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United States Department of Agriculture

20 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens
Hana T. Millen 1, Jordan C. Gonnering 1, Ryan K. Berg 2, Susan K. Spencer 3, William E. Jokela 3, John M. Pearce 4, Jackson S. Borchardt 1, Mark A. Borchardt 3
1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

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Biology

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model
Pablo Bermejo-Alvarez 1,2, Ki-Eun Park 1,2, Bhanu P. Telugu 1,2
1Animal Bioscience and Biotechnology Laboratory, United States Department of Agriculture, 2Department of Animal and Avian Sciences, University of Maryland

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

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Chemistry

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds
Isabelle A. Kagan 1, Michael D. Flythe 1
1Forage-Animal Production Research Unit, Agricultural Research Service, United States Department of Agriculture

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper ammonia-producing bacteria (HAB) native to the bovine rumen.

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Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water
Bledar Bisha 1, Jaclyn A. Adkins 2, Jana C. Jokerst 3, Jeffrey C. Chandler 1, Alma Pérez-Méndez 4, Shannon M. Coleman 4, Adrian O. Sbodio 5, Trevor V. Suslow 5, Michelle D. Danyluk 6, Charles S. Henry 2, Lawrence D. Goodridge 7
1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

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Immunology and Infection

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture
Anna M. Biesbrock 1, Christopher M. Powell 1, Wayne B. Hunter 2, Blake R. Bextine 1
1Department of Biology, University of Texas at Tyler, 2U. S. Horticultural Research Laboratory, USDA ARS

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

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Biology

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti
Jackson T. Sparks 1, Joseph C. Dickens 1
1Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Invasive Insect Biocontrol and Behavior Laboratory, United States Department of Agriculture

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

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JoVE Core

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers
Sarah M. Collier 1, Matthew D. Ruark 2, Lawrence G. Oates 3,4, William E. Jokela 5, Curtis J. Dell 6
1Office of Sustainability, University of Wisconsin-Madison, 2Department of Soil Science, University of Wisconsin-Madison, 3Department of Agronomy, University of Wisconsin-Madison, 4Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 5USDA-ARS Dairy Forage Research Center, 6USDA-ARS Pasture Systems Watershed Management Research Unit

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

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JoVE Journal

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections
Caleb Knepper 1, Beiquan Mou 1
1Agricultural Research Service, United States Department of Agriculture

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

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JoVE Core

Preparation and Testing of Plant Seed Meal-based Wood Adhesives
Zhongqi He 1, Dorselyn C. Chapital 1
1Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture

To facilitate the effort in seeking more economic and environment-friendly formulations of natural product-based wood adhesives, this work demonstrates the preparation and testing of plant seed-based wood adhesives. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for the substitution of synthetic-based wood adhesives.

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Immunology and Infection

Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
Mayara F. Maggioli 1,2, Mitchell V. Palmer 1, H. Martin Vordermeier 3, Adam O. Whelan 3, James M. Fosse 4, Brian J. Nonnecke 1, W. Ray Waters 1
1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 2Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, 3UK Veterinary Laboratories Agency, 4Visual Services, National Centers for Animal Health, Animal and Plant Health Inspection Service, United States Department of Agriculture

Long-term cultured interferon-γ enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

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Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa
Xiangyang Shi 1, Hong Lin 2
1Department of Plant Science, University of California, Davis, 2Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, United States Department of Agriculture

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce's disease in grapevine.

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Environment

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass
Gregory W. O'Neil 1, John R. Williams 1, Julia Wilson-Peltier 1, Gerhard Knothe 2, Christopher M. Reddy 3
1Department of Chemistry, Western Washington University, 2Agricultural Research Service, United States Department of Agriculture, 3Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

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Bioengineering

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System
Anjali Nandal 1,2, Barbara Mallon 3, Bhanu P. Telugu 1,2,4
1Department of Animal and Avian Sciences, University of Maryland, 2Animal Bioscience and Biotechnology Laboratory, ARS, USDA, 3NIH Stem Cell Unit, Bethesda, National Institutes of Health, 4RenOVAte Biosciences Inc

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

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Environment

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects
Saikat Kumar B. Ghosh 1, Wayne B. Hunter 2, Alexis L. Park 1, Dawn E. Gundersen-Rindal 1
1Invasive Insect Biocontrol and Behavior Laboratory, Agricultural Research Service, United States Department of Agriculture, 2Horticultural Research Laboratory, Agricultural Research Service, United States Department of Agriculture

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

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Environment

Detection of Viruses from Bioaerosols Using Anion Exchange Resin
Joshua W. Schaeffer 1, Jeffrey C. Chandler 2, Margaret Davidson 1,3, Sheryl L. Magzamen 1, Alma Pérez-Méndez 4, Stephen J. Reynolds 1, Lawrence D. Goodridge 5, John Volckens 6, Alan B. Franklin 2, Susan A. Shriner 2, Bledar Bisha 7
1High Plains Intermountain Center for Agricultural Health and Safety, Department of Environmental and Radiological Health Sciences, Colorado State University, 2National Wildlife Research Center, Wildlife Services, Animal and Plant Health Inspection Service, United States Department of Agriculture, 3Western Sydney University, 4Leprino Foods, Inc, 5Department of Food Science and Agricultural Chemistry, McGill University, 6Department of Mechanical Engineering, Colorado State University, 7Department of Animal Science, University of Wyoming

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

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Environment

Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon
Xia Li 1,2, Greg W. McCarty 2
1Department of Geographical Sciences, University of Maryland, 2Hydrology & Remote Sensing Laboratory, Agricultural Research Service, United States Department of Agriculture

Landscape processes are critical components of soil formation and play important roles in determining soil properties and spatial structure in landscapes. We propose a new approach using stepwise principal component regression to predict soil redistribution and soil organic carbon across various spatial scales.

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Genetics

Screening Cotton Genotypes for Reniform Nematode Resistance
John E. Erpelding 1, Salliana R. Stetina 1
1Crop Genetics Research Unit, Agricultural Research Service, United States Department of Agriculture

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

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Environment

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments
Joseph Funderburk 1, Xavier Martini 1, Josh Freeman 1, Iris Strzyzewski 1, Edward Traczyk 1, Thomas Skarlinsky 2, Scott Adkins 3
1North Florida Research and Education Center, University of Florida, 2Animal and Plant Health Inspection Services, Plant Protection Quarantine, Miami Plant Inspection Station, United States Department of Agriculture, 3Agricultural Research Service, US Horticultural Research Laboratory, United States Department of Agriculture

Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs.

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Bioengineering

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay
Jana A. Hassan 1, Ilea J. Chau-Ly 1, Jennifer D. Lewis 1,2
1Department of Plant and Microbial Biology, University of California, Berkeley, 2Plant Gene Expression Center, United States Department of Agriculture

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

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Genetics

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira
L. G. V. Fernandes 1,2, R. L. Hornsby 1, A.L.T.O. Nascimento *2, J. E. Nally *1
1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 2Laboratório de Desenvolvimento de Vacinas, Instituto Butantan

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

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