Comparison of ileum microflora of pigs fed corn-, wheat-, or barley-based diets by chaperonin-60 sequencing and quantitative PCR

Abstract

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

FIG. 1.

Taxonomic composition of corn (C), barley (B), and wheat (W) clone libraries. A. Percentage of clones recovered from corn, barley, and wheat libraries classified by taxonomic group, determined by comparison to the reference database of chaperonin-60 sequences. Total library sizes were 909, 930, and 915 clones for corn, barley, and wheat, respectively. B. Comparison of log₁₀ numbers of clones in each taxonomic category recovered from the corn, barley, and wheat libraries.

FIG.2.

Phylogenetic analysis of 372 unique nucleotide sequences recovered from the corn, barley, and wheat libraries. The tree was calculated with a maximum-likelihood distance formula and neighbor joining. Clusters of similar sequences within the tree are named according to the identity of the nearest database neighbors of the group (C = Clostridiales, B = Bacillales, G = gamma-proteobacteria, L = Lactobacillaceae, S = Streptococcus). The scale bar indicates 0.1 substitution per site. Numbers following the group names indicate the number of clones falling into that sequence group recovered from the corn:barley:wheat libraries. For example, C1 group sequences were recovered 88 times from the corn library, 18 times from the barley library, and 69 times from the wheat library.

FIG. 3.

Determination of the prevalence of L10 genomes in each individual animal fed diets based on corn (n = 15), wheat (n = 15), or barley (n = 15). A. Number of genomes (log₁₀) detected in each individual per gram of ileal digesta. The means of the 15 observations within each group were: corn-fed individuals, 7.58; barley-fed individuals, 8.83; and wheat-fed individuals, 9.53.