Detection Methods for Lipopolysaccharides: Past and Present

Lipopolysaccharide (LPS) is the primary component of the outer membrane of Gram‐negativebacteria. LPS aids in protecting bacterial cells, and also defines the unique serogroups used to classify bacteria. Additionally, LPS is an endotoxin and the primary stimulator of innate immune cells in mammals, making it an ideal candidate for early detection of pathogens. However, the majority of methods for detection of LPS focus on detection of the endotoxic component of the molecule, lipid A. Since lipid A is largely conserved among bacterial species and serogroups, these detection approaches are highly nonspecific. Thus, the importance of identifying the O‐polysaccharide antigenic portion of LPS, which confers serogroup specificity, has received a great deal of attention in recent years. However, methods that are highly selective to the O‐antigens are typically less sensitive than those that target the endotoxin. Here we present a history and comparison of the sensitivity of these methods and their value for detecting bacteria in a variety of different sample types

Resistance to Gray Leaf Spot of Maize: Genetic Architecture and Mechanisms Elucidated through Nested Association Mapping and Near-Isogenic Line Analysis

Citation: Benson, J. M., Poland, J. A., Benson, B. M., Stromberg, E. L., & Nelson, R. J. (2015). Resistance to Gray Leaf Spot of Maize: Genetic Architecture and Mechanisms Elucidated through Nested Association Mapping and Near-Isogenic Line Analysis. Plos Genetics, 11(3), 23. https://doi.org/10.1371/journal.pgen.1005045Gray leaf spot (GLS), caused by Cercospora zeae-maydis and Cercospora zeina, is one of the most important diseases of maize worldwide. The pathogen has a necrotrophic lifestyle and no major genes are known for GLS. Quantitative resistance, although poorly understood, is important for GLS management. We used genetic mapping to refine understanding of the genetic architecture of GLS resistance and to develop hypotheses regarding the mechanisms underlying quantitative disease resistance (QDR) loci. Nested association mapping (NAM) was used to identify 16 quantitative trait loci (QTL) for QDR to GLS, including seven novel QTL, each of which demonstrated allelic series with significant effects above and below the magnitude of the B73 reference allele. Alleles at three QTL, qGLS1.04, qGLS2.09, and qGLS4.05, conferred disease reductions of greater than 10%. Interactions between loci were detected for three pairs of loci, including an interaction between iqGLS4.05 and qGLS7.03. Near-isogenic lines (NILs) were developed to confirm and fine-map three of the 16 QTL, and to develop hypotheses regarding mechanisms of resistance. qGLS1.04 was fine-mapped from an interval of 27.0 Mb to two intervals of 6.5 Mb and 5.2 Mb, consistent with the hypothesis that multiple genes underlie highly significant QTL identified by NAM. qGLS2.09, which was also associated with maturity (days to anthesis) and with resistance to southern leaf blight, was narrowed to a 4-Mb interval. The distance between major leaf veins was strongly associated with resistance to GLS at qGLS4.05. NILs for qGLS1.04 were treated with the C. zeae-maydis toxin cercosporin to test the role of host-specific toxin in QDR. Cercosporin exposure increased expression of a putative flavin-monooxygenase (FMO) gene, a candidate detoxification-related gene underlying qGLS1.04. This integrated approach to confirming QTL and characterizing the potential underlying mechanisms advances the understanding of QDR and will facilitate the development of resistant varieties

Site Conditions Determine a Key Native Plant’s Contribution to Invasion Resistance in Grasslands

Many plant invasion studies in grasslands suggest that resident plants that share functional traits with invaders can reduce invasion by competing for limiting resources. However, since invasion studies often occur in highly controlled plots or microcosms, it is unclear how heterogeneous site conditions alter competitive interactions under realistic scenarios. To explore how landscape heterogeneity affects biotic resistance provided by competitive resident plants, we conducted a field‐based experiment across four sites in California grasslands. Plots contained naturally occurring populations of native Hemizonia congesta, but differed in other characteristics, including litter cover, annual grass cover, soil moisture, and species richness. We invaded plots with the functionally similar nonnative Centaurea solstitalis (yellow starthistle) and, at one site, supplemented one‐half of the established plots with water to test the effects of increasing a limiting resource. As in simplified plots and microcosms, increasing H. congesta abundance reduced starthistle biomass by competing for limited soil moisture, but only in plots with high starthistle germination. We conclude that higher abundances of native H. congesta can reduce starthistle invasion in heterogeneous grasslands, but competition is also affected by both abiotic (soil moisture) and biotic (starthistle germination number) conditions that vary across sites

Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNABulge Size and Sequence Dependence

In this report, we investigate the efficiency and selectivity of a Zn2+-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7-8 h as compared to 11-12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50-60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21-24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7-8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site

Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides

Citation: Chopyk, J., Moore, R. M., DiSpirito, Z., Stromberg, Z. R., Lewis, G. L., Renter, D. G., . . . Wommack, K. E. (2016). Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides. Microbiome, 4, 11. doi:10.1186/s40168-016-0155-4Background: Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Results: Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. Conclusions: The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide bacterial communities and populations may provide strategies to limit EHEC in cattle and provide biomarkers for EHEC risk assessment

Gene Expression Patterns Specific to the Regenerating Limb of the Mexican Axolotl

Salamander limb regeneration is dependent upon tissue interactions that are local to the amputation site. Communication among limb epidermis, peripheral nerves, and mesenchyme coordinate cell migration, cell proliferation, and tissue patterning to generate a blastema, which will form missing limb structures. An outstanding question is how cross-talk between these tissues gives rise to the regeneration blastema. To identify genes associated with epidermis-nerve-mesenchymal interactions during limb regeneration, we examined histological and transcriptional changes during the first week following injury in the wound epidermis and subjacent cells between three injury types; 1) a flank wound on the side of the animal that will not regenerate a limb, 2) a denervated limb that will not regenerate a limb, and 3) an innervated limb that will regenerate a limb. Early, histological and transcriptional changes were similar between the injury types, presumably because a common wound-healing program is employed across anatomical locations. However, some transcripts were enriched in limbs compared to the flank and are associated with vertebrate limb development. Many of these genes were activated before blastema outgrowth and expressed in specific tissue types including the epidermis, peripheral nerve, and mesenchyme. We also identified a relatively small group of transcripts that were more highly expressed in innervated limbs versus denervated limbs. These transcripts encode for proteins involved in myelination of peripheral nerves, epidermal cell function, and proliferation of mesenchymal cells. Overall, our study identifies limb-specific and nerve-dependent genes that are upstream of regenerative growth, and thus promising candidates for the regulation of blastema formation

Natural Analogue Constraints on Europa's Non-ice surface Material

Non-icy material on the surface of Jupiter’s moon Europa is hypothesised to have originated from its subsurface ocean, and thus provide a record of ocean composition and habitability. The nature of this material is debated, but observations suggest that it comprises hydrated sulfate and chloride salts. Analogue spectroscopic studies have previously focused on single phase salts under controlled laboratory conditions. We investigated natural salts from perennially cold (<0 °C) hypersaline springs, and characterised their reflectance properties at 100 K, 253 K and 293 K. Despite similar major ion chemistry, these springs form mineralogically diverse deposits, which when measured at 100 K closely match reflectance spectra from Europa. In the most sulfate-rich samples, we find spectral features predicted from laboratory salts are obscured. Our data are consistent with sulfate-dominated europan non-icy material, and further, show that the emplacement of endogenic sulfates on Europa’s surface would not preclude a chloride-dominated ocean

Prevalence and Level of Enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e in Culled Dairy Cows at Harvest

The primary objective of this study was to determine the prevalence and level of enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, and O145 (collectively EHEC-6) plus EHEC O157 in fecal, hide, and preintervention carcass surface samples from culled dairy cows. Matched samples (n=300) were collected from 100 cows at harvest and tested by a culture-based method and two molecular methods: NeoSEEK STEC (NS) and Atlas STEC EG2 Combo. Both the culture and NS methods can be used to discriminate among the seven EHEC types (EHEC-7), from which the cumulative prevalence was inferred, whereas the Atlas method can discriminate only between EHEC O157 and non-O157 EHEC, without discrimination of the serogroup. The EHEC-7 prevalence in feces, hides, and carcass surfaces was 6.5, 15.6, and 1.0%, respectively, with the culture method and 25.9, 64.9, and 7.0%, respectively, with the NS method. With the Atlas method, the prevalence of non-O157 EHEC was 29.1, 38.3, and 28.0% and that of EHEC O157 was 29.1, 57.0, and 3.0% for feces, hides, and carcasses, respectively. Only two samples (a hide sample and a fecal sample) originating from different cows contained quantifiable EHEC. In both samples, the isolates were identified as EHEC O157, with 4.7 CFU/1,000 cm2 in the hide sample and 3.9 log CFU/g in the fecal sample. Moderate agreement was found between culture and NS results for detection of EHEC O26 (k=0.58,

Prevalence of internal parasites in beef cows in the United States: Results of the National Animal Health Monitoring System’s (NAHMS) beef study, 2007–2008

During the United States Department of Agriculture (USDA) National Animal Health Monitoring System’s (NAHMS) 2007–2008 beef study, 567 producers from 24 US States were offered the opportunity to collect fecal samples from weaned beef calves and have them evaluated for the presence of parasite eggs (Phase 1). Participating producers were provided with instructions and materials for sample collection. Up to 20 fresh fecal samples were collected from each of the 99 participating operations. Fresh fecal samples were submitted to one of 3 randomly assigned laboratories for evaluation. Upon arrival at the laboratories, all samples were processed for the enumeration of strongyle, Nematodirus, and Trichuris eggs using the modified Wisconsin technique. The presence or absence of coccidian oocysts and tapeworm eggs was also noted. In submissions where the strongyle eggs per gram exceeded 30, aliquots from 2 to 6 animals were pooled for DNA extraction. Extracted DNA was subjected to genus level polymerase chain reaction (PCR) identification for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. In this study, 85.6% of the samples had strongyle type, Nematodirus, and Trichuris eggs. Among the samples evaluated, 91% had Cooperia, 79% Ostertagia, 53% Haemonchus, 38% Oesophagostomum, 18% Nematodirus, 7% Trichuris, and 3% Trichostrongylus. The prevalence of coccidia and tapeworm eggs was 59.9% and 13.7%, respectively

Effectiveness of current anthelmintic treatment programs on reducing fecal egg counts in United States cow-calf operations

During the United States Department of Agriculture (USDA) National Animal Health Monitoring System’s (NAHMS) 2007–2008 beef study, producers from 24 states were offered the opportunity to evaluate their animals for internal parasites and for overall responses to treatment with anthelmintics. A lapse of 45 d was required between initial sampling and any previous treatments. Choice of anthelmintic (oral benzimidazoles, and both injectable and pour-on endectocides) was at the discretion of the producer so as not to alter the local control programs. Fresh fecal samples were collected from 20 animals, or from the entire group if less than 20, then randomly assigned to 1 of 3 participating laboratories for examination. Analyses consisted of double centrifugation flotation followed by enumeration of strongyle, Nematodirus, and Trichuris eggs (the presence of coccidian oocysts and tapeworm eggs was also noted). Where strongyle eggs per gram (epg) exceeded 30, aliquots from 2 to 6 animals were pooled for egg isolation and polymerase chain reaction (PCR) analysis for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. Results from 72 producers (19 States) indicated that fecal egg count reductions were , 90% in 1/3 of the operations. All operations exhibiting less than a 90% reduction had used pour-on macrocyclic lactones as the anthelmintic treatment. While some of these less than expected reductions could have been the result of improper drug application, PCR analyses of the parasite populations surviving treatment, coupled with follow-up studies at a limited number of sites, indicated that less than expected reductions were most likely due to anthelmintic resistance in Cooperia spp. and possibly Haemonchus spp