Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR

Abstract

A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.

FIG. 1

Amplification of control rumen bacterial DNA (strains are listed in Materials and Methods) with the primer set detailed in Table 1. A DNA size marker is in the extreme left lane.

FIG. 2

Phylogenetic placement of 16S rDNA sequences generated by an E. ruminantium primer set from total rumen DNA (day 3 of high-grain diet). The numbers represent the confidence levels (percentage) generated from 1,000 bootstrap trees. The scale bar is in fixed nucleotide substitutions per sequence position. Sequences 670 bp long were used in this analysis.

FIG. 3

Phylogenetic placement of 16S rDNA sequences generated by an S. ruminatium-M. multiacida primer set from total rumen DNA on days 0 (0d), 3 (3d), and 28 (28d) of the switch to a high-grain diet. The numbers represent the confidence levels (percentage) generated from 1,000 bootstrap trees. The scale bar is in fixed nucleotide substitutions per sequence position. Sequences 513 bp long were used in this analysis.

FIG. 4

Qualitative PCR detection of 12 bacteria in the rumens of cows for which the diet had been changed from hay to grain. Day 0, before the experiment, animals maintained on basal hay diet; day 3, animals fed a high-grain diet for 3 days; day 28, animals fed a high-grain diet for 28 days. Lanes: 1, P. ruminantium; 2, P. bryantii; 3, P. albensis; 4, F. succinogenes; 5, R. amylophilus; 6, S. ruminantium-M. multiacida; 7, S. bovis; 8, T. bryantii; 9, E. ruminantium; 10, A. lipolytica; 11, S. dextrinosolvens; and 12, R. flavefaciens. Lane M, DNA size marker.

FIG. 5

Differential amplification of rumen bacterial DNA templates with universal bacterial primers 27f and 1525r (17). Real-time PCR amplification was conducted essentially as described in Materials and Methods with 30 ng of each bacterial DNA template. PCR cycling was performed as follows: 95°C for 10 min of initial denaturation, then 40 cycles of 95°C for 15 s, 60°C for 5 s, and 72°C for 1 min. The fluorescence was captured at the end of the extension phase. The threshold fluorescence values were calculated with the LightCycler software and were as follows: S. bovis, 6.736 cycles; S. ruminantium, 8.375 cycles; A. lipolytica, 8.412 cycles; P. bryantii, 8.758 cycles; R. flavefaciens, 8.821 cycles; T. bryantii, 9.071 cycles; P. albensis, 9.592 cycles; P. ruminicola, 10.98 cycles; E. ruminantium, 10.28 cycles; S. dextrinosolvens, 12.59 cycles; R. amylophilus, 13.39 cycles; and F. succinogenes, 15.85 cycles.