Recipient Organization
AGRICULTURAL RESEARCH SERVICE
1815 N University
Peoria,IL 61604
Performing Department
Office of the Area Director
Non Technical Summary
Sustainable dairy and beef production are a major goal of US agricultural research. In order to increase producer profits and reduce environmental impacts, we must identify inefficiencies in the system and provide data-driven recommendations for animal husbandry. Much progress has been made in the fields of cattle genetics and animal nutrition towards sustainable production; however, a major component of beef and dairy systems remains largely uncharacterized: the rumen microbiome. Responsible for all of the digestion of feed and plant materials for the host cow, the microbes of the rumen represent a composite of thousands of different species each with different impact on the cow's feed efficiency. Bacteria are typically the "digesters" of fiber in the rumen, whereas archaea are the primary methane producers -- the greenhouse gas that is over 35 times more potent than carbon dioxide. Mixed in with bacterial and archaeal cells in the rumen microbiome are ciliated protozoa cells that navigate rumen fluid to consume bacteria, plant materials or chemicals.Being approximately 100 times larger than the average rumen bacterial cells, these protozoa are the equivalent of "whales" in the ocean of the rumen digesta; however, we know very little about their biology and impacts on the system. There are many signs that the protozoa are more diverse in function and genetics than we have presently observed, but we lack sufficient tools to identify differences in each species on a genetic level. Unlike the bacteria, protozoa are not essential to cattle rumen function so they can be removed from the system without major deterimental effects to the cow.Observations have been made that protozoa increase the amount of methane emitted by the cow, but only anecdotal evidence exists as to why this is the case. To begin to unravel the impacts of protozoa on cattle rumen function, we need to develop genetic tools to identify them in the cow and her environment. Using the latest in DNA sequencing technologies, we plan to create detailed genetic maps of each protozoa species so that they can be investigated for their unique chemical and biological impacts on the system. If successful, we believe outcomes from our work will have impacts on dairy and beef production efficiency by reducing greenhouse gas emissions and converting them into improved nutrition for the cow herself.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
0%
Developmental
50%
Goals / Objectives
Rumen ciliated protists have measurable impacts on the production efficiency and greenhouse gas emissions of dairy and beef cattle. However, the exact metabolic contribution of the myriad species of protists on animal digestion remains uncharacterized due to the lack of suitable genetic tools to study them in detail. Short-read sequencing data under-samples the low GC content, highly repetitive genomes of rumen protists, making them recalcitrant to traditional metagenomic assembly surveys. In order to address this problem, we will sequence centrifuge enriched samples of rumen protists with the latest in long-read DNA sequencing technologies (ONT and PacBio). Using the latest in genome assembly algorithms, we will create reference-quality genome assemblies of individual protist species. We will then mine the resultant genome assemblies for insights into the metabolic contributions and symbiotic relationships with rumen prokaryotes that individual protist species provide to the rumen. Finally, we will generate species-specific PCR assays to faciliate the accurate classification of rumen protists in future high-throughput surveys. We will then use these markers in a proof-of-principle study to identify environmental reservoirs that may contain protist populations that infect Holstein dairy cattle. These markers will be compared to the protist profile of the rumen of each cow to estimate the accuracy of the assay and the transmissibility of the protist profile to the environment.In summary, our project consists of the following specific objectives:Generate high quality reference genome assemblies for ruminal protozoa species using the latest in DNA sequencing technologies.Identify correlations between rumen protozoa species and prokaryotic populations in the rumen, and classify potential downstream metabolic implications.Develop species-specific biomarker assays for rumen protozoa classification.The products of this project are likely to be used in all future rumen metagenomic surveys and may prompt the creation of novel therapeutics or management techniques to manipulate the rumen protist community for reduced methane emissions.
Project Methods
We will generate rumen protozoa samples from Holstein dairy cows housed at two different locations near Madison, Wisconsin. To collect protozoa samples sufficient for rapid formalin fixation and subsequent sequencing, we will sample cannulated dairy cows at the University of Wisconsin Dairy Research herd attached to the University of Wisconsin, in Madison.In order to preserve potential biological interaction between protozoa and prokaryotes, half of the volume of our rumen samples was immediately fixed with a 5% formalin solution as previously recommended (Burton et al., 2014). Fixation will be conducted within minutes of drawing samples from the rumen cannula with the explicit intent to preserve bacterial DNA within protozoa lysosomes or food vacuoles. These samples will be sent to Phase Genomics (Seattle, WA) for Proximeta library creation and sequencing. Raw Hi-C reads will be requested from the service provider for use in downstream analysis, as has been done previously by our group.To provide a suitable foundation to improve these assemblies, we will sequence existing protozoa enrichment DNA samples with PacBio Sequel CCS technology to generate high fidelity (HiFi) reads with lower error rates. We will then assemble HiFi reads into contigs using the latest version of metaFlye (Kolmogorov et al., 2019), which uses a graph-based approach to assemble reads into contigs. We will then co-assemble the HiFi contigs with our existing ONT read dataset to expand coverage and separate genetically similar protozoa species into resolved assemblies. We will then polish these assemblies with existing Illumina whole genome shotgun (WGS) reads generated from each of these cows using Pilon. Finally, we will bin contigs into protozoa assembled genomes (PAG) using Hi-C inter-contig link data from the Proximeta pipeline (Stalder et al., 2019) and tetranucleotide frequency metrics from MetaBat (Kang et al., 2015). Prokaryotic- and Eukaryotic-origin bins will be separated, and the former will be subjected to dereplication using the DAS_tool (Sieber et al., 2018) pipeline.The paucity of other rumen protozoa assemblies will present a challenge for assembly validation, so read alignment metrics from similar datasets (Bickhart et al., 2019; Stewart et al., 2019) will be used to identify misassemblies on protozoa contigs. In order to prevent the removal of legitimate horizontal gene transfer sequence, we will identify chimeric sequence using base-modification data from ONT read datasets and anomalies in Hi-C contig link alignment data. Base-modification data will be generated using DeepMod (Liu et al., 2019a) and will be used to identify genes that show a higher proportion of methylated adenine than cytosine, thereby indicating signatures of bacterial maintenance as opposed to host protozoal control. Additionally, Hi-C link alignments that show a higher proportion of inter-contig to intra-contig link connections within the suspected chimeric region will provide further evidence of potentially chimeric sequence.Concurrently with our metabolic analysis of genome content, we will select genomic loci suitable to distinguish protozoa species in subsequent amplicon-sequencing experiments. The multi-copy nature of the 18S rDNA locus in many protozoa species presents a substantial barrier to protozoa strain-separation and relative abundance estimation using typical ribotyping methods (Schloss et al., 2009). With the generation of high quality references for individual PAGs, we will have the opportunity to identify putative single-copy genomic loci that are present in all bins or a taxonomically-relevant subset of bins (ie. species of ciliate protozoa). The work proposed in this aim will result in the creation of species-specific single-locus or multi-locus sequence assays to distinguish between protozoa populations present in animal fluid samples or environmental swabs. As a proof of principal study, we will apply these assays to environmental samples taken around infected animals to attempt to answer open questions regarding protozoa transmission from infected to uninfected animals.Primers developed in this project will be tested on buccal swabs and rumen samples taken from a cohort of eight cannulated Holstein cows that were not previously sampled to generate the original protozoa assemblies. Swab (Kittelmann et al., 2015) and cannula (Stevenson and Weimer, 2007) samples will be collected and DNA will be extracted as previously described. First round comparisons will focus on the ability of primer-gene sets to generate operational taxonomic unit (OTU) counts that correspond to visual inspection data and are at least as discriminatory as 18S sequence data counts. Illumina WGS data will be aligned to protozoa assemblies generated in aim 1 to estimate the relative abundance of each species based on sequence read depth. These estimates will be compared against amplicon sequence-derived relative abundance values from each primer-gene set to determine the correlation of results between the two methods. Suitable primer-gene sets will be chosen based on their correlation with visual inspection counts and WGS data read-depth data. Phylogenies from variation discovered in amplified SCG regions will be constructed using neighbor-joining trees.We plan to conduct a proof-of-principle survey of the potential environmental vectors for protozoa infection that may inform future dairy management decisions. Validated markers will be used to test for the presence of protozoa samples in buccal swabs taken from ruminating cows and from environmental samples within the study site and immediately outside. Specific locations that will be swabbed at the study site will include the water trough, pen bars and bedding near each of the 8 cannulated cows.Prevalence estimates of rumen protozoa from these environmental samples will be compared back to the prevalence estimates derived via rumen cannula samples to identify if the cow's profile matches those of her immediate environment. Additionally, we will assess rumen protozoa contents of rumen cannula samples via light microscopy.Finally, swabs will be taken on barn external doors and in five equally spaced locations in designated pasture areas to identify the presence of rumen protozoa outside of the study areas. Attempts will be made to identify prevalence of specific genera of protozoa in each area, or to determine if there is a surface-specificity of colonization.