental conditions to be monitored.
We have elected to use the T-RFLP technique for chicken gut microbial community analysis because of its high-throughput potential. T-RFLP, like all PCR based methods, is influenced by template and therefore DNA extraction procedure. Choice of extraction method can greatly influence the complexity of the resulting profile (Figure), and impact on the knowledge gained.
Peaks in the Figure above represent taxonomically related groups and/or species of bacteria found in the chicken ileum. Numbers above peaks indicate peak position. Peak heights are representative of the proportion of different bacterial groups found in the population.
Note that more peaks and, therefore, bacterial groups are detected in the gut sample extracted with method A. Only dominant peaks present in extraction method A are also represented in extraction method B, indicating that extraction method B is less efficient in recovering all bacterial DNA. This demonstrates the importance of using an appropriate and efficient extraction method. The extraction method used on chicken gut and digesta samples is a modification of a proprietary method developed by SARDI researchers for analysis of soil-borne organisms.
PCR bias is also another important issue; therefore, PCR primer choice and standardized conditions are important when developing a high throughput method.
To this end we have: (i) compared various DNA extraction procedures and developed an effective DNA extraction protocol suitable for chicken gut samples, which is also conducive to high throughput; (ii) compared various universal 16S ribosomal DNA PCR primers to ascertain those which perform best; (iii) tested the reproducibility of our method; and (iv) developed quality controls to be included in each T-RFLP run.
The selection of restriction enzymes chosen for the T-RFLP analyses were based on those, which showed the greatest theoretical discrimination potential between available bacterial ribosomal 16S sequences in databases. These have given us good preliminary results in our T-RFLP analysis of chicken gut microbial samples. The T-RFLP data will be analysed with multivariate statistical models, such as multidimensional scaling.
Industry perspectives
The use of our developed T-RFLP tool will allow us to define what constitutes an optimum intestinal microflora in an "elite" chicken, and to utilise this technology and benchmark to conduct comparative studies of the effects of dietary manipulations on changes in the "good" and "bad" bacterial population in the gut of chickens. This tool will contribute to an increased knowledge of the chicken gut microbiota, and hence, a better understanding in its role in chicken nutrition, which will form the basis for practical recommendations on novel nutritional strategies for the chicken meat and egg industries in Australia.
Acknowledgments
Dr Valeria Torok is a Senior Research Officer supported by the Australian Poultry CRC.
References
Bedford, M.R. and Apajalahti, J. (2001). Enzymes in Farm Animal Nutrition. Eds M.R. Bedford and G.G. Partridge. CABI Publishing, Wallingford.
Choct, M., Hughes, R.J., Wang, J., Bedford, M.R., Morgan, A.J. and Annison, G. (1996). British Poultry Science, 37: 609-621.
Grimes, T.M. (2001). Proceedings Australian Poultry Science Symposium, 12: 10-16.
Klasing, K.C., Johnstone, B.K. and Benson, B.N. (1999). Recent Developments in Poultry Nutrition 2. Eds P.C. Garnsworthy and J. Wiseman. Nottingham University Press, Nottingham.
Muyzer, G. (1999). Current Opinion in Microbiology, 2: 317-322.
Osborn, A., Moore, E., and Timmis, K. (2000). Environmental Microbiology, 2: 39-50.
Smits, H.M., Veldman, A. Verstegen, M.W.A. and Beynen, A.C. (1997). Journal of Nutrition, 127: 483-487.
Steenfeldt, S., Knudsen, K.E.B. Borsting, C.F. and Eggum, B.O. (1995). Animal Feed Science and Technology, 54: 249-265.
Van Leeuwen, P., Mouwen, J.M.V.M., Van der Klis, J.D. and Verstegen, M.W.A. (2004). British Poultry Science, 45: 41-48.
From Proceedings of the "17th Australian Poultry Science Symposium", New South Wales, Australia.
