To investigate the impact of different housing conditions on the intestinal microbiota in ileum and caecum in laying hens, samples obtained from two experimental inoculation studies previously reported by De Vylder et al. [18, 19] have been further characterised using T-RFLP and 454 pyrosequencing. We found that individuals living in the same environment also tend to develop similar microbiota. Despite of being raised in the same environment and likely having similar microbiota to begin with, we found, that when hens were transferred to different cages types (conventional cages, furnished cages or aviary) for 2 weeks, minor but uniform changes in the T-RFLP profiles of the microbiota in ileum and caecum occurred. By comparing T-RFLP fingerprints from individual hens, we found highly similar ileal and caecal profiles in hens from same cage, which could be discriminated from other cages in the same experiment. However, the differences were not cage type specific, as when samples from two independent experiments were compared by PCA, the largest component were observed between experiments, meaning that cage type only had minor influence on the variance. This indicates that the intestinal microbiota may be influenced on the contact to the surrounding microbiological environment in the cage.
The differences in the evolution of the microbiota were further analysed by deep sequencing of 16S rDNA libraries from pooled caecal samples. When 16 week old laying hens were moved from a floor system and into conventional cages, their caecal microbiota changed towards a less diverse microbiota compared to hens from the same flock that were allocated to aviary and furnished cages. Sequencing of rDNA libraries revealed that hens housed in conventional cages showed a progressive decrease in the number of different OTUs in their caecal microbiota, compared to hens housed in aviary or furnished cages. The decline was already observed after 2 weeks in the cage, and it was even more pronounced after 4 weeks. The same reduction was not observed in the other cage systems. The OTUs that were not recovered in conventional cages were all represented in the other cages, however in low numbers reflecting that they belong to the group of less abundant species. As each OTU represents unique genera or even species, this reflects an overall decrease in diversity of their caecal microbiota towards fewer and more dominating species. Alternative cage systems are characterized by having larger cages due to flock sizes and facilities for enhancing natural behaviour. These facilities may, however, hinder the removal of manure compared to conventional cages, and an overall higher bacterial level has been noted in these systems . It is likely that the laying hens housed in a more contaminated environment, as in the alternative systems, may be more exposed to faeces from the other layers, and thereby continuously being reinoculated, thereby maintaining a higher species variety in the microbiota.
The vast amount of sequence reads generated from each sample by 454 pyrosequencing allowed quantification of the individual OTU in relation to the total flora. Only minor differences were observed in the relative distribution of phyla and classes of bacteria in the caecal microbiota between cages, but quantitative variations that were not cage specific were observed between different genera. However, when OTUs were grouped according to phyla and classes, comparable groups were found in all samples. This indicates that the cage system itself did not influence the balance between the large classes, but pinpoints the caecal microbiota as a dynamic, highly competitive organ where a decrease in one genus may be compensated by an increase in a closely related species, or other species belonging to the same functional guild that shares the same requirement for substrates.
When the consensus sequences from 197 OTUs were aligned with the RDP database, more than 91% were identifiable at least to phylum level, and more than 55% could be identified to genus level. The most prevalent phyla in the caecal microbiota were Bacteroidetes, with Firmicutes being the second most prevalent. The ratios between these two phyla (F/B) remained fairly equal between the CC and AC, but a decrease was observed for CC. A major reason for this difference was promoted by a shift from Faecalibacterium to Butyricimonas. Whether this change was mediated by the cage system of a coincidence remains to be established, but we did not find that it changed the susceptibility for Salmonella, probably because both species produces butyric acid. There are indications that the feed may have large influence the F/B ratio. In domestic and wild turkeys, Scupham et al.  found similar ratios between these phyla; however this is in contrast to the caecal microbiota found in broilers. In a number of studies [8, 13, 21, 22], the microbiota in broilers were heavily dominated by Firmicutes, with Bacteroidetes only present at much lower level. An explanation for this may be the different feeding strategies that are used. Broilers are normally fed a high energy diet that sustains fast growth, which possibly leaves more digestible nutrients for the intestinal microbiota. In contrast, laying hens are fed a much more restricted diet containing less energy and higher amounts of digestive fibers, which instead may favour genera from Bacteroidetes. The same phenomena has been described for the microbiota in obese humans, where Ley et al.  observed an increase in Bacteroidetes during long term restricted diet.
The two most dominating genera found in this study were Faecalibacterium and Butyricimonas constituting more than one third of the total microbiota in all sequenced caecal samples. The first species is a well known colonizer of the caecal microbiota of poultry; however Butyricimonas has just recently been described in rats , and has to our knowledge not been described in poultry before. Both bacteria are important contributors to the production of butyric acid in the lower intestine, and may represent an important functional guild in the microbiota. Studies have shown that especially butyric acid may have a prominent role in the reduction of invasion , and colonization of Salmonella in the caecal microbiota . Butyricimonas was the most dominant genus in caecum samples from conventional cages, but this difference was not reflected in any variations found in the colonization level of S. Enteritidis as reported by De Vylder et al. , who found no difference in excretion level and time between cage systems.
We did not find evidence that the introduction of S. Enteritidis to the intestinal microbiota were able to change the species diversity in ileum or caecum. When individual T-RFLP profiles from Salmonella positive layers were compared with cage mates that had cleared the infection no differences were observed. When comparing the distribution of OTU in each group before and after inoculation, the balance between different classes and genera were also maintained throughout the study. The low impact on the intestinal microbiota may be explained by the fact that inoculation only induced a subclinical infection, in contrast to experimental studies where a more profound disturbance of the microbiota has been observed in cases where diarrhoea has followed infection [27, 28].
In the early studies of Nurmi and Rantala , it was shown that a highly diverse intestinal microbiota in broilers is one of the best barriers towards colonization with Salmonella (competitive exclusion). However, we did not find that decreased diversity in the layers had a significant impact on the colonization and elimination of Salmonella. It is likely that this colonisation resistance is highly important in broilers where a mature flora has not been established yet, but in layers this may not be as important. Furthermore, in the second inoculation study where seeder birds were housed together with non-infected birds, De Vylder et al.  found that the transmission of S. Enteritidis was higher among hens housed in aviary or floor system than in conventional and furnished cages. A likely explanation for our observation is that direct contact to faecal material from infected hens is very important for the transmission of S. Enteritidis in a flock, and that the higher species diversity found in layers with more contact with faecal material does not prevent colonization, but keeps it at a relatively low level.