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At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus’s ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Exploring Trait Trade-Offs for Fungal Decomposers in a Southern California Grassland

Fungi are important decomposers in terrestrial ecosystems, so their responses to climate change might influence carbon (C) and nitrogen (N) dynamics. We investigated whether growth and activity of fungi under drought conditions were structured by trade-offs among traits in 15 fungal isolates from a Mediterranean Southern California grassland. We inoculated fungi onto sterilized litter that was incubated at three moisture levels (4, 27, and 50% water holding capacity, WHC). For each isolate, we characterized traits that described three potential lifestyles within the newly proposed “YAS” framework: growth yield, resource acquisition, and stress tolerance. Specifically, we measured fungal hyphal length per unit litter decomposition for growth yield; the potential activities of the extracellular enzymes cellobiohydrolase (CBH), β -glucosidase (BG), β -xylosidase (BX), and N-acetyl- β - D -glucosaminidase (NAG) for resource acquisition; and ability to grow in drought vs. higher moisture levels for drought stress tolerance. Although, we had hypothesized that evolutionary and physiological trade-offs would elicit negative relationships among traits, we found no supporting evidence for this hypothesis. Across isolates, growth yield, drought stress tolerance, and extracellular enzyme activities were not significantly related to each other. Thus, it is possible that drought-induced shifts in fungal community composition may not necessarily lead to changes in fungal biomass or decomposer ability in this arid grassland.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Impact of intense sanitization procedures on bacterial communities recovered from floor drains in pork processing plants

Pork processing plants in the United States (US) cease operations for 24–48 h every six or twelve months to perform intense sanitization (IS) using fogging, foaming, and further antimicrobial treatments to disrupt natural biofilms that may harbor pathogens and spoilage organisms. The impact such treatments have on short-term changes in environmental microorganisms is not well understood, nor is the rate at which bacterial communities return. Swab samples were collected from floor drains to provide representative environmental microorganisms at two US pork processing plants before, during, and after an IS procedure. Samples were collected from four coolers where finished carcasses were chilled and from four locations near cutting tables. Each sample was characterized by total mesophile count (TMC), total psychrophile count (TPC), and other indicator bacteria; their biofilm-forming ability, tolerance of the formed biofilm to a quaternary ammonium compound (300 ppm, QAC), and ability to protect co-inoculated Salmonella enterica. In addition, bacterial community composition was determined using shotgun metagenomic sequencing. IS procedures disrupted bacteria present but to different extents depending on the plant and the area of the plant. IS reduced TPC and TMC, by up to 1.5 Log 10 CFU only to return to pre-IS levels within 2–3 days. The impact of IS on microorganisms in coolers was varied, with reductions of 2–4 Log 10 , and required 2 to 4 weeks to return to pre-IS levels. The results near fabrication lines were mixed, with little to no significant changes at one plant, while at the other, two processing lines showed 4 to 6 Log 10 reductions. Resistance to QAC and the protection of Salmonella by the biofilms varied between plants and between areas of the plants as well. Community profiling of bacteria at the genus level showed that IS reduced species diversity and the disruption led to new community compositions that in some cases did not return to the pre-IS state even after 15 to 16 weeks. The results found here reveal the impact of using IS to disrupt the presence of pathogen or spoilage microorganisms in US pork processing facilities may not have the intended effect.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Evaluation of aflatoxin contamination in protein-rich pulses using a GFP-expressing Aspergillus flavus strain

Background Mycotoxigenic fungi pose significant threats to food safety and marketability. Crop-specific differences in susceptibility to these fungi can influence contamination levels. Objectives The resistance or susceptibility of protein-rich pulse crops—chickpeas (Cicer arietinumL. cv. CDC Frontier), lentils (Lens culinarisMedik cv. Eston), peas (Pisum sativumL. cv. LeRoy), and corn (Zea maysL. cv. H97C) to infection byAspergillus flavuswere evaluated using a kernel screening assay (KSA). Methodology A. flavusstrain 70 (AF-70) expressing green-fluorescent protein (GFP) was used to quantify fungal spread and mycotoxin production. Fungal infection and toxin levels, including aflatoxins (AFB 1 , AFB 2 ), cyclopiazonic acid (CPA), and α-aflatrem, were monitored at 2-day intervals over a 10-day period post inoculation. Results Although all seeds were infected byA. flavus, corn produced significantly higher levels of AFB 1 and AFB 2 compared to pulses. However, pulses accumulated relatively higher levels of CPA and α‑aflatrem. Conclusion While pulses may be less susceptible to aflatoxin contamination than corn, the elevated concentrations of CPA and α‑aflatrem underscore the need for further toxicological evaluation and mechanistic studies. Future research should explore the underlying resistance mechanisms from field to storage to better ensure crop safety.

Microbiology↗
DOE OSTI2022

Proteomics reveals pathways linked to septoria canker resistance and susceptibility in Populus trichocarpa

A major threat to forest ecosystems and plantation forestry is the introduction of a non-native pathogen. Among non-domesticated populations with relatively high levels of genetic diversity, a measurable range of susceptibility to resistance can be expected. Identifying genetic determinants of resistant and susceptible individuals can inform the development of new strategies to engineer disease resistance. Here we describe pathogen-induced changes in the proteome of Populus trichocarpa stem tissue in response to Sphaerulia musiva (Septoria canker). This hemibiotrophic fungal pathogen causes stem canker disease in susceptible poplar genotypes. Proteomics analyses were performed on stem tissue harvested across 0-, 12-, 24- and 48-h post-inoculation with Septoria from three genotypes including one resistant (BESC-22) and two susceptible [BESC-801; Nisqually-1 (NQ-1)]. In total, 11,897 Populus proteins at FDR <0.01 were identified across all time points and genotypes. Analysis of protein abundances between genotypes revealed that the resistant poplar genotype (BESC-22) mounts a rapid and sustained defense response involving pattern recognition receptors, calcium signaling proteins, SAR inducers, transcriptional regulators, resistance proteins, and proteins involved with the hypersensitive response. One susceptible genotype (BESC-801) had a downregulated and delayed defense response whereas the second susceptible genotype (NQ-1) lacked a distinct pattern. Overall, the proteome-wide and protein-specific trends suggest that responses to the Septoria canker infection are genotype-specific for the naïve host, Populus trichocarpa .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evaluating two live-attenuated vaccines against Salmonella enterica serovar Reading in turkeys: reduced tissue colonization and cecal tonsil transcriptome responses

Vaccines that cross-protect across serovars ofSalmonella enterica(Salmonella) would be a beneficial intervention against emerging and persistentSalmonellaisolates of concern for the turkey industry. The 2017–2019 foodborne outbreak ofSalmonella entericaserovar Reading (S. Reading) revealed the need for effective control of this serovar in turkey production. This study evaluated two live-attenuatedSalmonellavaccines, an internally developed cross-protective vaccine and a commercially available vaccine, against an outbreak-associated strain ofS. Reading in turkeys. At 1 day and 3 weeks of age, male turkey poults were either mock-vaccinated with phosphate buffered saline (PBS) or given one of the vaccines by oral gavage (primary and booster) or aerosol spray (primary) then drinking water (booster). At 7 weeks of age, poults were challenged with 10 9 colony forming units (CFU) ofS. Reading; a mock-vaccinated group was mock-challenged with PBS. Colonization of the cecal contents and cecal tonsil was 1.5–3 log 10 CFU/g lower in vaccinated birds than mock-vaccinated birds at 7 and/or 14 days post-inoculation (DPI).Salmonelladissemination to the spleen was significantly reduced by both vaccines. Gene expression of intestinal transporters (such asSCNN1BandSLC10A2) and tight junction proteins was significantly decreased in the turkey cecal tonsil transcriptome at 2 DPI withS. Reading. Vaccination with either vaccine mitigated most cecal tonsil gene expression responses toS. Reading challenge. Therefore, both the internally developed vaccine and commercial vaccine were cross-protective against colonization and dissemination, and both were able to limit transcriptional changes from challenge in intestinal health-related genes in the cecal tonsil, thereby providing vaccination efficacy and impact data againstS. Reading in turkeys.

Veterinary Sciences↗
DOE OSTI2022

Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) in Minho (Northern Portugal): Bioecology, Native Parasitoid Communities and Biological Control with Torymus sinensis Kamijo (Hymenoptera: Torymidae)

The Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu, native to China, was first detected in Europe in Italy in 2002. In Portugal it was declared in 2014, and it has since affected the Portuguese chestnut production. The introduction of its natural parasitoid Torymus sinensis Kamijo started through inoculative releases according to the National Action Plan for the Control of Dryocosmus kuriphilus, established by the Direção Geral de Alimentação e Veterinária (DGAV), in 2015. This research was carried out during 2018 and 2019, in five chestnut orchards in the Minho region (Northern Portugal). Between January and March 2018, at each location, three monthly harvests of 100 buds were carried out to determine the infestation level. Between April and June 2018, 240 galls were harvested every two weeks, with the purpose of studying the bioecology of D. kuriphilus. In each sampling site, 160 galls were placed in emergence boxes to follow adults of the D. kuriphilus population, and 80 galls were dissected with a binocular magnifying glass, to count and record the evolution of the larval stages of D. kuriphilus and of the parasitoids. The identification of T. sinensis and native parasitoids was carried out. The parasitism rates and Simpson diversity indices were determined. The percentage of buds infested by D. kuriphilus was high in all studied places, varying between 67% and 80%, regardless of the number of T. sinensis releases made and of the chestnut cultivars present in each orchard. The highest rate of parasitism by T. sinensis was 0.5044% and it was recorded in an orchard where there were two releases of T. sinensis in 2016 and 2017 (Arcos de Valdevez—Rio Cabrão—P1). Considering that the exotic parasitoid has already been released in the region since 2016, its establishment in all the chestnut orchards studied was confirmed, although with very low parasitism rates. The natural parasitism associated with the native parasitoid species of D. kuriphilus was confirmed in this work, recording values between 34% and 51% of the parasitized chestnut galls, with 11 species of native parasitoids having been identified: Eupelmus azureos, Eupelmus uruzonus, Eurytoma brunnivientris, Eurytoma pistaciae, Megastigmus dorsalis, Mesopolobus sp., Ormyrus pomaceus, Sycophila biguttata, Sycophila iracemae, Sycophila variegatta and Torymus flavipes.

Amorim, Amaro↗
DOE OSTI2020

Bacteriophage-Induced Lipopolysaccharide Mutations in Escherichia coli Lead to Hypersensitivity to Food Grade Surfactant Sodium Dodecyl Sulfate

Bacteriophages (phages) are considered as one of the most promising antibiotic alternatives in combatting bacterial infectious diseases. However, one concern of employing phage application is the emergence of bacteriophage-insensitive mutants (BIMs). Here, we isolated six BIMs from E. coli B in the presence of phage T4 and characterized them using genomic and phenotypic methods. Of all six BIMs, a six-amino acid deletion in glucosyltransferase WaaG likely conferred phage resistance by deactivating the addition of T4 receptor glucose to the lipopolysaccharide (LPS). This finding was further supported by the impaired phage adsorption to BIMs and glycosyl composition analysis which quantitatively confirmed the absence of glucose in the LPS of BIMs. Since LPSs actively maintain outer membrane (OM) permeability, phage-induced truncations of LPSs destabilized the OM and sensitized BIMs to various substrates, especially to the food-grade surfactant sodium dodecyl sulfate (SDS). This hypersensitivity to SDS was exploited to design a T4–SDS combination which successfully prevented the generation of BIMs and eliminated the inoculated bacteria. Collectively, phage-driven modifications of LPSs immunized BIMs from T4 predation but increased their susceptibilities as a fitness cost. The findings of this study suggest a novel strategy to enhance the effectiveness of phage-based food safety interventions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Dynamic Reconfiguration of Switchgrass Proteomes in Response to Rust ( Puccinia novopanici ) Infection

Switchgrass (Panicum virgatum L.) can be infected by the rust pathogen (Puccinia novopanici) and results in lowering biomass yields and quality. Label-free quantitative proteomics was conducted on leaf extracts harvested from non-infected and infected plants from a susceptible cultivar (Summer) at 7, 11, and 18 days after inoculation (DAI) to follow the progression of disease and evaluate any plant compensatory mechanisms to infection. Some pustules were evident at 7 DAI, and their numbers increased with time. However, fungal DNA loads did not appreciably change over the course of this experiment in the infected plants. In total, 3830 proteins were identified at 1% false discovery rate, with 3632 mapped to the switchgrass proteome and 198 proteins mapped to different Puccinia proteomes. Across all comparisons, 1825 differentially accumulated switchgrass proteins were identified and subjected to a STRING analysis using Arabidopsis (A. thaliana L.) orthologs to deduce switchgrass cellular pathways impacted by rust infection. Proteins associated with plastid functions and primary metabolism were diminished in infected Summer plants at all harvest dates, whereas proteins associated with immunity, chaperone functions, and phenylpropanoid biosynthesis were significantly enriched. At 18 DAI, 1105 and 151 proteins were significantly enriched or diminished, respectively. Many of the enriched proteins were associated with mitigation of cellular stress and defense.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Impact of Oxalic Acid Consumption and pH on the In Vitro Biological Control of Oxalogenic Phytopathogen Sclerotinia sclerotiorum

The phytopathogenic fungus Sclerotinia sclerotiorum has a wide host range and causes significant economic losses in crops worldwide. This pathogen uses oxalic acid as a virulence factor; for this reason, the degradation of this organic acid by oxalotrophic bacteria has been proposed as a biological control approach. However, previous studies on the potential role of oxalotrophy in biocontrol did not investigate the differential effect of oxalic acid consumption and the subsequent pH alkalinisation on fungal growth. In this study, confrontation experiments on different media using a wild-type (WT) strain of S. sclerotiorum and an oxalate-deficient mutant (strain Δoah) with the soil oxalotrophic bacteria Cupriavidus necator and Cupriavidus oxalaticus showed the combined effect of media composition on oxalic acid production, pH, and fungal growth control. Oxalotrophic bacteria were able to control S. sclerotiorum only in the medium in which oxalic acid was produced. However, the deficient Δoah mutant was also controlled, indicating that the consumption of oxalic acid is not the sole mechanism of biocontrol. WT S. sclerotiorum acidified the medium when inoculated alone, while for both fungi, the pH of the medium changed from neutral to alkaline in the presence of bacteria. Therefore, medium alkalinisation independent of oxalotrophy contributes to fungal growth control.

Estoppey, Aislinn (ORCID:0000000347410835)↗
DOE OSTI2022

Effect of Micro-Structural Dispersity of SiMo Ductile Iron on High Temperature Performance during Static Oxidation

High silicon and molybdenum (SiMo) ductile iron is commonly used for car exhaust systems, and its micro-structural dispersity depends on intrinsic parameters, which include alloy composition and inoculation efficiency, as well as extrinsic factors, such as casting wall thickness and molding material, which define cooling rate during solidification. Micro-structural dispersity is referred to as the degree of heterogeneity of sizes of structural constituencies within the microstructure. A variation in the micro-structural dispersity could impact the high temperature performance of SiMo ductile iron during static oxidation and transient thermo-mechanical loading conditions. In this study, static high temperature tests were performed on SiMo ductile iron solidified in a casting with varying wall thicknesses from 5 mm to 100 mm. The faster solidified specimens (taken from near chilled casting surfaces) had extremely high micro-structural dispersity as compared to the thicker section samples. After thermal exposure, each of the samples were characterized using 2D sections and 3D µCT images, and the results indicated an order of magnitude difference in graphite phase dispersity. The surface degradation was quantified after static oxidation experiments were implemented at temperature intervals between 650 °C and 800 °C. Non-destructive µCT 3D analysis and SEM/EDS were performed on cross sections and used to quantify the scale topology and structure. Carbon analysis was used to decouple the scale formation and decarburization phenomena that occurred within the samples. These methods enabled the quantification of the oxidation of the SiMo cast iron with different micro-structural dispersity levels after being exposed to high temperature static oxidation. Additionally, the complex material behavior during oxidation-assisted transient thermo-mechanical loading will be presented in a separate article.

36 MATERIALS SCIENCE↗
DOE OSTI2024

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones

Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Accessing Fungal Contributions to the Birch Effect: Real-Time Respiration from Pore-Scale Microfluidics

Drying and rewetting of soil stimulates soil carbon emission. The Birch effect, driven by these cycles, leads to CO2 efflux, which can be monitored using real-time mass spectrometry (RTMS). Although soil fungi retain water during droughts, their contribution to CO2 release during drying–rewetting cycles remains unclear. In this study, we present the first demonstration of integrating micromodels with RTMS to monitor the Birch effect by simulating drought and rewetting. Micromodels were inoculated with axenic fungal culture and dried to assess moisture retention. After drying, RTMS quantified CO2 release upon rewetting with H218O mixtures. Our results showed that soil fungi released CO2 upon rehydration and immediately utilized the external water source at the pore scale by generating subsequent 46CO2. This work is the first to integrate RTMS with microsystems to investigate pore-scale biogeochemistry and the involvement of fungi in the Birch effect.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Saprotrophic Fungus Induces Microscale Mineral Weathering to Source Potassium in a Carbon-Limited Environment

Plants rely on potassium for many critical biological processes, but most soils are potassium limited. Moving potassium from the inaccessible, mineral-bound pool to a more bioavailable form is crucial for sustainably increasing local potassium concentrations for plant growth and health. Here, we use a synthetic soil habitat (mineral doped micromodels) to study and directly visualize how the saprotrophic fungus, Fusarium sp. DS 682, weathers K-rich soil minerals. After 30 days of fungal growth, both montmorillonite and illite (secondary clays) had formed as surface coatings on primary K-feldspar, biotite, and kaolinite grains. The distribution of montmorillonite differed depending on the proximity to a carbon source, where montmorillonite was found to be associated with K-feldspar closer to the carbon (C) source, which the fungus was inoculated on, but associated with biotite at greater distances from the C source. The distribution of secondary clays is likely due to a change in the type of fungal exuded organic acids; from citric to tartaric acid dominated production with increasing distance from the C source. Thus, the main control on the ability of Fusarium sp. DS 682 to weather K-feldspar is proximity to a C source to produce citric acid via the TCA cycle.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

The Presence of Virus Neutralizing Antibodies Is Highly Associated with Protection against Virulent Challenge in Domestic Pigs Immunized with ASFV live Attenuated Vaccine Candidates

African swine fever virus (ASFV) is currently producing a pandemic affecting a large area of Eurasia, and more recently, the Dominican Republic in the Western Hemisphere. ASFV is a large and structurally complex virus with a large dsDNA genome encoding for more than 150 genes. Live attenuated virus strains can induce protection in domestic swine against disease produced by homologous virulent parental viruses. The roles of the different immune mechanisms induced by the attenuated strains in protection still need to be understood. In particular, the role of ASFV neutralizing antibody in protection still is an important controversial issue to be elucidated. Here we present the development of a novel methodology to detect virus neutralizing antibodies based on the reduction of virus infectivity in a Vero cell adapted ASFV strain. The described method was used to assess levels of virus neutralizing antibodies in domestic swine inoculated with live attenuated ASFV. Results demonstrated a high association between the presence of virus neutralizing antibodies and protection in 84 animals immunized with the recombinant vaccine candidates ASFV-G-Δ9GL/ΔUK or ASFV-G-ΔI177L. To our knowledge, this is the first report demonstrating an association between virus neutralizing antibodies and protection against virulent challenge in such a large number of experimental individuals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

ASF Vaccine Candidate ASFV-G-ΔI177L Does Not Exhibit Residual Virulence in Long-Term Clinical Studies

African swine fever (ASF) is an important disease in swine currently producing a pandemic affecting pig production worldwide. Except in Vietnam, where two vaccines were recently approved for controlled use in the field, no vaccine is commercially available for disease control. Up to now, the most effective vaccines developed are based on the use of live-attenuated viruses. Most of these promising vaccine candidates were developed by deleting virus genes involved in the process of viral pathogenesis and disease production. Therefore, these vaccine candidates were developed via the genomic modification of parental virus field strains, producing recombinant viruses and reducing or eliminating their residual virulence. In this scenario, it is critical to confirm the absence of any residual virulence in the vaccine candidate. This report describes the assessment of the presence of residual virulence in the ASFV vaccine candidate ASFV-G-ΔI177L in clinical studies conducted under high virus loads and long-term observation periods. The results demonstrated that domestic pigs intramuscularly inoculated with 10 6 HAD 50 of ASFV-G-ΔI177L did not show the presence of any clinical sign associated with ASF when observed daily either 90 or 180 days after vaccination. In addition, necropsies conducted at the end of the experiment confirmed the absence of macroscopic internal lesions associated with the disease. These results corroborate the safety of using ASFV-G-ΔI177L as a vaccine candidate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Comparison of the Transcriptome Response within the Swine Tracheobronchial Lymphnode Following Infection with PRRSV, PCV-2 or IAV-S.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory pathogen of swine that has become extremely costly to the swine industry worldwide, often causing losses in production and animal life due to their ease of spread. However, the intracellular changes that occur in pigs following viral respiratory infections are still scantily understood for PRRSV, as well as other viral respiratory infections. The aim of this study was to acquire a better understanding of the PRRS disease by comparing gene expression changes that occur in tracheobronchial lymph nodes (TBLN) of pigs infected with either porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2), or swine influenza A virus (IAV-S) infections. The study identified and compared gene expression changes in the TBLN of 80 pigs following infection by PRRSV, PCV-2, IAV-S, or sham inoculation. Total RNA was pooled for each group and time-point (1, 3, 6, and 14 dpi) to make 16 libraries—analyses are by Digital Gene Expression Tag Profiling (DGETP). The data underwent standard filtering to generate a list of sequence tag raw counts that were then analyzed using multidimensional and differential expression statistical tests. The results showed that PRRSV, IAV-S and PCV-2 viral infections followed a clinical course in the pigs typical of experimental infection of young pigs with these viruses. Gene expression results echoed this course, as well as uncovered genes related to intersecting and unique host immune responses to the three viruses. By testing and observing the host response to other respiratory viruses, our study has elucidated similarities and differences that can assist in the development of vaccines and therapeutics that shorten or prevent a chronic PRRSV infection.

Miller↗
DOE OSTI2023

Optimization of Molecular Methods for Detecting Duckweed-Associated Bacteria

The bacterial colonization dynamics of plants can differ between phylogenetically similar bacterial strains and in the context of complex bacterial communities. Quantitative methods that can resolve closely related bacteria within complex communities can lead to a better understanding of plant–microbe interactions. However, current methods often lack the specificity to differentiate phylogenetically similar bacterial strains. In this study, we describe molecular strategies to study duckweed–associated bacteria. We first systematically optimized a bead-beating protocol to co-isolate nucleic acids simultaneously from duckweed and bacteria. We then developed a generic fingerprinting assay to detect bacteria present in duckweed samples. To detect specific duckweed–bacterium associations, we developed a genomics-based computational pipeline to generate bacterial strain-specific primers. These strain-specific primers differentiated bacterial strains from the same genus and enabled the detection of specific duckweed–bacterium associations present in a community context. Moreover, we used these strain-specific primers to quantify the bacterial colonization of duckweed by normalization to a plant reference gene and revealed differences in colonization levels between strains from the same genus. Lastly, confocal microscopy of inoculated duckweed further supported our PCR results and showed bacterial colonization of the duckweed root–frond interface and root interior. The molecular methods introduced in this work should enable the tracking and quantification of specific plant-microbe associations within plant-microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗