Phylogenetic analysis of protozoa in the rumen contents of cow based on the 18S rDNA sequences

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.To process fibrous plant materials, the rumen harbors a collection of diverse microorganisms, including bacteria, archaea, fungi, and protozoa (reviewed in references 33 and 45). While the diversity and functions of the thousands of microbial species of this unique ecosystem (32) are interesting from both the evolutionary and functional perspectives, the rumen also represents a rich resource of enzymes for converting lignocellulosic feedstocks into biofuel (35, 43) and other applications (19). A range of inexpensive, robust enzymes with a broad range of specificities will likely be required for efficient industrial processing of highly complex plant polysaccharides. Identification of such enzymes that microorganisms use to break down plant materials has been greatly facilitated by metagenomics (42), both in the form of activity-based screens (20, 52) and also through increasingly powerful, high-throughput genomic DNA sequencing approaches (28, 57). As evident in numerous studies (28,39,41), metagenomics has proven to be particularly effective for identification of carbohydrate-active genes of fiber-adherent bacterial species of the rumen.In addition to bacteria and archaea, the rumen also hosts eukaryotic species, namely, anaerobic fungi and ciliate protozoans (reviewed in reference 33). Addressing the function of ruminal protozoans, in particular, has been a challenge due to the difficulty of maintaining these organisms in axenic cultures (55). Thus, assessing the diversity and dynamics of ruminal protozoans has been addressed historically in morphogenic studies (reviewed in reference 12) and by molecular phylogenetics (e.g., by using 18S rDNA markers [47]). Ruminal protozoans are known to contribute to fiber degradation in their hosts (21), and determination and characterization of their ability to directly process plant material have been addressed by diverse strategies, such as direct, biochemical detection of specific fibrolytic enzymes (e.g., cellulases) in extracts derived from individual protozoan species (38, 54), by molecular cloning studies to directly identify genes encoding enzymes capable of degrading cellulose or hemicellulose (49, 50) and, most recently, by sequencing of protozoan-derived expressed sequence tag (EST) libraries (41). Early studies to establish the capacity of protozoan ...

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confidence: 99%

Activity-Based Metagenomic Screening and Biochemical Characterization of Bovine Ruminal Protozoan Glycoside Hydrolases

Findley

Mormile

Sommer-Hurley

et al. 2011

“…However, there are some drawbacks to using microscopiccounting methods to quantify rumen protozoa, such as cell lysis, sensitivity, and variation in sample consistency. Furthermore, there is evidence that separation of rumen fluid from the solids can be misleading, with regard to both total numbers and generic distribution of rumen protozoa (7,27). For these reasons, we have developed a real-time PCR assay to quantify Entodinium (and Dasytricha) populations in total rumen contents.…”

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confidence: 99%

Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet

Skillman

Toovey

Williams

et al. 2006

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR () was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R 2 ‫؍‬ 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10 0 and 10 6 entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P ‫؍‬ 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.Ciliates are the most abundant protozoa found in the rumens of both domesticated and wild ruminants. Rumen ciliates are involved in host metabolism and digestion of plant material (37) and play an important role in the rumen microbial ecosystem by producing hydrogen as a by-product of plant digestion. The hydrogen is then used by methanogenic archaea (i.e., methanogens) to reduce carbon dioxide to methane, a potent greenhouse gas. Removal of protozoa from the rumen (i.e., defaunation) has been shown to reduce methane emission by an average of 13% (14). A more efficient use of nutrients in ciliate-free animals, especially when given poor diets that limit animal production, has also been reported (11). Because of the current interest in methane mitigation (15), it is likely that methods to accurately quantify protozoa in the rumen will become increasingly important. However, estimating population sizes in the rumen is difficult because microbial populations fluctuate dramatically during the day (21) and between animals (19) and because of sample heterogeneity.Rumen ciliates have complex growth requirements, and most are therefore difficult to culture. Most previous studies have used microscopic counts to enumerate protozoa in rumen samples (7), but these methods may underestimate protozoal populations due to the tendency of some species to lyse or settle during sample collection and processing. Because of their large size (15 to 250 m) and visible internal structures, it is easier to identify protozoa than bacteria, for example, by microscopic observ...

“…Most studies of rumen ciliate protozoa are performed using microscopy and traditional culturing techniques (2, 4-10), quantitative PCR (11, 12), denaturing gradient gel electrophoresis (13), and full-length 18S rRNA clone libraries (13,14). A few studies of rumen ciliate protozoa use highthroughput sequencing, although primer selection remains a problem, as some studies use universal eukaryotic primers, primers which target only one ciliate protozoon signature region, or primers which produce long amplicons that are unsuitable for current high-throughput technology (15)(16)(17)(18)(19)(20).…”

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confidence: 99%

Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

Ishaq

Wright

2014

Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen. Rumen ciliate protozoa represent important functional members of the rumen environment, as most have some cellulolytic or amylolytic abilities (1-3). Most studies of rumen ciliate protozoa are performed using microscopy and traditional culturing techniques (2, 4-10), quantitative PCR (11, 12), denaturing gradient gel electrophoresis (13), and full-length 18S rRNA clone libraries (13,14). A few studies of rumen ciliate protozoa use highthroughput sequencing, although primer selection remains a problem, as some studies use universal eukaryotic primers, primers which target only one ciliate protozoon signature region, or primers which produce long amplicons that are unsuitable for current high-throughput technology (15)(16)(17)(18)(19)(20).18S rRNA genes range from 1.5 kb to more than 4.5 kb (21), and in rumen ciliate protozoa, they are generally 1.5 kb to 1.8 kb in length. Like the 16S rRNA genes of prokaryotes, the 18S rRNA genes of eukaryotes have nine hypervariable regions (V1 to V9) which can be used for genus/species identification. Four gut ciliate signature regions exist within rumen protozoal 18S rRNA genes which represent areas of high variability that can improve identification down to the species level (22, 23). Signature region 1 occurs between bp 440 and 460 (within V3), signature region 2 occurs between bp 590 and 620 (between V3 and V4), signature region 3 occurs between bp 1220 and 1260 (within V6), and signature region 4 occurs between bp 1560 and 1580 (after V8) (Fig. 1). Additionally, rumen ciliate protozoa have a slightly different 18S rRNA secondary structure from nonrumen ciliates, in that rumen protozoa are missing helix E23-5 from the V4 region and other hel...