K.E. Hagen1
T.L. Grayson1
G.W. Tannock2
D.R. Korver1
G.M. Fasenko1
G.E. Allison3
1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada;
2Department of Microbiology, University of Otago, Dunedin - New Zealand;
3 Faculty of Medicine, The Australian National University, Canberra, Australia
The bacterial community of the crop, ileum and ceca of broilers was investigated during the first week of life using nucleic acid based microbial ecology techniques and selective bacteriological culture of lactobacilli. The microbial ecology studies revealed that the Lactobacillus populations in the ileum and ceca of very young birds were similar to each other and among birds. By day 3, each bird had developed its own unique microbial community. The LAB population in the crop was always more complex than in the ileum and ceca. Species belonging to the Lactobacillus acidophilus complex were detected in all organs. Isolates obtained from the selective culture of lactobacilli from the crop and ceca were genetically fingerprinted which revealed that several strains were present in different regions of the gut in the same and different birds. The species and strains present in the distal region of the gut appear to be dictated by an initial colonisation of the crop.
Introduction
The diverse collection of bacteria in the digestive tract, collectively referred to as the gut microflora, plays an important role in the nutrition, health and disease of broiler chickens, other livestock, and humans. The composition of the microflora in the broiler chicken gastrointestinal tract (GIT) is organ-specific, with microbial colonisation occurring in the crop, ileum, and caeca (Sarra et al., 1992; Tannock, 1995). Lactobacilli dominate the microflora in the crop and are numerous in the ileum, whereas the microflora in the caeca is dominated by anaerobes. In the 1970s, Fuller hypothesised that the crop microflora provides a bacterial inoculum for the remainder of the gut (Fuller and Brooker, 1974). Different research groups have independently identified the same Lactobacillus species (specifically Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus reuteri) in the crop, ileum, and caeca, suggesting that lactobacilli may be a genus that supports Fuller's hypothesis. Understanding how and where lactobacilli colonise the GIT is necessary to rationally derive husbandry methods that utilise feed supplements other than antimicrobial drugs for the efficient and sustainable production of poultry.
Methods
A culture-independent technique, based on the species specific sequence of the 16S rRNA gene, was used to analyse the microbial ecology of lactobacilli in the broiler gut (Walter et al., 2000, 2001). DNA was extracted from the crop, ileum and caeca of broilers on day 1, day 3, and day 7, and used as template in a PCR reaction that was designed to amplify the 16S rRNA gene of lactobacilli and closely related species. The resulting PCR reactions were analysed on a denaturing gradient gel (DGGE) which separates similar sized fragments of different sequence to create a profile of the Lactobacillus species present in the gut sample.
Crop and caecal samples were plated onto selective media to propagate lactobacilli from the chicken gut, and 10 colonies were analysed from each section of each bird. The isolates were speciated using molecular techniques (Guan et al., 2003). Isolates of the same species were then identified at the strain level using pulsed field gel electrophoresis (PFGE). PFGE is the industry "gold standard" for genetically fingerprinting bacterial strains. Bacterial cells are captured in agarose, the chromosomal DNA is purified in situ, cut with restriction enzymes, separated in a PFGE apparatus, and the fragmentation patterns analysed.
Results
The aim of our work is to understand the microbial ecology of lactobacilli in the chicken GIT. The objective of the current study was to investigate the development of the Lactobacillus microflora in the crop, ileum and caeca of healthy broilers in the first week of life. We had previously determined that the microflora in the crop changed dramatically during this time frame (Guan et al., 2003). A sample PCR-DGGE profile of Lactobacillus species found in the chicken gut is shown in Figure 1. Analysis of the data revealed that Lactobacillus populations in the ileum and caeca of day 0 and day 1 birds were similar to each other and among birds. The crop community, though different from that of the intestinal organs, was similar between birds. By day 3 and again at day 7, each bird had developed its own unique microbial community.
The LAB population in the crop was always more complex than in the ileum and caeca. Species belonging to the Lactobacillus acidophilus complex (Lactobacillus acidophilus, L. crispatus, L. amylovorus, L. gallinarum, and L. johnsonii) were detected in all of the organs.
Lactobacilli were also cultured from crop and caecal samples. Isolates of the same species were then identified at the strain level using PFGE (Figure 2).
Comparison of the origin of isolates revealed that several strains of L. johnsonii, L. gallinarum and L. crispatus were present in different regions of the gut in the same and different birds (Table 1). Other strains were only isolated from either the crop or caeca. Remarkably, both sections of the gastrointestinal tract can be inhabited by multiple strains of the same and different species.
Discussion
The species and strains present in the distal region of the gut appear to be dictated by an initial colonisation of the crop. Our observations support Fuller's hypothesis that the crop community acts as a bacterial inoculum for the remainder of the gut. The microflora in the crop, therefore, is important in the development of the microflora in distal regions of the gastrointestinal tract.
Acknowledgments
This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (GEA), an NSERC Postgraduate Scholarship (KH), The Canadian Foundation for Innovation Infrastructure Grant Program (GEA), and the Canada Research Chair Program (GEA).
References
Fuller, R. and Brooker , B. E. (1974). American Journal of Clinical Nutrition, 27: 1305-1312.
Guan, L.L., Hagen, K.E., Tannock, G.W., Korver, D.R., Fasenko, G.M. and Allison, G.E. (2003). Applied and Environmental Microbiology, 69: 6750-6757.
Sarra, P. G., Morelli, L., et al. (1992). The Lactic Acid Bacteria in Health and Disease. B.J.B. Wood. London, Elsevier Applied Science. I: 3-19.
Tannock, G. W. (1995). An Introduction to Microbes Inhabiting the Human Body. Chapman and Hall, London.
Walter, J., Tannock, G. W., Tilsala-Timsjarvi, A., Rodtong, S., Loach, D.M., Munro, K. and Alatossava, T. (2000) Applied and Environmental Microbiology, 66: 297-303.
Walter, J., Hertel, C., Tannock, G.W., Lis, C.M., Munro, K. and Hammes, W.P. (2001). Applied and Environmental Microbiology 67: 2578-2585.
From Proceedings of the "17th Australian Poultry Science Symposium", New South Wales, Australia.
