Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “pathogen detection”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A Comparative Metagenomic Analysis of Specified Microorganisms in Groundwater for Non-Sterilized Pharmaceutical Products

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome’s taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436–557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Highly Active Carbon–Platinum-Based Nanozymes: Synthesis, Characterization, and Immunoassay Application

Nanozymes (nanomaterials with intrinsic enzyme-like characteristics) have gained much attention for diagnostics and therapy due to their excellent enzyme-mimicking capability, great stability in environments, and facile and low-cost production. However, developing nanozymes with a high catalytic constant, K cat , has been challenging. Herein, we report a class of nanozyme-mimicking peroxidases, which are formed by depositing ultrasmall platinum nanoparticles (Pt NPs) 1–2 nm in size on the surface of hydrophilic nitrogen-doped carbon nanoparticles (CN NPs). These nanozymes defined as CN-Pt NPs show a high peroxidase-like activity with K cat values of 1.27 M·mL/s·g for 3,3,5,5′-tetramethylbenzidine (TMB) and 1.97 M·mL/s·g for hydrogen peroxide (H 2 O 2 ), respectively, which are at least one or two orders higher than many other reported carbon–noble metal-based nanozymes. Our developed CN-Pt NPs were further utilized in a colorimetric immunoassay as signal amplifiers for the biomarker detection of Burkholderia pseudomallei, a Gram-negative bacterial pathogen classified as a tier 1 select agent by the US CDC. In conclusion, the assay achieved lower limits of detection of 0.11 ng/mL in phosphate-buffered saline (PBS) and 0.16 ng/mL in human serum, when compared to many other assays in detecting the same biomarker.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Redox cycling-based detection of phenazine metabolites secreted from Pseudomonas aeruginosa in nanopore electrode arrays

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa, which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal–insulator–metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM < n pore > ~ 0.082 per nanopore – a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. Here, the NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

A Size-Selectively Biomolecule-Immobilized Nanoprobe-Based Chemiluminescent Lateral Flow Immunoassay for Detection of Avian-Origin Viruses

Here, a signal-amplifiable nanoprobe-based chemiluminescent lateral flow immunoassay (CL-LFA) was developed to detect avian influenza viruses (AIV) and other contagious and fatal viral avian-origin diseases worldwide. Signal-amplifiable nanoprobes are capable of size-selective immobilization of antibodies (binding receptors) and enzymes (signal transducers) on sensitive paper-based sensor platforms. Particle structure designs and conjugation pathways conducive for antigen accessibility to maximum amounts of immobilized enzymes and antibodies have advanced. The detection limit of the CL-LFA using the signal-amplifiable nanoprobe for the nucleoprotein of the H3N2 virus was 5 pM. Sensitivity tests for low pathogenicity avian influenza H9N2, H1N1, and high pathogenicity avian influenza H5N9 viruses were conducted, and the detection limits of CL-LFA were found to be 10 3.5 50% egg infective dose (EID 50 )/mL, 10 2.5 EID 50 /mL, and 10 4 EID 50 /mL, respectively, which is 20 to 100 times lower than that of a commercial AIV rapid test kit. Moreover, CL-LFA demonstrated high sensitivity and specificity against 37 clinical samples. The signal-amplifiable probe designed in this study is a potential diagnostic probe with ultrahigh sensitivity for applications in the field of clinical diagnosis, which requires sensitive antigen detection as evidenced by enhanced signaling capacity and sensitivity of the LFAs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Interaction of amphiphilic lipoarabinomannan with host carrier lipoproteins in tuberculosis patients: Implications for blood-based diagnostics

Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion. The lipoprotein capture assay relies on capture of the host lipoproteins, exploiting the biological association of host lipoprotein with microbial amphiphilic biomarkers to “concentrate” LAM. In contrast, the membrane insertion assay is independent of the association between pathogen amphiphiles and host lipoprotein association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane, which forms the functional surface of an optical biosensor. In this manuscript, we explored the use of these assays for the detection of LAM in sera from adults whose tuberculosis status had been well-characterized using conventional microbiological tests, and endemic controls. Using the lipoprotein capture assay, LAM signal/noise ratios were >1.0 in 29/35 (83%) individuals with culture-confirmed active tuberculosis, 8/13 (62%) individuals with tuberculosis symptoms, but no positive culture for M . tuberculosis , and 0/6 (0%) symptom-free endemic controls. To evaluate serum LAM levels without bias associated with potential differences in circulating host lipoprotein concentrations between individuals, we subsequently processed available samples to liberate LAM from associated host lipoprotein assemblies followed by direct detection of the pathogen biomarker using the membrane insertion approach. Using the membrane insertion assay, signal/noise for detection of serum LAM was greater than that observed using the lipoprotein capture method for culture-confirmed TB patients (6/6), yet remained negative for controls (2/2). Taken together, these results suggest that detection of serum LAM is a promising TB diagnostic approach, but that further work is required to optimize assay performance and to decipher the implications of LAM/host lipoprotein associations for diagnostic assay performance and TB pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

How Cooperative Engagement Programs Strengthen Sequencing Capabilities for Biosurveillance and Outbreak Response

The threat of emerging and re-emerging infectious diseases continues to be a challenge to public and global health security. Cooperative biological engagement programs act to build partnerships and collaborations between scientists and health professionals to strengthen capabilities in biosurveillance. Biosurveillance is the systematic process of detecting, reporting, and responding to especially dangerous pathogens and pathogens of pandemic potential before they become outbreaks, epidemics, and pandemics. One important tool in biosurveillance is next generation sequencing. Expensive sequencing machines, reagents, and supplies make it difficult for countries to adopt this technology. Cooperative engagement programs help by providing funding for technical assistance to strengthen sequencing capabilities. Through workshops and training, countries are able to learn sequencing and bioinformatics, and implement these tools in their biosurveillance programs. Cooperative programs have an important role in building and sustaining collaborations among institutions and countries. One of the most important pieces in fostering these collaborations is trust. Trust provides the confidence that a successful collaboration will benefit all parties involved. With sequencing, this enables the sharing of pathogen samples and sequences. Obtaining global sequencing data helps to identify unknown etiological agents, track pathogen evolution and infer transmission networks throughout the duration of a pandemic. Having sequencing technology in place for biosurveillance generates the capacity to provide real-time data to understand and respond to pandemics. We highlight the need for these programs to continue to strengthen sequencing in biosurveillance. By working together to strengthen sequencing capabilities, trust can be formed, benefitting global health in the face of biological threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Metabolomics Analysis of Bacterial Pathogen Burkholderia thailandensis and Mammalian Host Cells in Co-culture

The Tier 1 HHS/USDA Select Agent Burkholderia pseudomallei is a bacterial pathogen that is highly virulent when introduced into the respiratory tract and intrinsically resistant to many antibiotics. Transcriptomic- and proteomic-based methodologies have been used to investigate mechanisms of virulence employed by B. pseudomallei and Burkholderia thailandensis, a convenient surrogate; however, analysis of the pathogen and host metabolomes during infection is lacking. Changes in the metabolites produced can be a result of altered gene expression and/or post-transcriptional processes. Thus, metabolomics complements transcriptomics and proteomics by providing a chemical readout of a biological phenotype, which serves as a snapshot of an organism’s physiological state. However, the poor signal from bacterial metabolites in the context of infection poses a challenge in their detection and robust annotation. In this work, we coupled mammalian cell culture-based metabolomics with feature-based molecular networking of mono- and co-cultures to annotate the pathogen’s secondary metabolome during infection of mammalian cells. These methods enabled us to identify several key secondary metabolites produced by B. thailandensis during infection of airway epithelial and macrophage cell lines. Additionally, the use of in silico approaches provided insights into shifts in host biochemical pathways relevant to defense against infection. Using chemical class enrichment analysis, for example, we identified changes in a number of host-derived compounds including immune lipids such as prostaglandins, which were detected exclusively upon pathogen challenge. Taken together, our findings indicate that co-culture of B. thailandensis with mammalian cells alters the metabolome of both pathogen and host and provides a new dimension of information for in-depth analysis of the host–pathogen interactions underlying Burkholderia infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds

Abstract Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Integrated Pest Management (IPM) for Early Detection Algal Crop Protection. Final Report

During large-scale, outdoor algal biomass growth, production strain(s) are subject to attack by pathogens, predators, and non-productive competitors, compromising the biomass yield. The project team at UCSD developed and demonstrated the effectiveness of Volatile Organic Compound, VOC, analysis via mass spectrometry (MS) as an early detection system for the presence of production algal ponds infection. The MS detection can then trigger high-resolution melt analysis (HRMA) and enhanced quantitative PCR (qPCR) to identify predatory or pathogenic species precisely. This study gathered the foundational data informing how wholly automated systems monitor the health of production ponds. By detecting the presence of low molecular weight VOCs in the air space above ponds, the instrument was able to mimic “sniffing” the health of the algae pond. This early warning system will be free growers from time-consuming and costly regular surveillance of ponds with the current techniques, such as qPCR and flow-cam assays, which are slower, less sensitive, and not easily fully automated, resulting in increased production efficiency through the reduction of crop loss. This project demonstrated that Chemical Ionization Mass Spectrometry, CIMS, could serve as a real-time monitor of ponds health tracking up to 100 different VOCs simultaneously for continuous periods up to 60 days without interruption, signaling infection within an algal crop 24 to 36 hours prior to techniques such as qPCR and microscopy. While mass spectrometry is an expensive tool, this simple VOC sampling system proved not only to be sensitive and robust but could to multiplexed to monitor several ponds, lowering the overall cost serially. This technology demonstrated a reduction in biomass production cost by approximately 50% compared to growers not employing any detection methodology.

09 BIOMASS FUELS↗
DOE OSTI2022

Construction of chicken infectious anemia virus infectious clone and study on its pathogenicity

Chicken infectious anemia virus (CIAV) can be transmitted through contaminated live poultry vaccine. However, the pathogenicity of contaminated CIAV strains is rarely reported. Previously, the chickens showed the typical symptoms of anemia after using the attenuated live fowl pox virus (FPV) vaccine. Therefore, exogenous CIAV contamination was suspected. We detected anti-CIAV antibodies in SPF chicks vaccinated with the FPV vaccine. CIAV contamination was confirmed in the FPV vaccine, and the CIAV strain was named JS2020-FPV. This study aims to rescue JS2020-FPV by reverse genetic assays and investigate its pathogenicity. Firstly, double-copies infectious clone of JS2020-FPV was constructed. For the pathogenicity study, infectious clone of JS2020-FPV was used to inoculate 1-day-old SPF chicks. The typical symptoms of anemia were observed in the JS2020-PFV group 14 days post inoculation. The hematocrit and body weight of chicks in the JS2020-PFV group were significantly lower than those in the mock group. Notably, the thymus development index and antibody levels of NDV were lower in chicks in the JS2020-PFV group than those in the mock group. Different degrees of apoptosis of MSB1 and DF-1 were observed after inoculated with the JS2020-FPV VP3 recombinant fusion protein expressed by E. coli system, indicating that VP3 induced apoptosis in the transformed cells. Overall, the pathogenicity of the CIAV detected in the contaminated vaccine was confirmed by inoculating SPF chicks with the double-copies infectious DNA clone in this study. Our findings indicate that the dangers of vaccine contamination cannot be ignored.

Wang, Jinjin↗
DOE OSTI2023

Development of a colloidal gold immunochromatographic assay strip using monoclonal antibody for rapid detection of porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) cause diarrhea and dehydration in newborn piglets and has the potential for cross-species transmission. Rapid and early diagnosis is important for preventing and controlling infectious disease. In this study, two monoclonal antibodies (mAbs) were generated, which could specifically recognize recombinant PDCoV nucleocapsid (rPDCoV-N) protein. A colloidal gold immunochromatographic assay (GICA) strip using these mAbs was developed to detect PDCoV antigens within 15 min. Results showed that the detection limit of the GICA strip developed in this study was 10 3 TCID 50 /ml for the suspension of virus-infected cell culture and 0.125 μg/ml for rPDCoV-N protein, respectively. Besides, the GICA strip showed high specificity with no cross-reactivity with other porcine pathogenic viruses. Three hundred and twenty-five fecal samples were detected for PDCoV using the GICA strip and reverse transcription-quantitative real-time PCR (RT-qPCR). The coincidence rate of the GICA strip and RT-qPCR was 96.9%. The GICA strip had a diagnostic sensitivity of 88.9% and diagnostic specificity of 98.5%. The specific and efficient detection by the strip provides a convenient, rapid, easy to use and valuable diagnostic tool for PDCoV under laboratory and field conditions.

Wang, Wei↗
DOE OSTI2023

Targeted metagenomic assessment reflects critical colonization in battlefield injuries

Current diagnostics and clinical management strategies for combat wounds are based on decisions made by expert clinicians. However, even in the hands of experienced surgeons, wounds from combat injuries can exhibit failed healing and complications related to limitations in the rapid and comprehensive generation of diagnostic information. Previous studies have demonstrated the possible use of genomic sequencing approaches to detect microbial signatures involved in combat casualty care. While effective, whole metagenome sequencing is limited by the depth required to confidently detect all relevant signatures. To address this, we developed a targeted capture sequencing panel to detect microbial signatures relevant to wound healing. These targets include known microbial nosocomial pathogens, wound colonizers, and genes involved in virulence and antimicrobial resistance. A bioinformatics pipeline was built to identify genomic regions of interest and over 8,000 oligonucleotide probes were designed for capture. The panel was synthesized and validated using control reference genomes in human background and on wound-effluent samples from a cohort of combat-injured U.S. service members. Our panel was sensitive against wound-colonizing species, Acinetobacter baumannii and Pseudomonas aeruginosa, and was specific in detecting corresponding virulence and antimicrobial-resistance genes as well as other pathogenic species present in microflora mixtures. Random forest feature permutation confirmed the prevalence of Acinetobacter and Pseudomonas in critically colonized wounds and wounds that failed to heal, respectively. Our results demonstrate the capability of targeted sequencing tools and analysis platforms to profile and deliver information on pathogenic factors influencing wound progression, thereby guiding therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa , which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa . The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

Zhu, Xiao-Xuan↗
DOE OSTI2021

Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The ectomycorrhizal fungus Pisolithus microcarpus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis

Significance Plant genomes encode hundreds of genes controlling the detection, signaling pathways, and immune responses necessary to defend against pathogens. Pathogens, in turn, continually evolve to evade these defenses. Small RNAs, such as microRNAs (miRNAs), are one mechanism used by pathogens to overcome plant defenses and facilitate plant colonization. Mounting evidence would suggest that beneficial microbes, likewise, use miRNAs to facilitate symbiosis. Here, we demonstrate that the beneficial fungus Pisolithus microcarpus encodes a miRNA that enters plant cells and stabilizes the symbiotic interaction. These results demonstrate that beneficial fungi may regulate host gene expression through the use of miRNAs and sheds light on how beneficial microbes have evolved mechanisms to colonize plant tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Community Input on the Need, Scope, and Development Roadmap of a Proposed National Virtual Biosecurity for Bioenergy Crops Center (NVBBCC)

Brookhaven National Laboratory (BNL) was awarded a pilot project in FY22 under the U.S. Department of Energy (DOE) Office of Science Biopreparedness Research Virtual Environment (BRaVE) initiative, to define research priorities, needs, and requirements for a national virtual center devoted to the biosecurity of bioenergy crops. The mission of the proposed center, referred to as the National Virtual Biosecurity for Bioenergy Crop Center (NVBBCC), would be to provide the scientific basis and tools to detect, characterize, model, and mitigate biothreats to bioenergy crops. This function will be essential to ensure the projected increased US reliance over the next few decades on key plant-based energy products, such as biojet fuel. The NVBBCC is envisioned as a distributed, virtual center with multiple national laboratories at its core to maximize the use of existing unique facilities and expertise across the DOE complex. A major goal of the pilot project was to develop a roadmap for establishing NVBBCC through a series of meetings to gather community input. A total of about 150 individuals, drawn from DOE laboratories, the USDA, academia, NIH, DHS and the private sector participated in six planning meetings held in FY23. Four of the meetings were focused on specific research topics (disease detection, dispersion and disease propagation, biomolecular characterization of plant-pathogen interaction, and mitigation strategies). These four meetings were followed by a meeting that focused on computational needs to support collaborative, data-intensive research within a distributed center as well as workforce development. A final meeting focused on establishing and maintaining preparedness within NVBBCC to respond to an emerging disease within bioenergy crops and how it would collaborate and coordinate with USDA and DHS.

09 BIOMASS FUELS↗
DOE OSTI2024

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Cell culture systems have greatly expanded our understanding of how bacterial pathogens target signaling pathways to manipulate the host and cause infection. Advances in genetic engineering have allowed for the creation of fluorescent protein readouts within signaling pathways, but these techniques have been underutilized in pathogen biology. Here, we genetically engineered a lung cell line with fluorescent reporters for extracellular signal-related kinase (ERK) and the downstream transcription factor FOS-related antigen 1 (Fra1) and evaluated signaling after inoculation with pathogenic and non-pathogenic bacteria. Cells were inoculated with 100 colony-forming units of Acinetobacter baylyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus agalactiae, or Staphylococcus epidermidis and imaged in a multi-mode reader. The alamarBlue cell viability assay was used as a reference test and showed that pathogenic P. aeruginosa induced significant (p < 0.05) cell death after 8 h in both wild-type and engineered cell lines compared to non-pathogenic S. epidermidis. In engineered cells, we found that Fra1 signaling was disrupted in as little as 4 h after inoculation with bacterial pathogens compared to delayed disruption in signaling by non-pathogenic S. epidermidis. Overall, we demonstrate that low levels of pathogenic versus non-pathogenic bacteria can be rapidly and sensitively screened based on ERK-Fra1 signaling

59 BASIC BIOLOGICAL SCIENCES↗