2011, Vol. 6 No. 1, Article 77

Molecular Characterization and Diversity of Rumen Bacterial Flora in Indian Goat by 16S rDNA Sequencing

Jayesh Kumar Manilal Patel¹, Mayur. K. Jhala², Prashant Soni¹, Nadeem Shabir*¹, P. R. Pandya⁴,
K. M. Singh¹, D. N. Rank³ and C. G. Joshi¹

¹Department of Animal Biotechnology

²Department of Veterinary Microbiology

³Department of Animal Genetics and Breeding

⁴Animal Nutrition Research Station

Anand Agricultural University, Anand (388 001), Gujarat, India

*Corresponding Author; e-mail address: [email protected]

ABSTRACT

Present study was aimed to characterize bacterial communities in goat rumen using a culture-independent approach for a pooled sample of rumen fluid from five adult surti goats. Due to their unique browsing habits including feeding on unconventional tree leaves, goats are expected to host distinct bacterial communities in their rumen. So, 16S rDNA sequences were amplified and cloned from the sample and 60 such clones were sequenced to evaluate the bacterial diversity in goat rumen. Sequence similarity search with Genbank databases revealed that five clones (8%) exhibited similarities with known bacterial species (viz. Ruminococcus albus, Ruminococcus flavefaciens, Prevotella multiformis {2 clones} and Butyrivibrio fibrisolvens) and five clones (8%) showed similarities with known bacterial genera (viz., Ruminococcus, Prevotella, and Bacillus {3 clones}). Taxonomic classification by Ribosomal Database Project (RDP) revealed that the 60 clones mainly belonged to two phyla viz. Bacteriodes (21 clones) and Firmicutes (20 clones), however, 17 clones fell under unclassified bacteria and two clones under unclassified root. Phylogenetic analysis using neighbour-joining method grouped three clones (5%) with species, two clones (3%) with genera, one clone (1.6%) with family, twenty seven clones (45%) with unidentified rumen bacteria and the remaining twenty seven clones (45%) grouped separately and showed distinct genetic grouping compared to the reported sequences in the Genbank database.

KEY WORDS

Goat rumen, 16S rDNA, clones, sequencing and bacterial diversity.

INTRODUCTION

The ruminants are fed on lignocellulosic agricultural by-products (like cereal straw, tree foliage, etc.) and cakes of oil seeds in tropical countries like India. The digestion of such plant materials occurs by virtue of the extensive microbial community, including bacteria, fungi, and protozoa [1], which are found in the rumen and provide the host with nutrients, predominantly in the form of volatile fatty acids and microbial protein. The microbes present in the rumen ecosystem can be classified into 3 domains: Bacteria, Archaea (methanogens), and Eucarya (protozoa and fungi) [2]. Classically, most of the knowledge of the microbial composition of rumen fluid has been derived using traditional methods, such as the roll-tube technique [3] or most probable number (MPN) estimates [4]. The rumen bacteria have been shown by traditional procedures to belong to some 22 predominant species [5]. Rumen microbes have been extensively studied both qualitatively [6] and quantitatively [7] using DNA-based technologies(16s RNA/ 18s RNA gene). These techniques have been further used to construct a library of 16S rDNA clones of rumen microbes and to demonstrate considerable diversity of rumen bacteria. Molecular research on microbial ecology of animals provides a broad perspective of application in the field of animal sciences [8]. India ranks second in the goat population of world contributing 25.6% to the total livestock population of the country [9]. Since Indian goats primly sustain on grazing and crop residues, the microbial population is expected to be more diverse. Thus the identification of the rumen flora, bacteria in particular, seems important in understanding the digestion potential of unconventional feed in goats. Surti is a popular breed of goat found in central and southern Gujarat. Surti goats are of medium size predominantly white in color, with a body weight of 32-35 kg. The present study includes the identification of rumen bacteria in surti goat by amplification, cloning and sequencing of 16S rRNA gene of bacteria, followed by sequence comparison and phylogenetic analysis.

MATERIALS AND METHODS

Sample collection and DNA extraction
The experiment was carried out on five adult surti goats, at approximately 3 years of age and with a mean live weight of 32±3.2 kg. They were reared at Instructional Farm, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand. All the animals were maintained under a uniform feeding regime [10] for at least 21 days. The diet comprised of green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum), non-conventional feed (Azadirachta indica) and concentrate mixture (20% crude protein, 65% total digestible nutrients). Approximately 50 ml of rumen fluid from each goat was collected via a stomach tube located in the middle part of the rumen and connected to a vacuum pump at 0, 2, 4, 6 h after feeding [11]. Samples were pooled and filtered through four layers of cheesecloth to remove particulate matter. Strained samples were used for the total microbial DNA extraction. Total DNA (0, 2, 4 and 6 hrs x 5 animals) extraction was done using Phenol: Chloroform method.
PCR amplification of the 16S rRNA gene
Bacteria specific primers [12]: a forward primer 27F (5’- AGAGTTTGATCMTGGCTCAG - 3’) and a reverse primer 1492R (5’- ATAGGYTACCTTGTTACGACT- 3’) were used. Subsquently, 16S rDNA was amplified using the metagenomic DNA and Master mix (Fermentas, UK). 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) using the following program: denaturing at 95°C for 5 min, followed by 30 cycles of 30 s of denaturing at 95°C, 30 s of annealing at 50°C and 2 min of elongation at 72°C, with a final extension at 72°C for 10 min for the first set. The anticipated product of approximately 1.4 kb was separated by agarose gel electrophoresis and cleaned by using a QIAquick DNA Gel Extraction Kit (QIAGEN, CA) in accordance with the directions of the manufacturer.
Cloning and sequencing
The purified PCR products were cloned in InstaTA cloning kit (Fermentas, UK) according to the instructions of the manufacturer. The recombinant plasmids were then extracted by the Mini-prep Plasmid Extraction Kit (QIAGEN, CA) (Fig. 1). Sequencing was performed for all the clones in the library with an ABI Prism 310 Genetic Analyser (Applied Biosystems Inc., CA) using BigDye Terminator (version 3.1) in the Animal Biotechnology Laboratory, AAU, Anand, Gujarat, India.
Restriction digestion of the colony PCR products (73 clones) obtained from clones was carried out by HAE-III to confirm the identity of PCR products and 12 common clones were removed from further processing to avoid sequencing of common clones (Fig. 2).
Sequence analysis and construction of phylogenetic tree
All reference sequences were obtained from the GenBank/EMBL/DDBJ/RDP [13]. Sequences (~650 bp) from the current study were analysed by the CHECK_CHIMERA program [14] to remove any chimeric rDNA clone. The similarity searches for sequences were carried out by BLAST [15] and alignment was done using CLUSTAL W [16]. Ambiguously and incorrectly aligned positions were aligned manually. Phylogenetic tree was constructed by the neighbour joining method using MEGA 4.0 software [17]. Bootstrap resampling analysis for 500 replicates was performed to estimate the confidence of tree topologies [18].

RESULTS

Sequence Similarity
A total of 60 16S rDNA clones of partial length were isolated from the rumen liquor of the goats. All the clones were subjected to sequence analysis, followed by homology search using databases: the GenBank and the Ribosomal Database Project (RDP) database. In our library, about 91.66% clones (55 clones) had ≥90% similarity to 16S rDNA data sequences from those databases. Furthermore, about 6.66% (4 clones) of the sequences were 85-89% similar to 16S rDNA data sequences, while for the remaining 1.66% (1 clone) the similarity was less than 85% (Table 1 and Table 2). Five clones (8%) displayed similarities with known bacterial species viz. Ruminococcus albus, Ruminococcus flavefaciens, Prevotella multiformis (2 clones) and Butyrivibrio fibrisolvens. Again five clones (8%) showed similarities with known bacterial genus viz., Ruminococcus spp, Prevotella spp and Bacillus spp (3 clones). Only a single clone had similarities with family Porphyromonadaceae (1.6%) and another clone (1.6%) exhibited similarities with phylum Firmicutes. Twenty clones (33.3%) presented similarities with Uncultured Rumen Bacteria (URB), twenty six clones (43.3%) had similarities with Uncultured Bacteria and two clones (3.3%) were similar to Uncultured Equine Intestinal Eubacteria (UEIE).
Phylogenetic Analysis
The collection of cloned 16S rRNA gene sequences of bacteria entailed several major bacterial lineages. Since the similarity for most of the sequences with those of known rumen bacteria was low to identify the sequence as representing a particular species, a phylogenetic tree was constructed to investigate their taxonomic affiliation. The phylogenetic tree was generated using MEGA 4 software to check the taxonomic identities of the 60 clones of our library. The bacterial sequences obtained from the rumen formed tightly clustered, deeply diverging groups affiliated to different bacterial phyla as well as unclassified groups. The phylogenetic tree was mainly divided in to four clusters (Fig. 3). In Cluster I, 27 clones were grouped separately and no cultured or uncultured representatives from GenBank database was grouped with these clones. Cluster II included nine reference sequences retrieved from NCBI nucleotide database which, however, did not group with any of the 60 sequences obtained in the present study. Interestingly, they were grouped adjacent to cluster I in comparison to rest of the clusters signifying lesser genetic distance between cluster I and Cluster II. Cluster III represented 14 clones which were further divided into five subgroups. Sub group-I consisted of three clones (FJ970705, FJ970706 and FJ970683) that clustered together, without showing genetic similarity with any reference sequences. Sub group-II consisted of three clones (FJ970693, FJ970703 and FJ970667) that again did not match with any of the reference sequences. Sub group-III contained one clone (FJ970677) clustered with uncultured group of family Porphyromonadaceae as well as with uncultured rumen bacterium. Sub group-IV included two clones (FJ970664 and FJ970675) clustered with uncultured rumen bacterium. Sub group-V included five clones (FJ970680, FJ970661, FJ970712, FJ970670 and FJ970681) clustered together with uncultured Prevotella as well as unidentified rumen bacterium, of which, the later two clones were at less genetic distance than the first three and thus more similar to the mentioned taxa. Cluster IV represented 19 clones that were further divided in to four subgroups. Sub group-I consisted of clones FJ970659 and FJ970665 that clustered with Ruminococcus albus and Ruminococcus flavefaciens respectively. The same clones were also identified as Ruminococcus albus and Ruminococcus flavefaciens by similarity search and thus confirms their identification. Sub group-II consisted of clone FJ970689 clustered with Butyrivibrio fibrisolvens. The same clone was identified as belonging to genus Butyrivibrio by taxonomic classification at RDP which strengthened its identification as Butyrivibrio fibrisolvens. Sub group-III consisted of clone FJ970700 clustered with genus Bacillus which was also identified up to genus (i. e. Bacillus) level by both similarity search and taxonomic classification and hence validated. Sub group-IV included remaining 15 clones, of which nine clones clustered along with one or many of themselves and six clones clustered with uncultured rumen bacteria.
Taxonomic classification using RDP
All the 60 obtained sequences were subjected to classification by using Taxonomic Classifier software available at RDP. From the highest to the lowest, the major formal taxonomic ranks obtained were domain, phylum, class, order, family, genus and species.
Classification by RDP revealed that 60 clones were mainly distributed into two phyla, namely Bacteroidetes with 21 clones (35.0 %), Firmicutes with 20 clones (33.0 %), 17 clones (29 %) fell under unclassified bacteria and two clones (3 %) were grouped under unclassified root (Table 3).
Nucleotide Sequence and Accession Numbers
The nucleotide sequence data reported in this study can be obtained from the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under Accession numbers FJ970656 to FJ970715 with the generic name of AVCGRB (Anand Veterinary College Goat Rumen Bacteria).

DISCUSSION

Species identification using sequence similarity demands sequences having similarity greater than 98% [19]. In our library, 5 clones were identified as species viz. Ruminococcus albus, Ruminococcus flavefaciens, Prevotella multiformis (2 clones) and Butyrivibrio fibrisolvens. 21 clones belonged to Phylum Bacteroidetes and 20 clones belonged to Phylum Firmicutes. The proportions of bacterial communities in Surti goat rumen are similar to those reported previously in other rumen ecology studies. In a metagenome analysis by pyrosequencing of rumen sample of Angus-Simmental cross steers (Bos taurus) fed a diet of grass-legume hay [20], 62% of sequences were identified as the Firmicutes and 21% of sequences as the Bacteroides fragilis group. A work published [21] reported that 52.4% of clones identified in the rumen liquor of Holstein cow (Bos taurus) fed a diet of hay belonged to the Firmicutes, and 38.1% to the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Another study [22], summarizing the published data for rumen bacteria, reported that on average 54% of rumen bacteria were members of the Firmicutes and 40% were from the CFB phylum. Yuhei et al. [23] found that 81.3% clones represented the Firmicutes, while 14.4% clones belonged to the Bacteroides fragilis group and Actinobacteria and Proteobacteria in fecal microbiota of cattle. A research group in year 2007 [24] assigned 57.1% of clones to the phylum Firmicutes, 42.2% of clones to the CFB phylum, and one clone (0.7%) to Spirochaetes in gayals (Bos gaurus frontalis, syn. B. frontalis) fed a diet composed of fresh bamboo leaves and twigs (Sinarundinaria sp.), with 50.5±3.16 % dry matter, 10.2± 0.40 % crude protein, 38.8±1.17 % crude fiber. Dowd et al. [25], using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), evaluated ubiquitous bacteria from the cattle faeces, which included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophaga, Anaerotruncus, and Acidaminococcus spp. In a similar study [26] conducted on rumen bacterial 16S rDNA of Chinese goat, five clones (31%) showed over 97% similarities with Genbank database sequences while as eight clones (50%) showed similarities in the range of 90-96% and the remaining three clones (19%) were less than 90 % similar to unidentified bacteria. The data is comparable to the present study wherein 77% clones showed similarity in range of 95-99% with Genbank database. Further in their study, one clone (6.25%) belonged to Prevotella spp. ,while as 3 clones (5%) in our study belonged to Prevotella spp. which is again in agreement with the mentioned study. Another study in Surti buffalo [27] revealed that out of 191 clones analyzed, 41 clones (21.4%) belonged to Firmicutes ( Low G+C group), 27 clones (14.1%) belonged to CFB phylum, 13 clones (6.8%) belonged to Spirochaetes, 1 clone (0.5%) belonged to Actinobacteria and 109 clones (57%) belonged to unidentified bacteria. In present study involving goats, Actinobacteria and Spirochaetes groups were not found. This may probably indicate species difference between goat and buffalo in terms of their rumen bacterial flora. However, a study suggested [28] that sample collection time, site, method and technical bias of PCR amplification may affect the results in this kind of experiment, which possibly demands repeated attempts involving these two species to conclude the presence/absence of Actinobacteria and Spirochaetes groups of bacteria in rumen of goats.
Despite the possible deficiencies of analysis, it is concluded that surti goat rumen harbours a diverse range of bacteria. The results are more or less consistent with the previous studies. However, further studies need to be conducted to validate and extend the present observations.

ACKNOWLEDGEMENTS

Authors are thankful to the Department of Biotechnology, Government of India, for the financial assistance provided by them.

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TABLES

Table 1. Similarity values of clones (1-30) based on 16S rDNA sequences retrieved from the rumen fluid of Indian Surti goat

Table 2. Similarity values of clones (31-60) based on 16S rDNA sequences retrieved from the rumen fluid of Indian Surti goat

Table 3. Summary of sequence similarity at 95% confidence level by Taxonomic Classifier (Ribosomal Database Project)

FIGURES

Fig. 1: Agarose gel electrophoresis of M13 PCR products showing recombinant as well as non recombinant clones. +ve clones represent the recombinant clones while as –ve clones represent the non-recombinant clones.

Fig. 2: RFLP by HAE-III digestion. Twelve clones with common banding pattern (clones no. 4, 11, 38, 23, 45, 31, 65, 32, 39, 61, 50 and 63) were removed to screen out the common clones.

Fig. 3: Phylogenetic relationships of partial 16S rDNA sequences of clones recovered from Surti goat rumen fluid samples. The unrooted tree was inferred by the neighbour joining method using the MEGA 4 software. The scale bar equals to an average of 5 nucleotide substitutions per 100 positions. The 16S rDNA sequences of the present study are represented by ‘FJ’. Cluster I involves 27 novel clones that grouped seperately, Cluster II involves 9 reference 16S rDNA sequences from Genbank that did not group with any of the 60 clones, Cluster III and IV involved 14 and 19 clones respectively that grouped or did not group with any of the representative sequences from genbank.