Introduction

Protozoa are unicellular eukaryotic microorganisms which are ubiquitous in nature and anthropogenic environments. The rumen ciliates are potentially an agriculturally important group of protozoa found in domestic and wild ruminants [30]. Several factors seem to influence the composition of the protozoan population in the rumen. These include type and amount of feed consumed, pH, turnover rate, and feed level. The rumen microorganisms utilize carbohydrates as a carbon source in energy-yielding processes, whereas hydrogen gas (H2) is released as byproduct during ATP generation. In the protozoa, in turn, benefit from hydrogen removal by the methanogens because hydrogen is inhibitory to protozoan metabolism [32]. Earlier reports have provided evidence of strong relationships, such as endosymbiosis between ruminal protozoa and methanogens [8, 28]. Ciliate protozoa play a diverse role in the ruminal metabolism of nutrients. To improve the efficiency of feed crude protein utilization, considerable effort has been made to find a means of total elimination of protozoa from the rumen (defaunation) and a massive reduction in the rumen protozoan population (reduced fauna). Chemical drenching of experimental animals has been found to improve milk production. Detection and identification of protozoa have commonly been achieved through microscopic examination of morphological features. It remains difficult and time-consuming to reliably detect or identify many protozoan species by these methods, as protozoa may be fragile and inconspicuous and as it may be difficult to determine whether a given morphological feature can be regarded as distinct or not [3, 9, 23]. The anaerobic ruminal protozoa have been well studied [31], but much of this work is based on microscopic examination [4]. Difficulties in cultivating protozoa, and their polymorphic nature, have delayed effective assessment of protozoan ecology and taxonomy [5]. The small subunit ribosomal RNA (SSU-rRNA) gene called 16S rRNA in prokaryotes and 18S rRNA in eukaryotes is widely used as molecular marker to identify morphologically indistinguishable species, to infer their phylogenetic relationships, and to elucidate diversity. PCR-sequencing methods have been extensively used to examine the protozoal diversity in rumen samples [13]. PCR–DGGE has also been used in the profiling of protozoal communities in the rumen [20, 24].

India possesses more than 50 % of world’s buffalo population; Indian buffalo produce more than 50 % milk in India [15]. Surti is a popular breed of buffalo found in central Gujarat state. The Surti buffaloes are of medium size having docile temperament and body weight 300–350 kg at maturity. Since our animals mainly sustain on crop residues, the protozoan population is expected much different than that of exotic cattle. The present study was conducted to examine the diversity of rumen protozoa in Surti buffalo offered diet, green fodder bajra (Pennisetum purpureum), mature pasture grass (Dichanthium annulatum), and compound concentrate mixture. The molecular techniques were used to construct a library of 18S rDNA clones of rumen protozoa, and a phylogenetic tree for the clones isolated.

Materials and Methods

Sampling and DNA Extraction

The permission of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) was obtained prior to initiation of the study. The experiments were carried out on three adult Surti buffaloes (Bubalus bubalis), approximately 3 years of age with a mean live weight of 201 ± 18 kg, which were reared at the Department of Animal Nutrition, College of Veterinary Science and A.H. Anand. All the animals were maintained under uniform feeding regime (ICAR 1998) for minimum 21 days for dietary adaptation. The diet comprised of green fodder bajra (P. purpureum), mature pasture grass (D. annulatum), and compound concentrate mixture (20 % CP, 65 % TDN). The animals were offered 10 kg green, ad-lib dry grass and 2.5 kg of concentrate mixture daily. Animals were let loose daily for 2 h in the morning and evening during which they had free access to drinking water. Approximately 500 ml of rumen fluid was collected after 21 days (dietary adaptation) using flexible tube at 0, 2, 4, and 6 h after feeding [14]. About 100 ml rumen fluid was passed through four layers of cheese cloth (autoclave) in laminar air flow to remove particulate matter. Remaining rumen fluid was stored at −80 °C for further study. Total DNA (0, 2, 4, 6 h × 3 animals) were extracted from pooled sample by using a commercially available kit according to the manufacturer’s instructions (QIAGEN Stool kit; QIAGEN, CA). The total DNA mixture was used as a template in PCR to amplify 18S r DNA.

PCR Primers and Amplification

A protozoa-specific forward primer (5′-ACTTTCGATGGTAGTGTATTGGACTAC-3′) was used with a Eukarya-specific reverse primer (5′-ATGATCCTTCTGCAGGTTCACCTAC-3′) [19]. Subsequently, 18S r DNA fragment were amplified by PCR using the metagenomic DNA. A total of 25 μl of reaction mixture consisted of 10 pmol of each primer, 30 ng of template DNA, and 12.5 μl of master mix (Fermentas, UK). The PCR amplification was performed by Thermal Cycler (ABI, USA) and the PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 30 cycles of amplification consisting of a 1-min denaturation step at 94 °C, a 1-min annealing step at 37 °C, a 3-min extension step at 72 °C, and a final 10-min extension at 72 °C. The anticipated product of approximately 1.36 kb was cleaned using a Qiagen DNA Gel Extraction Kits (QIAGEN, CA) in accordance with the directions of the manufacturer.

Cloning and Sequencing

The purified PCR products were cloned in InstaT/A cloning kit (Fermentas, UK) as per the instructions of the manufacturer. The recombinant plasmids after were extracted by the Qiagen mini-prep plasmids extraction kit (QIAGEN, CA). Plasmid inserts were amplified with primers M13F (5′-GTAAAACGACGGCCAG-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′) and nucleotide sequences of cloned genes were determined by sequencing with M13F/M13R primer in ABI Prism 310 Genetic analyser (Applied Biosystems Inc., CA) using BigDye Terminator (version 3.1) at the Animal Biotechnology Laboratory, AAU, Anand, Gujarat, India. Finally a total of 172 clones were sequenced.

Sequence Analysis

All reference sequences were obtained from the GenBank/EMBL/DDBJ/ [2]. Sequences (~550 bp) from the current study were analysed by the CHECK_CHIMERA program [18] to remove any chimera rDNA clone. The similarity searches for sequences were carried out by BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/Blast.cgi [17] and alignment was done using CLUSTAL W (http://www.ebi.ac.uk/Tools/clustalw2/index.html [26]. Ambiguously and incorrectly aligned positions were aligned manually. The distance matrix was calculated using the DNADIST program included in PHYLIP [7] and used to assign sequences in various operational taxonomic units (OTUs) or phylotypes by DOTUR [21] with 95 % confidence intervals to quantify the diversity of phylotypes and total of 53 OTUs were distinguished, based on unique 18S rDNA sequences. The percentage of Good’s coverage was calculated as [1 − (n/N)] × 100, where ‘n’ is the number of single-clone OTUs and ‘N’ is the library size. Phylogenetic tree was constructed by the neighbor-joining method using MEGA 4.0 [25]. Bootstrap re-sampling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies [7].

Nomenclature and Nucleotide Sequence Accession Numbers

The prefix IBRP was used to denote OTU identified and nucleotide sequences have been deposited in the GenBank database under the accession numbers EU345005 to EU345176.

Results

Sequence Analysis

All the sequences (172) were subjected to online homology search in GenBank [2] which implements the BLAST algorithm [17]. The summary of Blast result are given in Table 1. The sequences generated in this study showed 79–96 % sequence similarities with the sequences of protozoa available in the GenBank. The clones sequenced in this study were grouped into 53 OTUs, based on rDNA sequences. Of the 53 OTUs, 12 had >90 % similarity with the 18S rDNA sequences available in the GenBank, 27 had 86–90 % similarity and 14 had <85 % similarity.

Table 1 Similarity values of 18S rRNA sequences retrieved from the rumen fluid of Surti buffaloes
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Phylogenetic Analysis

The phylogenetic analysis of the sequences (Fig. 1) showed that 40 OTUs, representing 124 clones, belonged to unidentified protozoa. Twelve OTUs (44 clones) belonged to the class Kinetofragminophorea. Within the Kinetofragmi nophorea, seven OTUs, representating 27 clones, grouped with Dasytricha ruminantium-like clones and five OYUs representing 17 clones grouped with Isotricha prostoma-like clones. The phylogenetic analysis indicate that the OTU19 (4 Clones) belonged to haptorida. However, Blast results hit with known protozoa i.e. Troglodytella abrassarti. This is due to very low similarity (Table 2). Thus, 18S rRNA sequences obtained from rumen formed tightly-clustered affiliated to the different groups. The total 11 single-clone OTUs, so Good’s coverage (93.75 %) of 18S rDNA libraries indicated that the sequences identified in libraries represent the protozoan diversity in the rumen.

Fig. 1
figure 1

Phylogenetic relationships of partial 18S rRNA sequences of clones recovered from Surti buffalo rumen samples. The rooted tree was inferred by the neighbor-joining method with 1,000 bootstrap replicates using the MEGA 4 tree building program. The Paramecium tetraurelia (X03772) are used as the out-group for rooting the tree. The scale bar represents 5 % sequence divergence. The symbol filled square indicates the reference sequences, open circle indicates Dasytricha ruminantium-like clone, filled circle indicates Isotricha prostoma-like clone, open square indicates haptorida protozoa, and filled triangle indicates unidentified protozoa; reference sequence (AB437347: Troglodytella abrassarti)

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Table 2 Analysis of 18S rDNA phylotypes diversity retrieved from the rumen fluid of Surti buffaloes
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Discussion

The molecular inventory of protozoa revealed in present study showed the occurrence of complex protozoa communities in buffalo rumen ecosystem. The number and distribution of phylotypes indicates the protozoa diversity in rumen of Surti buffalo. Compared to other ecosystems, there is no previous information about the rumen protozoa of Indian buffalo. The relative lack of information on ruminal protozoa may be due to difficulties with isolation, culture, or maintenance. Rumen isolates often lose viability for unknown reasons during purification or sub culturing of pure isolates. More than 24 genera of ruminal protozoa have been described based on cultivation and morphological studies [4, 29]). Although, sequences of few genera are available in sequence databases. Most genera are representatives of typical bovine rumen populations viz Entodinium, Diplodinium, Eudiplodinium, Ostracodinium, Metadinium, Enoploplastron, Polyplastron, Epidinium, Ophryoscolex, Isotricha, and Dasytricha [13, 30]. The present study revealed the phylogenetic diversity of the protozoan community in the rumen fluid of Surti buffalo by analyzing protozoan 18S rDNA sequences. BLASTn searches showed that sequenced clones shared similarity (79–96 %) with ruminal protozoan sequences with GenBank database. The rumen fluid library was classified into three phylogenetic groups. The largest group was affiliated with the unidentified protozoa (40 OTUs, 124 clones), second group affiliated with the Kinetofragminophorea protozoa (12 OTUs, 44 clones), and third the group affiliated with the Haptorida protozoa (01 OTU, 04 clones). The predominant protozoa identified in this study were the unidentified group (Table 2). These results agree with the report of Isotricha and Dasytricha genera in the cattle rumen [13, 16]. High number of Entodinium sp. has been reported by Akbar et al. [1] in Ghizel Sheep fed in pasture and nourished by dried grape by-product and Leng et al. [16] in Yunnan Yellow Cattle fed malt meal. Interestingly, we could not detect the sequences related to Entodinium sp.; this may be due to the diet composition. Dehority and Odenyo [6] suggest that a selection for highly concentrated feed stuffs would lead to an Entodinium only fauna, as they observed in Grant’s gazelle. Karnati et al. [13] reported that their protozoan-specific primers (used here) had a single mismatch with the 18S rDNA of Entodinium sp., but matched exactly with the 18S rDNA sequences of other protozoan species. They suggested that a PCR primer degenerates at the mismatched position would help minimize PCR bias, allowing for more representative retrieval of ruminal protozoan 18S rDNA from complex ruminal samples. The diversities of different clone libraries of rumen protozoa have been given in Table 3.

Table 3 The diversity comparing of different clone libraries of rumen protozoa
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However, Isotricha sp. and Polyplastron sp. were identified in the washed ciliate suspension and many methanogens were detected on ciliate cells by F420 auto fluorescence [27]. Similar results were also observed in our studies. Biochemical differences between Isotricha and Dasytricha have been examined by [11, 12]. Dasytricha is more versatile than Isotricha, fermenting cellobiose, galactose, and maltose. Fermentation products from galactose were the same as those formed from glucose. From glucose, the holotrich produced lactic, acetic, and butyric acids, carbon dioxide, hydrogen, and traces of propionic acid [12]. A single OTU (IBRP19) located within the haptorida protozoa and may represent a dietary transient.

Conclusions

In conclusion, Surti buffalo rumen harbors protozoal community in the rumen that is composed of several genera including, Dasytricha, Isotricha, Ostracodinium, Polyplastron, Cycloposthium), Teuthophrys, and Troglodytella. While majority of the sequences were unidentified. An advanced set of protozoan-specific phylogenetic probes and quantitative real time PCR assay are needed to their distribution throughout rumen microbial communities. Future studies to understand the effects of varying rumen protozoa on different animal feeding habits, digestion and methanogens will also be important.