The extreme complexity of the rumen microbiota has been uncovered in numerous publications employing isolation of pure cultures and description of their physiology. Based on this information, the putative contribution of these isolates to the overall metabolism and function of the rumen could be suggested. However, the next step—confirmation of the putative functionality by monitoring various functional groups by a cultivation-based approach—is very time- and labor-consuming, and the results, which are based on phenotypic characteristics, are not precise or conclusive. During the last decade, the use of molecular probes has become a popular approach in different fields of microbial ecology, and rumen microbiology is not exceptional in this regard. However, taking into consideration the enormous complexity of the rumen, the number of molecular probe tools specifically designed for monitoring the specific groups of microorganisms in this ecosystem is very limited. These few available molecular probes essentially target only seven bacterial species and one archeon (see the introduction). In this work, we designed and validated PCR primers for detection of 13 additional species of rumen bacteria. These primers were used in conjunction with real-time PCR, allowing accurate quantification of a target in a mix of total community DNA.
PCR primers for detection of 13 species of rumen bacteria were designed to satisfy the specificity and usability requirements of real-time PCR quantification. In the case of S. ruminantium
and M. multiacida
, it was not possible to design species-specific primers that would have sufficiently high melting temperatures for use in the LightCycler system. Therefore, this primer pair targets both species, but, if necessary, discrimination is still possible after sequence and phylogenetic analyses of the libraries produced with these primers (Fig. ). Libraries obtained after amplification with E. ruminantium
primers demonstrated a slightly higher level of diversity (96.8% of sequence similarity) than the ≥97% similarity sufficient for the integration into the same species nomination (28
). Sequence and phylogenetic analyses (Fig. ) demonstrated the existence of two clusters suggesting the necessity of additional species nominations. The second cluster, however, is represented exclusively by PCR-retrieved sequences, and further isolation and phenotypic characterization are necessary for new taxonomic designation. On the contrary, the 16S rDNA sequences of streptococci appeared to be less diverse, with many sequences having ≥97% sequence similarity, but which nevertheless have been elevated to the species level. An on-line similarity search demonstrated, for example, that S. bovis
has 99% similarity to S. caprinus
and 98% similarity to S. infantarius, S. waius,
and S. alactolyticus,
as well as being 97% similar to S. gallolyticus
and S. intestinalis.
Thus, the possibilities for designing only S. bovis-
specific primers were limited, and our primer pair reacted with streptococci of other ecological origin, e.g., with S. infantarius
and S. equinus.
The close similarity of 16S rDNA sequences within the Streptococcus bovis-Streptococcus equinus
complex has also been established in other investigations (8
), which suggests that less-conserved sequences, such as the intergenic region between 16S and 23S rDNAs, may be necessary for more accurate discrimination between them. In the present study, we assumed that the streptococci of other ecological origin are unlikely to be present in the rumen, but this requires further research. Another aspect of primer design was determining the primers' appropriateness for the LightCycler (or other real-time PCR) system. P. albensis
and R. amylophilus
primer pairs performed well with pure control cultures in both conventional and real-time PCR systems. Also, these two primer sets performed well in conventional PCR with total community DNA (Fig. ). However, the results of their application in real-time PCR with total rumen DNA samples were inconsistent, supposedly because of very fast ramps between the denaturing and annealing temperatures and because of a short incubation time (5 s) during the annealing phase.
After validation, these primers were used to monitor and to quantify 11 rumen bacterial species during the diet shift from forage to grain. The dynamics of truly fibrolytic rumen bacteria were in good correlation with the diet change. The quantities of R. flavefaciens
and F. succinogenes
DNAs in total rumen DNA, which were arbitrarily taken as 100% on a hay diet, dropped correspondingly to more than 10- and 20-fold those found in animals on a grain diet. The R. flavefaciens
data are also in good agreement with data from our previous experiments, in which we analyzed the 16S rDNA clone libraries produced from the same rumen DNA samples (33
). In these experiments, R. flavefaciens-
related sequences comprised 5.88% of all retrieved sequences from animals on the hay diet, but were undetectable (with a detection limit of ~2%) for those on the grain diet (33
). However, only a single F. succinogenes-
related sequence has been found in several clone libraries reported to date (32
), while our quantitative data do suggest that F. succinogenes
may reach at least the same quantities as R. flavefaciens
populations in animals on the hay diet. The clone libraries reported in these studies (32
) were constructed with the universal bacterial primers, and we hypothesized that DNA of F. succinogenes
may be amplified less efficiently than other bacterial templates present in a mix. Real-time PCR with various rumen bacterial templates and the universal bacterial primer set 27f and 1525r (17
) confirmed that, under otherwise identical amplification conditions, this particular template has a prolonged lag phase compared with those of other templates (Fig. ), which may be the reason for its underrepresentation in several clone libraries reported (32
). Because the real-time PCR procedure uses the calibration curve obtained from pure cultures, more accurate quantification is possible in comparison with the PCR-generated clone libraries. Several factors that may contribute to differential amplification have been discussed (35
). Presently recognized contributors are (i) genome size and rrn
gene copy number, (ii) choice of primers and number of cycles, (iii) annealing efficiency and specificity of primers, (iv) G+C content, (v) DNA concentration, and (vi) DNA-associated molecules. In the case of F. succinogenes,
this is definitely not a gene copy effect. In a previous work (20
), we established that F. succinogenes
possesses at least three rRNA operons, whereas an online search with the “fastest” S. bovis
template checked against the genome of the taxonomically similar S. equi
produced only one high-scoring hit, suggesting this group may possess a single rRNA operon. Also, the efficiency of annealing and extension seems not to be a factor, as judged by the exponential increase in its fluorescence comparable to those of the other templates (Fig. ). Poor annealing or extension appears to be a problem with the other bacterial template, E. ruminantium
, which displays slower and nonexponential fluorescence kinetics compared with those of the other templates (Fig. ). Since the difference in F. succinogenes
amplification is largely attributed to the beginning of PCR cycling, the problem may be associated with the original DNA template, perhaps due to DNA-associated molecules.
Ruminal prevotella are known to possess oligosaccharolytic and xylanolytic activities and to occupy the ecological niches of the second line degraders (9
). Comparative quantification of P. ruminicola
on a hay diet suggested that this population is the most numerous among the populations studied. On a grain diet, the P. ruminicola
count declines, but it still remains one of the predominant populations. The other representative of the genus, P. bryantii,
demonstrated the opposite kinetics, suggesting its role in starch degradation. Both of these species demonstrated a tremendous increase during the transition period on day 3, and this observation correlates with our previous data from the clone libraries (33
The saccharolytic spirochete T. bryantii
has been shown to be associated with the fibrolytic bacteria of the rumen and, albeit not possessing any fibrolytic activity, could enhance fiber degradation in a coculture with fibrolytic bacteria (16
). In our experiment, the quantification of this bacterial DNA demonstrated kinetics similar to those of two fibrolytic bacteria, F. succinogenes
and R. flavefaciens.
The dynamic of two taxonomically different xylanolytic bacteria, E. ruminantium
(belonging to low-G+C, gram-positive bacteria) and S. dextrinosolvens
(belonging to the gamma subclass of Proteobacteria
) also followed a similar decline during the diet switch, with the latter species not detectable on day 28. Based on the rate of lipolysis by pure cultures, A. lipolytica
has been suggested to be an organism that may play an important role in the lipolytic activity of the rumen (23
). However, no statistically significant changes were detected in the A. lipolytica
DNA concentration during the shift to a grain diet containing increased amounts of lipids.
In our previous analysis of clone libraries generated from the rumen microbiota during the diet switch, we detected the numerical prevalence of low-G+C, gram-positive bacteria belonging to the Selenomonas-Succiniclasticum-Megasphaera
group in Clostridium
cluster IX in grain diet microbiota (33
). The simultaneous quantification of two species in this group, S. ruminantium
and M. multiacida,
is in agreement with our earlier findings and demonstrates that these two bacteria represent the most numerous group in animals on a grain diet (Table ). An amylolytic bacterium, S. bovis
, has been considered as a major culprit in the development of lactic acidosis (19
), and selective inactivation of this bacterium by immunization results in reduced symptoms of lactic acidosis (11
). However, the absolute numbers of this bacterium seem to be low, and it was undetectable in our previous clone libraries with a detection limit of ~2% (33
). With the more sensitive approach implemented in this work, we were able to monitor its dynamics. Similarly with other amylolytic bacteria of the rumen, such as the prevotellas, S. bovis
responded to the grain diet switch with a tremendous 67-fold increase. However, surprisingly, on the grain-adapted system, the numbers were twofold lower than on the hay diet. This suggests that, besides the amylolytic activity, this bacterium may possess other functional activities important for rumen digestion of plant polysaccharides.
To our knowledge, this is the first demonstration of the applicability of real-time PCR for quantification of bacterial species in a complex microbial ecosystem. Previous applications of this technique have been limited to detection and quantification of specific transcripts and, in clinical and veterinary microbiology, detection and quantification of pathogens, contaminants, and antibiotic resistance genes. We demonstrated that, with the availability of calibration strains, their dynamics could be accurately monitored in a complex mix, such as rumen content. DNA calibration curves could be based on the actual cell numbers, thus linking the cultivation and molecular detection methods. The approach implemented in this work can be applied to other microbial systems as well. In addition, the set of primers developed during this study not only is suitable for quantification purposes, but can also be used for rapid preliminary identification of other bacterial strains isolated from the rumen.