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Item Dryland Cropping Systems, Weed Communities, and Disease Status Modulate the Effect of Climate Conditions on Wheat Soil Bacterial Communities(2020-08) Ishaq, Suzanne L.; Seipel, Tim F.; Yeoman, Carl J.; Menalled, Fabian D.Little knowledge exists on how soil bacteria in agricultural settings are impacted by management practices and environmental conditions in current and predicted climate scenarios. We assessed the impact of soil moisture, soil temperature, weed communities, and disease status on soil bacterial communities in three cropping systems: (i) conventional no-till (CNT) systems utilizing synthetic pesticides and herbicides, (ii) USDA-certified tilled organic (OT) systems, and (iii) USDA-certified organic systems with sheep grazing (OG). Sampling date within the growing season and associated soil temperature and moisture exerted the greatest effect on bacterial communities, followed by cropping system, Wheat streak mosaic virus (WSMV) infection status, and weed community. Soil temperature was negatively correlated with bacterial richness and evenness, while soil moisture was positively correlated with bacterial richness and evenness. Soil temperature and soil moisture independently altered soil bacterial community similarity between treatments. Inoculation of wheat with WSMV altered the associated soil bacteria, and there were interactions between disease status and cropping system, sampling date, and climate conditions, indicating the effect of multiple stressors on bacterial communities in soil. In May and July, cropping system altered the effect of climate change on the bacterial community composition in hotter conditions and in hotter and drier conditions compared to ambient conditions, in samples not treated with WSMV. Overall, this study indicates that predicted climate modifications as well as biological stressors play a fundamental role in the impact of cropping systems on soil bacterial communities.Item Pelleted-hay alfalfa feed increases sheep wether weight gain and rumen bacterial richness over loose-hay alfalfa feed(2019-06) Ishaq, Suzanne L.; Lachman, Medora M.; Wenner, Benjamin A.; Baeza, Amy; Butler, Molly; Gates, Emily; Olivo, Sarah; Geddes, Julie Buono; Hatfield, Patrick G.; Yeoman, Carl J.Diet composed of smaller particles can improve feed intake, digestibility, and animal growth or health, but in ruminant species can reduce rumination and buffering – the loss of which may inhibit fermentation and digestibility. However, the explicit effect of particle size on the rumen microbiota remains untested, despite their crucial role in digestion. We evaluated the effects of reduced particle size on rumen microbiota by feeding long-stem (loose) alfalfa hay compared to a ground and pelleted version of the same alfalfa in yearling sheep wethers. In situ digestibility of the pelleted diet was greater at 48 h compared with loose hay; however, distribution of residual fecal particle sizes in sheep did not differ between the dietary treatments at any time point. Both average daily gain and feed efficiency were greater for the wethers consuming the pelleted diet. Observed bacterial richness was very low at the end of the adaptation period and increased over the course of the study, suggesting the rumen bacterial community was still in flux after two weeks of adaptation. The pelleted-hay diet group had a greater increase in bacterial richness, including common fibrolytic rumen inhabitants. The pelleted diet was positively associated with several Succiniclasticum, a Prevotella, and uncultured taxa in the Ruminococcaceae and Rickenellaceae families and Bacteroidales order. Pelleting an alfalfa hay diet for sheep does shift the rumen microbiome, though the interplay of diet particle size, retention and GI transit time, microbial fermentative and hydrolytic activity, and host growth or health is still largely unexplored.Item Ground Juniperus pinchotii and urea in supplements fed to Rambouillet ewe lambs Part 2: Ewe lamb rumen microbial communities(2018-12) Ishaq, Suzanne L.; Yeoman, Carl J.; Whitney, T. R.This study evaluated effects of ground redberry juniper (Juniperus pinchotii) and urea in dried distillers grains with solubles-based supplements fed to Rambouillet ewe lambs (n = 48) on rumen physiological parameters and bacterial diversity. In a randomized study (40 d), individually-penned lambs were fed ad libitum ground sorghum-sudangrass hay and of 1 of 8 supplements (6 lambs/treatment; 533 g/d; as-fed basis) in a 4 x 2 factorial design with 4 concentrations of ground juniper (15%, 30%, 45%, or 60% of DM) and 2 levels of urea (1% or 3% of DM). Increasing juniper resulted in minor changes in microbial β-diversity (PERMANOVA, pseudo F = 1.33, P = 0.04); however, concentrations of urea did not show detectable broad-scale differences at phylum, family, or genus levels according to ANOSIM (P > 0.05), AMOVA (P > 0.10), and PERMANOVA (P > 0.05). Linear discriminant analysis indicated some genera were specific to certain dietary treatments (P < 0.05), though none of these genera were present in high abundance; high concentrations of juniper were associated with Moraxella and Streptococcus, low concentrations of urea were associated with Fretibacterium, and high concentrations of urea were associated with Oribacterium and Pyramidobacter. Prevotella were decreased by juniper and urea. Ruminococcus, Butyrivibrio, and Succiniclasticum increased with juniper and were positively correlated (Spearman\'s, P < 0.05) with each other but not to rumen factors, suggesting a symbiotic interaction. Overall, there was not a juniper x urea interaction for total VFA, VFA by concentration or percent total, pH, or ammonia (P > 0.29). When considering only percent inclusion of juniper, ruminal pH and proportion of acetic acid linearly increased (P < 0.001) and percentage of butyric acid linearly decreased (P = 0.009). Lamb ADG and G:F were positively correlated with Prevotella (Spearman\'s, P < 0.05) and negatively correlated with Synergistaceae, the BS5 group, and Lentisphaerae. Firmicutes were negatively correlated with serum urea nitrogen, ammonia, total VFA, total acetate, and total propionate. Overall, modest differences in bacterial diversity among treatments occurred in the abundance or evenness of several OTUs, but there was not a significant difference in OTU richness. As diversity was largely unchanged, the reduction in ADG and lowerend BW was likely due to reduced DMI rather than a reduction in microbial fermentative ability.Item Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet(2018-04) Zeng, Huawei; Ishaq, Suzanne L.; Liu, Zhenhua; Bukowsky, Michael R.The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat western diet. To test the hypothesis that a high fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or a HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin 6 (IL6), tumor necrosis factor α (TNF α), monocyte chemoattractant protein 1 (MCP1), and inducible nitric oxide synthase (iNOS) in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that a HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.Item Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract(2018-02) Yeoman, Carl J.; Ishaq, Suzanne L.; Bichi, Elena; Olivo, Sarah K.; Lowe, James; Aldridge, Brian M.The impact of maternal microbial influences on the early choreography of the neonatal calf microbiome were investigated. Luminal content and mucosal scraping samples were collected from ten locations in the calf gastrointestinal tract (GIT) over the first 21 days of life, along with postpartum maternal colostrum, udder skin, and vaginal scrapings. Microbiota were found to vary by anatomical location, between the lumen and mucosa at each GIT location, and differentially enriched for maternal vaginal, skin, and colostral microbiota. Most calf sample sites exhibited a gradual increase in α-diversity over the 21 days beginning the first few days after birth. The relative abundance of Firmicutes was greater in the proximal GIT, while Bacteroidetes were greater in the distal GIT. Proteobacteria exhibited greater relative abundances in mucosal scrapings relative to luminal content. Forty-six percent of calf luminal microbes and 41% of mucosal microbes were observed in at-least one maternal source, with the majority being shared with microbes on the skin of the udder. The vaginal microbiota were found to harbor and uniquely share many common and well-described fibrolytic rumen bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing rumen and reticulum with microbes important to the nutrition of the adult animal.Item Feed efficiency phenotypes in lambs involve changes in ruminal, colonic, and small-intestine-located microbiota(2017-06) Perea, K.; Perz, Katharine A.; Olivo, Sarah K.; Williams, Andrew F.; Lachman, Medora M.; Ishaq, Suzanne L.; Thomson, Jennifer M.; Yeoman, Carl J.Several studies have revealed differences in rumen-located microbes between greatly efficient and inefficient animals; however, how the microbiota vary in the hind gastrointestinal tract (GIT) has only been sparsely explored and how they vary in the small intestine remains to be determined. We therefore sampled the microbiota of the duodenum, jejunum, ileum, colon, and colorectally-obtained feces, in addition to the rumen of 12 lambs that, in a residual feed intake trial, were found to be at either extreme of feed efficiency phenotypes. The 16S rRNA gene (V3-V4 region) profiles of all samples were analyzed and revealed unique microbiota in all GIT locations except the jejunum and ileum (ANOSIM R > 0.2, P < 0.001). Measures of beta-diversity revealed greater dissimilarity between more anatomically distant GIT locations (e.g., Rumen-Duodenum, ANOSIM R = 0.365, P < 0.001; Rumen-Colon, ANOSIM R = 1, P < 0.001) with the nearest distal region typically more similar than the nearest proximal location. The relative abundances of 13 operational taxonomic units (OTUs) from the duodenum, jejunum, colon, and feces, as well as the rumen, differed between efficient and inefficient animals (Bonferroni corrected, P < 0.05), while another 2 OTUs trended toward significance. These OTUs were classified as taxa with known roles in fibrolysis (Fibrobacteres, Ruminococcaceae, and Saccharofermentans) and others that are commonly associated with health (Bifidobacteriaceae, and Christensenellaceae) and dysbiosis (Proteobacteria). Our findings show biospatial delineations of microbiota throughout the GIT and suggest that feed efficiency extends beyond the rumen, transcending these regions, and involves increases in both rumen-and colon-located fibrolytic taxa, increases in bifidobacterial species in the small intestine, and reductions in small intestine and distal GIT-located Proteobacteria.Item Impact of Cropping Systems, Soil Inoculum, and Plant Species Identity on Soil Bacterial Community Structure(2017-02) Ishaq, Suzanne L.; Johnson, Stephen P.; Miller, Zachariah J.; Lehnhoff, Erik A.; Olivo, Sarah K.; Yeoman, Carl J.; Menalled, Fabian D.Farming practices affect the soil microbial community, which in turn impacts crop growth and crop-weed interactions. This study assessed the modification of soil bacterial community structure by organic or conventional cropping systems, weed species identity [Amaranthus retroflexus L. (redroot pigweed) or Avena fatua L. (wild oat)], and living or sterilized inoculum. Soil from eight paired USDA-certified organic and conventional farms in north-central Montana was used as living or autoclave-sterilized inoculant into steam-pasteurized potting soil, planted with Am. retroflexus or Av. fatua and grown for two consecutive 8-week periods to condition soil nutrients and biota. Subsequently, the V3-V4 regions of the microbial 16S rRNA gene were sequenced by Illumina MiSeq. Treatments clustered significantly, with living or sterilized inoculum being the strongest delineating factor, followed by organic or conventional cropping system, then individual farm. Living inoculum-treated soil had greater species richness and was more diverse than sterile inoculum-treated soil (observed OTUs, Chao, inverse Simpson, Shannon, P<0.001) and had more discriminant taxa delineating groups (linear discriminant analysis). Living inoculum soil contained more Chloroflexi and Acidobacteria, while the sterile inoculum soil had more Bacteroidetes, Firmicutes, Gemmatimonadetes, and Verrucomicrobia. Organically farmed inoculum-treated soil had greater species richness, more diversity (observed OTUs, Chao, Shannon, P<0.05), and more discriminant taxa than conventionally farmed inoculum-treated soil. Cyanobacteria were higher in pots growing Am. retroflexus, regardless of inoculum type, for three of the four organic farms. Results highlight the potential of cropping systems and species identity to modify soil bacterial communities, subsequently modifying plant growth and crop-weed competition.Item Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of in vitro and in vivo Protease SpeB Expression and Virulence(2017-01) Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Li, Jinquan; Ishaq, Suzanne L.; Yeoman, Carl J.; Lei, BenfangGroup A Streptococcus (GAS) acquires mutations of virulence regulator CovRS in human and mouse infections that upregulate virulence genes and downregulate protease SpeB. To identify in vivo mutants with novel phenotype, GAS isolates from mouse infection were screened by enzymatic assays for SpeB and platelet-activating factor acetylhydrolase Sse, identifying a new type of variants that had enhanced Sse expression and normal SpeB production (Sse(A+)SpeB(A+)). Sse(A+)SpeB(A+) variants have transcripts levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse(A+)SpeB(A+) isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse(A+)SpeB(A+) isolates also had nonsense mutations or small indels of rocA RocA and CovS mutants have similar enhancement in expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA, but not CovS, do not downregulate speB transcription at stationary growth phase and in subcutaneous infection of mice. RocA and CovS mutations have greater enhancement in expression of hasA than spyCEP in mouse skin infection in comparison with wild type GAS. RocA mutants rank between wild type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infection of mice and exhibit distinct gene expression pattern and virulence from CovS mutants. The findings provide novel information for the understanding of GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.