Aims: This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep.
Methods and Results: DGGE was used to study the ciliate diversity in the rumen of sheep. There was considerable variation between sheep which were co-housed, and fed the same diet. However, no difference in the major banding patterns was detected, when samples were collected from a single sheep sampled at different points. Following dietary changes, use of a pair-wise comparison of lanes, demonstrated that although there was still diversity between the ciliate population of sheep, the effects as a result of dietary changes were greater.
Conclusions: The technique generated molecular profiles which are sufficiently different to allow comparison between samples, and to permit molecular ecological studies on the rumen ciliate population.
Significance and Impact of the Study: The outcome of this study means that ciliate diversity in the rumen may now be studied by those unfamiliar with morphological identification of these organisms.
Ruminants are large, herbivorous animals and include some of the most economically important domestic animals (e.g. cattle and sheep). They are characterized by the presence of a fore-stomach or rumen, which is the site of initial digestion of the animal's food, and allows them to efficiently digest low-protein plant diets. Fermentation in this organ is performed by an extremely diverse microbiota consisting of archaea, eubacteria, anaerobic fungi and protozoa. Depending on the diet, the ciliated protozoa can account for 50% or more of the total microbial biomass of the rumen (Harrison and McAllan 1980), with around 105–106 protozoal cells per millilitre of rumen fluid (Dehority 1978). With a rumen volume of about 100 l, a typical cow can have as many as 1011 individual protozoal cells present at any one time. On the basis of morphological studies, it has been estimated that these cells can be accounted for by as many as 20 distinct species populating the rumen of a single cow at any one time (Williams and Coleman 1992).
By classical morphological criteria more than 250 species of ciliates have been described which live in the rumen of various feral and domesticated ruminants (Williams and Coleman 1992). However, the validity of these observations has been questioned, because many of the ‘species’ described exhibit a substantial morphological plasticity (Dehority 1994). Moreover, the number of species in an individual host is highly variable, and it has been suggested that many ciliate species are shared by the various hosts (Williams and Coleman 1992). Recent research, using molecular characterization has suggested that the protozoal diversity within the rumen is even greater than that first anticipated (Moon-van der Staay et al. 2002), but despite recent progress with molecular ecological studies (Karnati et al. 2003), the level of diversity present between individuals remains unclear.
Molecular profiling methods have been used to answer similar questions regarding molecular diversity in a number of other ecological niches (e.g. Labbe et al. 2003; Green et al. 2004). This paper reports the use of denaturing gradient gel electrophoresis (DGGE) to assess the diversity of the major species of ciliates present in the rumen of sheep housed together and fed the same diet, the diversity of ciliates present in a single sheep on consecutive days and the diversity of ciliates present in four different sheep on two different diets, with respect to the level of diet diversity vs inter-sheep diversity.
Materials and methods
Sheep were fed on a diet of hay and concentrates as described previously (Eschenlauer et al. 1998) except in the case of dietary variation, where sheep were fed on a high and low protein diet, with grass silage as the roughage source and the protein concentration varied by changes in the wheat composition of the diet (McEwan et al. 2002).
Isolation and purification of DNA
Ruminal contents were removed via a cannula and a ciliate-enriched fraction was collected by allowing cells to sediment (Eschenlauer et al. 1998). DNA was extracted using a QIAamp® DNA Stool Mini Kit (Qiagen Ltd, West Sussex, UK) following the manufacturer's instructions.
Approximately 200 bp of the 18S rDNA gene were amplified using the primers: forward – 5′-GGTGGTGCATGGCCG-3′, reverse – AATTGCAAAGATCTATCCC with a 45 nucleotide GC-clamp linked to the 5′ terminus of the reverse primer: 5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCGGGGCC-3′. Use of a GC-clamp has been shown to help obtain a better profile on the denaturing gel (Muyzer et al. 1998). The primers were designed to be specific to the rumen ciliates using recently derived sequence information (Moon-van der Staay et al. 2002). PCR was performed using the following steps: 1 cycle (94°C for 4 min, 60°C for 30 s, 72°C for 1 min); 35 cycles (94°C for 1 min, 60°C for 30 s, 72°C for 1 min); 1 cycle (94°C for 1 min, 60°C for 30 s, 72°C for 10 min). Amplicons were analysed by electrophoresis on a 2·5% agarose gel and visualized after staining with ethidium bromide.
DGGE was performed on the DCodeTM Universal Mutation Detection system (16 cm system; Bio-Rad, Hemel Hempstead, UK). DGGE parallel gradient gels ranged from 20 to 35% (6% acrylamide), run at 130 V, 200 mA, 250 W for 3·5 h at 60°C. DNA was visualized by staining with SYBR®Gold nucleic acid gel stain (Molecular Probes, Eugene OR, USA).
As a check of the above primers three clones of previously amplified 18S genes from each of five different species of ciliates were run on a gel. In all 15 lanes only a single band was detected, although mobility on lanes varied with species and when DNA was isolated, cloned into the PCR®2·1-TOPO vector (Invitrogen BV, Leek, the Netherlands) following the manufacturer's instructions and sequenced from these bands, all showed greatest similarity to the sequence corresponding to the original ciliate from which it had been isolated. This demonstrated the potential of this technique to produce amplicons from a number of different ciliate species.
Samples collected simultaneously from eight sheep which had been fed on the same diet, and housed together demonstrated that there was considerable inter-animal variation in the composition of the major ciliate species present in the rumen (Fig. 1). However, when samples were collected on four successive days from a single sheep, no variation was detected between samples (Fig. 2). These two observations imply that, at least in the short term, there is relatively little difference in the major ciliate population of an individual sheep with time, but that there is considerable variation between animals, despite them being co-housed and fed an identical diet.
Although the primers had been shown to be capable of being used to amplify ciliate 18S genes, it was necessary to verify that the observations seen in Figs 1 and 2 were not the result of amplifying either 18S genes from other sources, or 16S genes from rumen bacteria. The validity of the amplicons was investigated by extracting DNA from 10 bands from the gel shown in Fig. 1, and cloning the DNA into the PCR®2·1-TOPO vector (Invitrogen BV, Leek, The Netherlands) following the manufacturer's instructions. Plasmids were isolated from recombinant colonies using a plasmid extraction kit (Qiagen Ltd) following the manufacturer's instructions, and the nucleotide sequence of the insert was determined using an ABI PrismTM 377XL DNA sequencer (Applied Biosystems, Warrington, UK). BLASTN analysis indicated that the ‘best hit’ in all cases corresponded to known 18S rDNA sequences from rumen ciliates.
Given the apparent day-to-day stability in the composition of the most abundant ciliate species within a single sheep this suggests that the technique has potential for monitoring a population as a result of an environmental change such as alteration of diet. Figure 3 shows the profiles obtained from four sheep fed on either high or low protein diets in a 2 × 2 switch-over design. In similar types of research from other ecosystems, pair-wise comparison between individual lanes was performed by calculating Euclidean distance relationships between lanes (e.g. Eiler et al. 2003). Here bands were scored as either present or absent on the gel and all lanes were compared against each other and a pair-wise matrix was constructed for all comparisons. This effectively means that the analysis was performed by one of the other Minkowski Metrics (of which Euclidean Distance analysis is an example) – Hamming Distances.
This matrix was then used as the input file for the Neighbor program within PHYLIP (Felsenstein 1989). The relationship between lanes can be summarized according to the pattern observed in Fig. 4. This demonstrates that although there is variation in the pattern observed between samples collected from different sheep, that the biggest change in the population dynamics is as a result of dietary change.
The fact that the cloned samples yielded single bands on DGGE gels allowed these primers to be used with confidence. In addition, the diversity seen between the populations from different sheep (Fig. 1) demonstrates that the primers have the ability to detect several sequences within a sample, and that they also have the potential to be used as detectors of biodiversity. Equally it is clear from Fig. 2 that the reproducibility of the technique is stable from day-to-day – based on the assumption that there is unlikely to be a major change in the ciliate population of a sheep's rumen from day-to-day on a constant diet.
Due to the potential ability to detect variability, coupled with the stability within the rumen as an ecosystem over a short period of time, makes DGGE analysis a perfect system to monitor population shifts as a result of changes to the rumen ecosystem. An example of the potential use of this approach is seen in Fig. 3, where samples have been collected from the same four sheep each on different diets at different time points. The interpretation of this figure, as calculated by Hamming Distances, is seen in Fig. 4, where it is clearly demonstrated that there is less animal-to-animal variation than there is variation seen on diet.
In conclusion, DGGE analysis is a powerful tool for detecting and analysing the biodiversity in the major rumen ciliate population between animals fed on a single diet, as over a short time there is relatively little change within a single animal. Furthermore, it can be used to measure the response resulting from a dietary change, allowing it to determine if the postdietary change is greater than that already naturally observed between sheep.
The Rowett Research Institute receives funding from the Scottish Executive Environmental and Rural Affairs Department. This investigation has been supported by a European Union funded thematic research programme – ERCULE (European rumen ciliate collection QLRI-CT 2000 01455). Martina Regensbogenova was the recipient of a Training Studentship under the Rowett Research Institute's Marie Curie Training award (HPMT-CT-2001-00409).