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A metagenomic study of the rumen virome in domestic caprids

  • Samuel Namonyo
  • Maina Wagacha
  • Solomon Maina
  • Lillian Wambua
  • Morris Agaba
Brief Report

Abstract

This project sought to investigate the domestic caprid rumen virome by developing a robust viral DNA isolation and enrichment protocol (utilizing membrane filtration, ultra-centrifugation, overnight PEG treatment and nuclease treatment) and using RSD-PCR and high throughput sequencing (HTS) techniques. 3.53% of the reads obtained were analogous to those of viruses denoting Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, Microviridae, Poxviridae, Tectiviridae and Marseillevirus. Most of the sequenced reads from the rumen were similar to those of phages, which are critical in maintaining the rumen microbial populations under its carrying capacity. Though identified in the rumen, most of these viruses have been reported in other environments as well. Improvements in the viral DNA enrichment and isolation protocol are required to obtain data that are more representative of the rumen virome. The 102,130 unknown reads (92.31%) for the goat and 36,241 unknown reads (93.86%) for the sheep obtained may represent novel genomes that need further study.

Introduction

There is an increasing interest in the microbial community resident in the alimentary canal of vertebrates due to the recognition that they play critical roles in the nutrition and health of their hosts [1]. Research in rumen microbiology has fundamentally involved traditional culture-based procedures, including isolation, enumeration and nutritional characterization, presumably accounting for 10 to 20% of the rumen microbiome [1]. To overcome the bias and deficiencies inherent with cultivation-based methodologies, molecular techniques have been adopted to gain a better understanding of the structure of microbial ecosystems [2]. The most promising of these non-culture based methodologies is metagenomics. This is the analysis of the collective genomes extant in a defined environment or ecosystem, thus giving an insight into functions of non-cultivable microbiota [3]. Initial metagenomic research was used to elucidate the microbial structure of aquatic and soil ecosystems [3].

Rumen phages and archaeal viruses play an important role in the population dynamics of rumen bacteria and archaea by causing lysis [4]. Lysis of crucial bacteria involved in vital metabolic pathways inside the rumen could alter the foregut fermentation and affect the total ruminant performance. However, very little is known about the diversity of phages inside the rumen and their effects on bacteria, archaea and protozoa. Only two previous studies report on rumen virome based on high throughput sequencing of virus enriched samples using the bovines (Bos taurus) as a model [5, 6]. This project sought to add to this with the study focusing on goats (Caprus hircus) and sheep (Ovis aries) species.

The fastq files with the sequences from 454 pyrosequencing were deposited in the Sequence Read Archives under the accession numbers SRR7686970 and SRR7686971.

Materials and methods

Whole rumens from 8 sheep and 8 goats were collected from the Dagoretti abattoir in Nairobi, Kenya. The rumens were separately dissected under aseptic conditions. Using a large stainless steel sieve (approximately 12 cm in diameter), individual samples were sieved into other sterile containers in a laminar flow biological safety cabinet (CSC-chemoflow, Ireland). The filtrates were pooled into large 800 ml centrifugation tubes. The filtrates were then centrifuged at 4,000X g at 10 °C for 30 minutes in an RC-3B refrigerated centrifuge (Sorvall instruments, USA). The supernatants were carefully pipetted from the individual tubes and subjected to another round of centrifugation at 6,000X g at 10 °C for 30 minutes in the Allegra 25R centrifuge (Beckman coulter, USA) using the 5-5.1 rotor.

To enrich for phages in the rumen samples the filtrates were first processed to remove the remaining small debri by centrifugation at 8,000X g at 10 °C for 10 minutes in the 5424 Eppendorf desktop centrifuge (Eppendorf, Germany). The supernatants were then pooled and filtered through a 0.45 µm-membrane filter (Sartorius, Germany) to remove protozoa and most bacteria and subsequently further filtration was done using the 0.22 µm-membrane filter (Sartorius, Germany) to remove the remaining small bacteria. The samples were aliquoted in 1 ml quantities into 2 ml tubes. To each, 20% v/v of chloroform was added, vortexed briefly using the vortex genie 2 (Scientific industries, USA) then mixed well at 37 °C for 15 minutes before being centrifuged at 13,000X g for 10 minutes in the 5424 Eppendorf centrifuge (Eppendorf, Germany).

To precipitate some of the proteins in the solution but not the phages, 2.9% w/v sodium chloride was added and incubated on ice for 30 min. The tubes were centrifuged at 5,000X g for 10 minutes at 10 °C in a 5424 Eppendorf centrifuge (Eppendorf, Germany). The clear supernatant was transferred onto new tubes.

To the supernatant, 25% v/v of PEG (polyethylene glycol) was added to each of the samples and incubated at 4 °C in a refrigerator for 22 hours. Finally the samples were centrifuged at 13,000X g for 30 minutes in a 5424 Eppendorf centrifuge (Eppendorf, Germany). The supernatant was discarded and the phage pellet dissolved in 1 ml phosphate buffer saline (PBS). Nuclease treatment was done by adding 10 units of RNase and 20 units of DNase per 1 ml of the sample, and then followed by incubation in a water bath at 37 °C for 10 minutes to activate the two enzymes. The enzymes were deactivated by addition of 1 µl of EDTA. This was followed by incubation at 65 °C for 10 minutes in a Grant sub aqua 12 water-bath (Grant Instruments, UK).

DNA was extracted from the enriched samples using the Roche high pure viral nucleic acid large volume kit (Roche, Switzerland) according to the manufacturers instructions. The extracted DNA was purified, then submitted to the BecA-Ilri Hub Genomics and Sequencing Unit for sequencing using 454 pyrosequencing (Roche, Switzerland). The sequencing team performed a Eco-R1 Restriction site dependent-PCR using the primer 5’-(t/c)(t/c/g)(t/c)(a/c/g)(a/c/g)(a/t/g)n5GAATTC-3’ and Jiang and Co-workers’ protocol [7] before the library preparation. Quality control was done using 454QC 2.3.2 [8] and the reads analysed using MG-RAST (with the M5nr database) [9] with a maximum e-value of 1e-5, minimum percentage identity cut off of 80% and an alignment length cut off of 15. To corroborate the results obtained by MG-RAST, the taxonomic content of the trimmed and filtered reads obtained above was analysed by MEGAN V (with the NCBI nr database) [10].

Results

Initial trial runs during optimization of the protocol yielded an average of 15.7, 61.7, 91.9, 10.2, 75.8, 14.6, 27.9, 38.3 ng/μl in the goats and an average of 24.7, 4.4, 25.4, 24.9, 4.7, 17.2, 1.9, 4.7 ng/μl in the sheep. Pooling these samples considerably increased the yield to 622.4 ng/μl for the goat and 538.9 ng/μl for the sheep with an absorbance ratio (A280/260) of 1.73 and 1.83 respectively and A260/230 of 1.68 and 1.71 respectively. From an eighth of a 454-pyrosequencing run, a total of 176,375 reads were obtained. Of these, the total reads from the sheep were 45,763 (38,710 high quality reads) and those of the goat were 130,612 (110,608 high quality reads). The high quality reads ranged between 100 bp and 646 bp (86.86%). Though the samples were enriched for viruses and extracellular DNA digested using DNase1, there was still a lot of non-viral DNA. Bacterial hits were 5,973 reads for the goat and 1,897 reads for the sheep, Unclassified but belonging to viruses (goat = 1,046 reads and sheep = 94 reads), Viruses (goat = 1,056 reads and sheep = 108 reads), Eukaryota (goat = 299 reads and sheep = 159 reads), Archaea (goat = 99 reads and sheep = 50 reads) and Unknown (goat = 102,130 reads and sheep = 36,241 reads) (Table 1).
Table 1

The families of viruses whose DNA signature was similar to the reads sequenced from the rumen of the sheep and goat samples enriched for viruses

Family of Virus

Abundance

Goat

Sheep

Marseillevirus

1

Microviridae

2

2

Mimiviridae

5

1

Myoviridae

60

26

Podoviridae

1

5

Poxviridae

2

4

Tectiviridae

1

Siphoviridae

1271

62

Unclassified (derived from viruses)

86

47

Unclassified (derived from Caudovirales)

29

29

Total

1456

179

Among viruses of the order Caudovirales, the Siphoviridae (goat = 94.15% and sheep = 92.79%), Myoviridae (goat = 3.19% and sheep = 2.97%) and Podoviridae (goat = 0.14% and sheep = 0.12%) were the most numerically dominant viral families whose DNA signature was similar to the reads obtained. Sequences bearing similarities with the Tectiviridae and Marseillevirus were detected in the goat but not the sheep (Table 2).
Table 2

The viruses having DNA signatures similar to those of the reads annotated by MG-RAST and confirmed by MEGAN in the sequenced goat and sheep samples

Virus

Abundance

Sheep

Goat

Lactococcus phage 1706

11

520

Rhodococcus phage ReqiPepy6

11

402

Lactobacillus A2

5

43

Bacillus virus 1

36

Bacillus phage Bam 35

1

1

Acanthamoeba polyphaga mimivirus

1

5

Tectivirus

1

Marseillevirus

1

Xanthomonas phage Xp15

2

2

Lactobacillus phage LP65

2

4

Phage ϕ-mru.

7

16

Discussion

Chloroform was applied to stabilize the nucleic acids and to precipitate some of the proteins [11] although sodium chloride (2.9% NaCl with an end concentration of 1.0 M) was used for protein precipitation [12]. Polyethylene glycol concentrates the phages, owing to its ability to induce attractive interactions that crystalize viruses in the interpolymer gaps between the PEG molecules. This augments the yield of the phages [13]. The phage DNA is covered in a tough and highly resistant nucleocapsid [14] and was not degraded during the nuclease treatment. Rosseel [15] noted that a combination of 454 pyrosequencing with various forms of PCR amplification before library preparation is efficacious in viral metagenomic studies. Although we used RSD-PCR [7] because of its low cost and ease of execution, results were skewed towards reads having EcoR1 site as opposed to other restriction sites that may have been be present along the sequence length thus reducing the overall sequence coverage.

HTS techniques have proved to be invaluable in studying the viral metagenomes of complex environments in mammalians such as the bovine rumen or human gut, that have a wide assortment of interacting microorganisms [5, 16, 17] The potential of metagenomics was noted forehand as a tool for studying complex communities such as the soil and ocean [18, 19]. Microbes, specifically viruses, from these environments have nucleotide sequences with little or no similarity to the sequences present in viral databases [17, 20]. This has been noted by Rohwer [20] and Berg Miller and coworkers [5] as being typical of viral metagenomic studies. They further noted that this was due to the vast diversity of viruses and their underrepresentation in the currently available databases. They also hypothesized that the majority of viral sequences reside in this large pool of sequences that do not have any significant matches in these existing databases. Trials with the GS De Novo Newbler Assembler [21] and CLC genomics workbench 10.5 https://www.qiagenbioinformatics.com/ to generate contigs had a low yield. We speculate that the relatively low number of reads could have played a part in this, since assembly requires overlap of reads and thus over-sampling [22], which was a short coming in this study. This method of assembly was thus discarded and subsequent downstream analysis was carried out using the individual reads.

Most of the reads were similar to phages that had been isolated in other environments than the rumen. Previous studies on the bovine rumen also showed a large variation [5] which could be attributed to external sources such as diet, lactation state, location and time of sampling [23]. Most of these reads are analogous to those of viruses that have been shown to be present in the feed and water of these animals or that attacking the normal flora in their mouth and alimentary canals.

There is a big difference in the number of the total reads obtained after sequencing for the sheep and goat samples as well as from the Siphoviridae. We cannot explain why this is so or if this variation is the norm between goats and sheep in general because there were no replicates for comparison since the samples were pooled. However, we speculate that the feeding habits may have had a role to play in this case. The goat (a browser) has a more diverse feeding habit than the sheep (a grazer) and would thus have a richer phage population gathered from these sources.

Though the diet of the animals used in this study was not determined forehand, the reads that map to those of phages targeting specific species of bacteria present in certain feeds can give an insight into the feeding characteristics of the studied caprines and ovines (Table 3).
Table 3

Some of the viruses or phages that were found are analogous to hosts and environments they were previously isolated in other studies

Virus/Phage

Host

Other environments isolated

Putative role

Lactococcus phage 1706

Lactococcus lactis

Raw\skimmed milk [24]

Phage

Lactobacillus phage LP65

Lactobacillus plantarum

Fermented vegetables, silage, alimentary canal of mammals [25]

Phage

Xanthomonas phage Xp15

Xanthomonas campestris

Black rot of many plants [26]

Phage

Bacillus phage Bam35

Bacillus thuringiensis (Bt)

Soil, leaf surfaces, flourmills and grain storage facilities [27]

Phage

Acanthamoeba polyphaga mimivirus

Acanthamoeba polyphaga

Water [28]

Amoeba virus

Phage ϕ-mru

Methanobrevibacter ruminantium M1

Bovine rumen [29]

Prophage

Lytic phages play an important role in regulation of bacteria and archaea while prophages play an important role in horizontal gene transfer [5]

Previous investigations into the rumen ecosystem have shown that bacteriophages constantly interact with microbial populations [5, 30] and is indicative of the important roles that viruses play in controlling microbial communities [5]. Comparisons of rumen metagenome sequences and the reference genome sequences to the rumen virome have shown that there are numerous sequence similarities detected between these and prophages [5].

As found in other studies [5, 6, 15] significant contamination with background DNA (bacterial and eukaryotic) has been observed. Phages are 10 times more prevalent than bacteria in most environments but only account for 2 to 5% of the total DNA due to their small genome size [30]. Edwards and co-workers estimate that approximately 60% of the annotated bacterial genome sequences contain a minimum of one proviral sequence and that on average about 3% of the bacterial DNA originated from a virus [31]. Contamination with bacterial DNA is therefore advantageous in the study of these prophages since studies by Qin and co-workers [32] noted phages’ preference of the temperate lifecycle in the human gut, in direct opposition to the active kill-the-winner viral-bacterial dynamics in the marine ecosystem. Berg Miller and co-workers [5] corroborate this report that the number of prophages’ is almost double that of the lytic phages in the bovine rumen ecosystem.

Reads that are similar to those of Phage ϕ-mru, an archaeaphage specific to the methanogen Methanobrevibacter ruminantium M1 were also present. Methanogens produce a lot of methane (CH4) a potent green house gas [33]. Ruminants contribute approximately 18-20% of the global methane produced each year [34]. Furthermore, 2-15% of these animals’ squandered ingested energy solely as methane, representing a significant inefficiency in nutrient conversion and utilization [35, 36]. If novel methanophages could be isolated, enriched or engineered they could help controlling these nuisance methanogens.

Conclusion

Though this study only utilized an eighth of a 454 pyrosequencing run, the data obtained provided an insight into the possible viruses inhabiting the rumen. However, this study as well as others of the bovine rumen [5, 6] revealed considerable contamination with non-viral DNA sequences as well as sequences showing little or no homology to those available on public databases. This necessitates ameliorated rumen virus enrichment and viral DNA extraction protocols so that the data obtained could be more representative of the rumen virome. The majority (92.31% for the goat and 93.86% for the sheep) of the reads (goat = 102,130 reads and sheep = 36,241 reads) are unknown, and some of these may be novel viral genomes absent in public databases. This opens up a potential research domain to understand not only the abundance and diversity but also the role rumen viruses play in their ecosystem. The annotated roles are general for most phages and further insight is needed to specifically elucidate their roles in the rumen ecosystem. More light could be shed on large-scale lysis of bacteria and archaea involved in beneficial processes in the rumen including digestion and energy production. An improved understanding of rumen viral diversity and viral gene pool in the gut microbiome could probably transform livestock feeding regimes in order to maximize feed efficiency and increase animal performance and might lead to a beneficial and healthier gut ecosystem.

Notes

Acknowledgements

We thank BecA-ILRI Hub, its staff and that of the University of Nairobi for their support.

Compliance with ethical standards

Funding

Funding was provided by the Africa Biosciences Challenge Fund which is financed by The Syngenta Foundation for Sustainable Agriculture, The Bill & Melinda Gates Foundation, The Australian Agency for International Development and The Swedish Ministry for Foreign Affairs through the Swedish International Development Cooperation Agency.

Conflict of interest

Samuel Namonyo, Maina Wagacha, Solomon Maina, Lillian Wambua and Morris Agaba declare that they have no conflict of interest.

Ethical approval

All the rules and guidelines in the treatment and handling of the animals used in this study were followed as outlined by the ministry of livestock (Kenya) and the International Livestock Research Institute.

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Samuel Namonyo
    • 1
    • 2
  • Maina Wagacha
    • 1
  • Solomon Maina
    • 2
  • Lillian Wambua
    • 1
    • 4
  • Morris Agaba
    • 2
    • 3
  1. 1.The University of NairobiNairobiKenya
  2. 2.BecA-ILRI HubNairobiKenya
  3. 3.The Nelson Mandela African Institute of Science and TechnologyArushaTanzania
  4. 4.International Centre for Insect Physiology and EcologyNairobiKenya

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