Oakley B.B.,University of Warwick |
Dowd S.E.,Research and Testing Laboratories |
Purdy K.J.,University of Warwick
FEMS Microbiology Ecology | Year: 2011
The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates. Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units, a richness that precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem, we have developed and validated a free software tool that automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and nontarget sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimized candidate assays derived from primer design software packages. ThermoPhyl derives its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMans® qPCR) application and potentially for oligonucleotide probes. Here, we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples. © 2011 Federation of European Microbiological Societies.
Rice W.C.,U.S. Department of Agriculture |
Galyean M.L.,Texas Tech University |
Cox S.B.,Research and Testing Laboratories |
Dowd S.E.,Molecular Research MR DNA |
Cole N.A.,U.S. Department of Agriculture
BMC Microbiology | Year: 2012
Background: The high demand for ethanol in the U.S. has generated large stocks of wet distillers grains (DG), a byproduct from the manufacture of ethanol from corn and sorghum grains. Little is known, however, about the potential influence of dietary DG on fecal microbial community structure. A better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. Five diets consisting of a traditional diet fed to finishing beef cattle in the Southern High Plains of Texas-CON (steam-flaked corn control with 0% DG), and four concentrations of DG in the dietary dry matter; 10 C (10% corn-based DG), 5S (5% sorghum-based DG), 10S (10% sorghum DG), and 15S (15% sorghum DG) were fed to steers at the Texas Tech University Burnett Animal Center. Diets were essentially isonitrogenous with a formulated crude protein value of 13.5%. Results: Fecal grab samples were obtained from 20 steers (n = 4 per diet) and the barcoded DNA pyrosequencing method was used to generate 127,530 16S operational taxonomic units (OTUs). A total of 24 phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla (core set) were observed in all animals regardless of dietary treatment. The average abundance and range of abundance, respectively of the core phyla were as follows: Firmicutes (61%, 19 to 83%), Bacteroidetes (28%, 11 to 63%), Proteobacteria (3%, 0.34 to 17.5%), Tenericutes (0.15%, 0.0 to 0.35%), Nitrospirae (0.11%, 0.03 to 0.22%), and Fusobacteria (0.086%, 0.017 to 0.38%). Feeding DG-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional CON. Four low abundance phyla significantly responded to dietary treatments: Synergistetes (p = 0.01), WS3 (p = 0.054), Actinobacteria (p = 0.06), and Spirochaetes (p = 0.06). Conclusions: This is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet DG. Comparison of our results with other cattle DNA sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. These three phyla, in order of relative abundance are; Firmicutes, Bacteroidetes, and Proteobacteria. The presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others. © 2012 Rice et al; BioMed Central Ltd.
Martynova-Van Kley M.A.,Stephen F. Austin State University |
Oviedo-Rondon E.O.,North Carolina State University |
Dowd S.E.,Research and Testing Laboratories |
Hume M.,Food and Feed Safety Research Unit |
And 2 more authors.
International Journal of Poultry Science | Year: 2012
Coccidiosis causes mucosal damage and predisposes birds to enteropathogen infection. In this study pyrosequencing was used to evaluate effects of coccidiosis on the intestinal microflora of broilers given diets without feed additives or supplemented with either a growth promotant antibiotic and an ionophore, or two essential oil blends. DNA samples were collected from the cecal contents of broilers before (19 d) and after (26 d) infection with mixed Eimeria spp. (E. acervulina, E. maxima and E. tenella). A 454 FLX pyrosequencer and 16S universal primers were used to obtain quantitative profiles of bacterial taxa present in each sample. The relative percent abundance of the identified taxa was analyzed using hierarchical clustering and principal component analysis. Samples from pre-infected broilers were dominated by bacterial species belonging to genera Subdoligranulum, Coprococcus, Alistipes, Lactobacillus and Faecalibacterium. Post-infection samples were dominated by species from the genera Escherichia/Shigella and Bacteroides. Eimeria infection did not significantly affect the richness of the microbial communities but rather its composition. The composition of the cecal microbiome correlated with the average feed conversion ratio. The methodology used in this study proved effective in understanding the effects of coccidia infection on intestinal microflora of broilers raised on diets supplemented with growth promoting antibiotics, ionophores and essential oils. © Asian Network for Scientific Information, 2012.
Acosta-Martinez V.,U.S. Department of Agriculture |
Cotton J.,U.S. Department of Agriculture |
Gardner T.,U.S. Department of Agriculture |
Gardner T.,North Carolina State University |
And 4 more authors.
Applied Soil Ecology | Year: 2014
Identification of microbial assemblages predominant under natural extreme climatic events will aid in our understanding of the resilience and resistance of microbial communities to climate change. From November 2010 to August 2011, the Southern High Plains (SHP) of Texas, USA, received only 39.6. mm of precipitation (vs. the historical average of 373. mm) and experienced the three hottest months (June-August 2011) since record keeping began in 1911. The objective of this study was to characterize soil bacterial (16 S rRNA gene) and fungal (internal transcribed spacer 1-4, ITS1-ITS4) species distribution and diversity via pyrosequencing during the peak of the drought/heat wave in July 2011 and when the Drought Index and temperatures were lower in March 2012. Samples were collected from two different soil types (loam and sandy loam) under two different dryland cropping histories (monoculture vs. rotation). Fungal Diversity Indexes were significantly higher after the drought/heat wave while Bacterial Indexes were similar. Bacterial phyla distribution in July 2011 was characterized by lower relative abundance of Acidobacteriaand Verrucomicrobia, and greater relative abundance of Proteobacteria, Chloroflexi, Actinobacteria and Nitrospirae than March 2012 samples. Further grouping of pyrosequencing data revealed approximately equal relative proportions of Gram positive (G+) and Gram negative (G-) bacteria in July 2011, while G- bacteria predominated in March 2012. Fungal class Dothideomycetes was approximately two times greater in July 2011 than in March 2012. while the class Sordariomycetes and a group of unidentified OTUs from Ascomycota increased from July 2011 to March 2012. Microbial community composition was less influenced by management history than by the difference in climatic conditions between the sampling times. Correspondence analysis identified assemblages of fungal and bacterial taxa associated with greater enzyme activities (EAs) of C, N, or P cycling found during the drought/heat wave. Microbial assemblages associated with arylsulfatase activity (key to S cycling), which increased after the drought/heat wave, were identified (Streptomyces parvisporogenes, Terrimonas ferruginea and Syntrophobacter sp.) regardless of the soil and management history. The distinct microbial composition found in July 2011 may represent assemblages essential to maintaining ecosystem function during extreme drought and intense heat waves in semiarid agroecosystems. © 2014.
Martynova-Van Kley A.,Stephen F. Austin State University |
Manoharan M.S.,Stephen F. Austin State University |
Bray J.,Stephen F. Austin State University |
Hume M.E.,U.S. Department of Agriculture |
And 2 more authors.
International Journal of Poultry Science | Year: 2013
Eimeria spp. invade and damage the intestinal cell lining of broilers resulting in cell necrosis and secondary bacterial infections. The current work investigates the effect of anticoccidial agents, salinomycin in combination with Roxarsone and Eimeria-challenge on the composition of broiler cecal microflora. Three hundred and twenty day-old male Cobb broilers were among four treatment groups: NN (no salinomycin and no Eimeria challenge) and NC (no salinomycin plus Eimeria challenge) received basal diet with no salinomycin, while SN (salinomycin and no Eimeria challenge) and SC (salinomycin plus Eimeria challenge) received basal diet with salinomucin. Broilers in groups NC and SC were orally gavaged on d 28 with a mixed Eimeria spp. challenge. Body weight and Eimeria lesion scores were determined at d 35. Cecal bacterial DNA from broilers at day 28 and day 35 were subjected to 454 pyrosequencing of 16S rDNA for sequence identification. Relative percent abundance and richness of the identified taxa were analyzed. Salinomycin had significant influence on the total number of taxa (p = 0.02) and on cecal microbial community structure (p = 0.002). The mixed Eimeria challenge marginally affected the total number of taxa (p = 0.06) and the composition of microbial communities (p = 0.09). Broiler age, body weight and Eimeria lesion score had no significant effect on the cecal microbial communities. Results from this study indicate that pyrosequencing is effective in understanding the dynamics and functionality of cecal microbial communities in relation to anticoccidial treatment, Eimeria challenge and broiler performances. © Asian Network for Scientific Information, 2013.