The rumen microbiome is a dynamic environment consisting of bacteria, protist, and fungi responsible for fiber degradation. Advances in molecular techniques have enabled description of bacterial microbiome via pyrosequencing. Cattle consuming low-quality forage are often supplemented with protein to increase forage intake and digestion, but effect on the rumen bacterial community is unknown. Thus, increasing post-extraction algal residue (PEAR) and cottonseed meal (CSM) supplementation was provided to steers consuming oat straw to observe the rumen microbiome within the liquid and solid fraction. Weighted UniFrac analysis indicated different fraction-associated communities with greater similarity across treatments in the solid fraction. Bacteroidetes was the predominant phyla detected in all samples (>65%). Within Bacteroidetes, Prevotella was the most abundant genus. In the liquid fraction, Lachnospiraceae, Ruminococcaceae, and Clostridiaceae increased with PEAR provision (P < 0.05). Similar proportions of bacteria between unsupplemented control and CSM supplemented steers indicate factors other than N supply may impact ruminal bacteria populations. A second experiment evaluated the effect of supplemental CSM or dried distillers’ grain (DDG) for Brahman steers consuming rice straw. Total digestible OM intake and total tract OM digestion increased linearly with additional CSM and DDG provision (P < 0.01). Provision of CSM increased ruminal ammonia linearly (P < 0.01), but DDG supplementation resulted in a quadratic response (P = 0.02). Overall, protein supplementation may increase LQF utilization via changes in the rumen microbiome.

Previous research has documented shifts in microbial hindgut populations resulting from dietary starch inclusion, and recent evidence indicates only 30% of equine cecal contents has been cultured successfully. Next generation sequencing (NGS) techniques allows detection of new species previously undetected. Therefore, the objective of this study was to determine community profiles equine cecal microbiota in response to abrupt dietary starch inclusion. Seven cecally cannulated Quarter horse geldings (497 to 580 kg) were utilized in a crossover design with two 28 d periods and a 28 d washout between each. Horses were randomly assigned to dietary treatments consisting of commercial concentrate offered individually at either 0.6% (LS) or 1.2% BW (HS; as fed) daily divided into 2 meals at 12 h intervals. Prior to start of each period horses were allowed ad libitum access to coastal bermudagrass (Cynodon dactylon) hay and concentrate was fed on d 1 with no adaptation. Samples of cecal fluid were collected on d 1 prior to 0 h and 3, 6, 9, and 12 h post morning meal and on d 1, 2, 3, and 7 at 6 h post morning meal. Cecal pH was determined immediately and a samples of cecal fluid were stored. Genomic DNA was extracted and the V4-V6 segment of 16s rRNA gene was PCR amplified using universal Eubacterial primers 530Fand 1100R and sequenced on the Roche 454 FLX platform. The reads were denoised, chimera checked, and Operational Taxonomic Units (OTUs) were identified using the reference Ribosomal Database Project 16S rRNA dataset. Data were analyzed using PROC MIXED procedure of SAS. Bacterial phyla were largely unaffected by dietary treatment for the first 12 h after the initial concentrate meal except for Verrucomicrobia which was greater in LS horses (P ≤ 0.04). Regardless of treatment, Bacteriodetes increased (P ≤ 0.02) over the first 12 h following initial addition of dietary starch. Adaptation to dietary treatments over 7 d resulted in decreased numbers of Tenericutes (P ≤ 0.07) in HS horses compared to LS. Cecal environment and microbial populations were altered after abrupt and long term exposure to dietary starch.

Clonal diversity in cancer has been proposed as a mechanism underlying patient-to-patient variability in therapeutic response, as well as the variability in the likelihood and the time to relapse of acute myeloid leukemia (AML) and other cancers. As a neoplasm develops it often continues to mutate, diversifying into differing clonal populations. Darwinian evolutionary pressures such as inherent fitness imbalances, immune system interactions, and chemotherapy treatments target sensitive clones and drive competition between the clonal populations; selecting for dynamic and resistant cell lines. In this way clonal diversity is conceivable as an impediment to a complete remission with more populations offering more opportunities for therapy resistance. Bulk next generation sequencing (NGS) is currently used to assess clonal composition in leukemia but requires several broad assumptions be made, which can result in incorrect assessments of diversity. Factors such as differences in zygosity of mutations, convergent evolution, or contamination with wild-type/non-cancerous cells can artificially raise or lower reported variable allele frequencies (VAF), leading to errors in clonal assessments. To examine discrepancies between the actual clonal structure and the clonal structures determined through bulk sequencing we developed a novel method of sampling the cell population to identify concurrent mutations. We first created an in silico model which randomly draws cell samples from a simulated tumor multiple times and calculates the VAF for each mutant allele in each sample. By tracking the correlation of mutations between sample replicates, a clonal composition that is not observable from the bulk NGS VAF becomes apparent. We then created in vitro model tumors from AML cell lines, isolated low cell number samples via flow cytometry, and applied a multiplex/nested PCR protocol with pyrosequencing to quantify VAFs in each sample. Again, by calculating the correlation of mutant alleles between replicates, previously unseen with NGS characteristics of the clonal structure becomes evident. Population sampling analysis may potentially offer a solution for clarifying how we can interpret NGS clonal analyses.

ABSTRACT Two experiments were conducted to determine the effects of various forage to concentrate ratios on the rumen microbial ecosystem and rumen fermentation parameters using culture-independent methods. In the first experiment, cattle were fed either a high concentrate (HC) or a high concentrate without forage (HCNF) diet. Comparison of rumen fermentation parameters between these two diets showed that duration of time spent below pH 5.2 and rumen osmolality were higher for HCNF. Calculations using Simpson’s index showed a greater diversity of dominant species for HCNF than in HC based on 16S rRNA PCR-DGGE. Real-time real-time PCR showed populations of Fibrobacter succinogenes (P=0.01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were present at higher (P≤0.05) concentrations in solid than in liquid digesta in both diets. The second experiment compared cattle as they adapted from a strictly forage to a concentrate diet, after which they were subject to an acidotic challenge and a recovery period (Forage, Mixed Forage, High Grain, Acidosis and Recovery). A total of 153,621 high-quality bacterial sequences were obtained from biopsied rumen epithelium, and 407,373 sequences from the solid and liquid phases of rumen contents. Only 14 epithelial genera representing >1.0% of the epimural population differed (P ≤ 0.05) among dietary treatments. However, clustering showed a closer relation in bacterial profiles for the Forage and Mixed Forage diets as compared to the High Grain, Acidosis and Recovery diets. Several epithelial identified genera including Atopobium, Desulfocurvus, Fervidicola, Lactobacillus and Olsenella increased as a result of acidosis. However, any changes in bacterial populations during the acidosis challenge were not sustained during the recovery period. This indicates a high level of stability within the rumen epimural community. An epithelial core microbiome was determined which explained 21% of the enumerable rumen population across all treatment samples. Cluster analysis of the solid and liquid phase rumen bacterial showed that these populations differed (P ≤ 0.10) between forage and grain-based diets. Rumen core microbiome analysis found 32 OTU’s representing 10 distinct bacterial taxa in whole rumen contents for all dietary treatments. Heifers that developed clinical acidosis vs the subclinical acidosis showed increases in the genera Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Variation in microbial taxa as an effect of both treatment and animal was evident in the solid and liquid fractions of the rumen digesta. However, impacts of a dietary treatment were transient and despite an acidotic challenge, rumen microbiota were able to recover within a week of perturbation. The bacterial populations in the rumen are highly diverse as indicated by DGGE analysis and showed clear distinction between not only dietary treatments, individual animals, but also between epithelial, liquid and solid associated populations on the same diet. Molecular techniques provide an increased understanding of the impact of dietary change on the nature of rumen bacterial populations and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.

Environmental microbiology has traditionally been performed using culture-based methods. However, in the last few decades, the emergence of molecular methods has changed the field considerably. The latest development in this area has been the introduction of next-generation sequencing, including pyrosequencing. These technologies allow the massively parallel sequencing of millions of DNA strands and represent a major development in sequencing technologies. The purpose of this study was to use both pyrosequencing and traditional culture-based techniques to investigate the diversity and dynamics of bacterial populations within milk and untreated sewage sludge samples. Pasteurized and raw milk samples were collected from grocery stores and dairies within Texas. Milk samples were analyzed by plating, pyrosequencing, and an assay for the presence of cell-cell signaling molecules. Samples were processed, stored, and then evaluated again for spoilage microflora. The results of this study showed that raw milk had a considerably higher bacterial load, more diversity between samples, and a significantly higher concentration of pathogens than pasteurized milk. Additionally, this study provided evidence for varying spoilage microflora between raw and pasteurized milk, as well as evidence for the production of cell-cell signaling molecules by bacterial organisms involved in milk spoilage. Four samplings of untreated sewage sludge were collected from wastewater treatment plants in seven different municipalities across the United States. Samples were subjected to quantification of selected bacterial organisms by culture and a pyrosequencing analysis was performed on extracted community DNA. The results of this study showed that untreated sewage sludge is inhabited by a huge diversity of microorganisms and that certain municipalities may have distinct bacterial populations that are conserved over time. Additionally, this study provided some evidence for seasonal differences in several of the major bacterial phyla. Lastly, this study emphasized the challenges of comparing results obtained by culture and pyrosequencing. In conclusion, this study showed that both milk and sewage are highly diverse, dynamic environments that can contain organisms of public health concern. The use of both culture-based methods and pyrosequencing in this study proved a complementary approach, providing a more comprehensive picture of both microbial environments.

Microorganisms found in the indoor environment of retail stores are not well studied, despite potentially serious human exposure implications. In this study, filters from central heating, ventilation, and air-conditioning (HVAC) units were used to collect a time-integrated sample of the airborne indoor microbiome in 14 U.S. retail stores. The microbial communities recovered from the filter dust samples were analyzed with pyrosequencing to characterize the fungal and bacterial microbiome present. The objectives of this work were to: (1) characterize the microbial communities present in retail stores, (2) delineate relationships between the indoor microbiome and building or environmental parameters, and (3) evaluate the use of HVAC filter dust as a sample location for indoor microbial investigations. The microbiome in retail stores was observed to be diverse with 4,771 and 1,577 unique operational taxonomic units for bacteria and fungi, respectively. The diverse microbial community detected over time in the same store could not be fully explained by seasonal trends. That indicates that even when utilizing a long-term sampling approach like HVAC filter samples, the indoor microbiome cannot be completely characterized by a single sampling event. The bacterial community in retail stores was influenced by the outdoor microbiome and microbiota commonly associated with human skin. Physical location of the retail stores had some influence on the bacterial microbial community present and strongly impacted the fungal community recovered. Air exchange rate did not influence the observed bacterial or fungal communities. In a comparison to five other indoor air samplers, passive samplers including HVAC filter dust and settled dust captured a higher diversity of the microbial community. The collection of a more diverse sample may allow detection of potentially pathogenic microorganisms. However, there was significant difference in the microbial community structures recovered between samplers in the same sampling event which suggests sampling methodology has an impact on the inferred microbiome recovered. The overall results from this study indicate that retail stores harbor a diverse microbial community that varies over time. / text

Biological mechanisms that potentially contribute to residual feed intake (RFI) have not been fully understood in the grazing animal. The objective of this study was to determine the differences of RFI measured in confinement (RFIc) or grazing (RFIg) on animal performance. Animals were previously classified in confinement as high RFI (HRFIc), or low RFI (LRFIc) and subsequently under grazing as high (HRFIg) or low (LRFIg). Effects of forage quantity on dry matter intake (DMI), and biological mechanisms that contribute to variations in RFI were investigated using ultrasound, carcass traits and bacterial populations. Bulls were allotted to replicate bermudagrass pastures at low (LSTK) or high (HSTK) stocking intensities and heifers grazed one Ryegrass pasture. Ruminal microbial content was collected and profiled using bacterial tag-encoded FLX amplicon pyrosequencing technique. In 2009, bulls were harvested directly off the pasture and ultrasound and carcass measurements were determined. Data were analyzed using PROC GLIMMIX of SAS. Linear regressions were obtained using PROC REG to estimate RFI. In 2009, there was a difference for LRFIc bulls in F:G (P=0.032), and HRFIg bulls on LSTK had an interaction for ADG (P=0.043). HRFIg bulls had greater intakes regardless of STK (P=0.003). In 2010, HRFIc bulls remained heavier throughout with the greatest DMI (P=0.0095). There were no differences for any traits for 2010 RFIg bulls. At a LSTK, HRFIg bulls tended (P>0.05) to have a lighter gastrointestinal tract (GIT) weight (P=0.093) while liver weight (P=0.072) tended to be heavier for all bulls. The small intestine was heavier for LRFIg bulls (P=0.09) on a HSTK. There was an interaction for microbial bacteria identified in the rumen in 2009 on hemicellulolytic (P=0.048), starch (P=0.025), and pectinolytic (P=0.057) degrading bacteria. HRFIg bulls at a LSTK had a greater percentage for amylolytic and pectinolytic degrading bacteria (P=0.008 and P=0.051, respectively) in the large intestine. There were no interactions for any substrates in 2010. DMI (P<0.0001) was greater each year for HRFIc heifers and was greater (P=0.0168, P=<0.0001, P=<0.0001) each year respectively for LRFIg heifers. No differences were found for initial BW, final BW, MetBW, and ADG in the RFIc or RFIg classes. HRFIg bulls with the greatest forage availability consumed more and had heavier GIT, but it is still unclear how the microbial fauna affected the efficiency among RFI phenotyped bulls.

The application of animal manure as a fertilizer source is a common practice in Western Canada. Manure acts as an organic amendment, while providing an economic means of restoring soil nutrients imperative to plant growth. This being said, manure is also microbiologically active, capable of introducing new bacteria into the soil and groundwater environments. The development of high-throughput molecular techniques has provided a means of characterizing the bacterial communities of animal manures, and ecosystems affected by their presence. This study examined the impact of pig slurry amendments on the bacterial communities of soil, groundwater, and the hindgut of grazing cattle over the growing season using three common molecular methods (Terminal-restriction fragment length polymorphism (T-RFLP), Sanger sequencing, and pyrosequencing). T-RFLP results show that sample occasion had more of an impact on the bacterial communities in cattle, soil and groundwater than slurry application. T-RFLP, Sanger sequencing and pyrosequencing characterized the pig slurry as being dominated by Firmicutes, specifically Clostridium spp. Sanger sequencing confirmed that very few classified genera present in the pig slurry were common to the three other environments. Pyrosequencing provided a more in-depth characterization of soil and groundwater over the growing season following the application of slurry. Firmicutes were again dominant in the pig slurry, and were comprised mainly of Clostridium spp. Slurry treated soils had greater relative abundance of Firmicutes, specifically Clostridium spp., compared to unamended soil. Pyrosequencing identified only one groundwater sequence out of 13,578 sequences that was common to the pig slurry, which indicates very little transfer from slurry to groundwater. Pyrosequencing was then performed on an annual cropping system to compare the impact of manures (solid dairy, and solid pig), and synthetic N on the bacterial community of soil in the short- (within a growing season) and medium-term (after three successive years). Solid pig manure treatments revealed greater diversity compared to synthetic and control treatments and diversity was also higher at post-harvest than post-planting. Bacterial communities between treatments were distinct in the short-term but returned to their original structure by the end of the growing season indicating a resilient soil bacterial community.

The cpn60 gene is a DNA barcode for bacteria. Recently, the PCR primers that have been used extensively to amplify the cpn60 Universal Target (UT) region of bacteria were redesigned to improve their utility for fungal taxa. Additional novel primers were designed to amplify other regions of the cpn60 gene, specifically from fungal genomes. Design of the redesigned and novel primers was based on 61 nucleotide full-length cpn60 reference sequences available in 2012, including Ascomycota (51), Basidiomycota (5), Chytridiomycota (2), Glomeromycota (1), and Oomycota (2). The research described here investigated the utility of these primers for detecting and identifying fungal taxa and for profiling mixed communities of bacteria and fungi. The redesigned primers were used to discover cpn60 UT sequences for Ascomycota (1), Basidiomycota (2), and Chytridiomycota (1). The novel primers were used to discover new cpn60 sequence data for Ascomycota (3), Basidiomycota (1), and Zygomycota (1). To be adopted for use in studies of microbial communities that are predominantly bacterial, the redesigned cpn60 UT primers must perform at least as well as the original primers for bacterial profiling. Bacterial profiles, created using the original and redesigned primers and two DNA template samples created by pooling DNA extracts from vaginal swabs from individual women, were compared. These included comparisons of diversity indices, rarefaction curve analysis and Operational Taxonomic Unit abundances. Diversity indices and rarefaction curve analysis for bacterial profiles with original and redesigned primers were similar. OTU abundance estimates with the original and redesigned primers were compared at higher and lower taxonomic levels. The overall patterns produced were similar. For one template only, the phylum Bacteroidetes had a greater apparent abundance with the original primers than with the redesigned primers. The greater apparent abundance of Bacteroidetes taxa was balanced by a lesser apparent abundance of taxa that were not assigned to a phylum. These differences may reflect differences in the performance of the two primer sets. At lower taxonomic level, most OTU were represented with apparently equal abundances with redesigned and original primers in same template. Very few OTU were represented with different proportional abundances with redesigned and original primers. Different OTU having same reference cpn60 UT sequence as best hit were sometimes represented by different proportional abundance with same primer in same template that made the analysis difficult. On the whole, the redesigned cpn60 UT primers behaved at least as good as the original cpn60 UT primers. The overall results showed that the redesigned and novel primers used in this study had substantial utility for the identification of fungal samples and mixed microbial communities.

Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.