USDA/ARS 2005 Report: Mid West Area (Peoria, Ames) Reports

On this page... 3602-32000-005-00D 3620-42000-024-00D 3620-42000-026-00D 3620-42000-027-00D 3620-42000-030-00D 3620-22000-007-00D 3620-22000-008-00D 3620-42000-028-00D 3620-42000-031-00D 3625-32000-056-00D 3625-32000-069-00D 3625-31320-001-00D 3625-32000-051-00D 3625-32000-052-00D 3625-32000-053-00D Table of Contents Next 3602-32000-005-00D Project Title: Handling and Transport stress interactions with Pathogen Biology in Livestock Project No: 3602-32000-005-00D SY(s): Lay Jr, Donald; Eicher, Susan; Rostagno, Marcos Location: West Lafayette, Indiana Livestock Behavior Research What was the single most significant accomplishment this past year? The Stress Response Alters Salmonella Biology: In collaboration with researchers at NADC, we discovered that Salmonella can can grow in an acid environment after they have been exposed to the hormone norepinephrine. This is the first evidence to suggest that transportation stress in swine can actually enhance the virulence of salmonella infection, and thus increase the opportunity of post-harvest contamination. A complete understanding of this phenomenon will allow the development of strategies to decrease the amount of Salmonella found on the carcasses at slaughter. Back to top 3620-42000-024-00D Project Title: Control of Fusarium Verticillioides, Fumonisins and Fusarium Diseases in corn and small grains Project No: 3620-42000-024-00D SY(s): Proctor, Robert; Desjardis, Anne; Brown, Daren; Kendra, David; Butchko, Robert; Busman, Mark Location: National Center for Agricultural Utilization Research Mycotoxin Research What was the single most significant accomplishment this past year? Title: Development of a F. verticillioides microarray. To better understand the genetic pathways that regulate fumonisin production in F. verticillioides and to identify genes that contribute to the ability of this fungus to infect corn and cause ear rot, we continued to develop genomic tools for F. verticillioides. In FY 05, we generated a F. verticillioides microarray in collaboration with the Institute for Genomic Research (TIGR). The microarray consists of >11,000 F. verticillioides gene sequences all of which are present on a glass slide. The 11,000 gene sequences present in the microarray were obtained from the F. verticillioides Expressed Sequence Tag (EST) database, which we completed during FY 04 in collaboration with TIGR. The microarray can be hybridized to ribonucleic acid (RNA) isolated from F. verticillioides grown under one or multiple conditions to determine which genes are expressed (turned on) or not expressed (turned off) under the different conditions. Thus, the microarray is a tool for monitoring the expression of all 11,000 gene sequences simultaneously. Previous technology allowed for analysis of expression of one gene at a time. Therefore, microarray analysis is a significant advancement in gene expression analysis that should enhance our ability to study the regulation of fumonisin biosynthesis and the interactions of F. verticillioides and corn. Such advancements should in turn aid in the development of strategies to control the presence of fumonisins in U.S. corn. List other significant accomplishments, if any. Title: Identification and characterization of a gene located next to the fumonisin biosynthetic gene cluster that regulates fumonisin production. Analysis of the F. verticillioides Expressed Sequence Tags (ESTs) database revealed the presence of a gene, designated FUM21, that is located adjacent to the fumonisin biosynthetic gene cluster in F. verticillioides. Functional analysis of FUM21 suggests it is a transcriptional regulator of fumonisin biosynthetic genes. Identification of FUM21 is critical breakthrough in understanding the regulation of fumonisin biosynthesis. Title: Identification of fumonisin-insensitive corn. To determine whether sensitivity of corn to fumonisin mycotoxins can affect fumonisin contamination in corn, 42 genetically diverse land races of corn were screened in a laboratory assay for their sensitivity to fumonisins. Seeds of each land race were germinated on water agar containing up to 300 mu-M fumonisin B1. Only two land races were highly insensitive (ED50 200 mu-M). Genetic analysis revealed that the high levels of insensitivity were an inheritable trait. Title: Functional analysis of regulatory genes from EST database. To better understand how fumonisin biosynthesis is regulated, the F. verticillioides Expressed Sequence Tag (EST) database was used to identify genes that potentially regulate the fumonisin biosynthetic gene (FUM) cluster. Thirty sequences with similarity to known fungal regulatory genes and that were present in the "FUM genes on" EST library but absent in the "FUM genes off" library were identified. Disruption plasmids for 20 candidate genes were engineered and transformed into F. verticillioides in order to inactivate each gene independently. Disruption mutants for six of the genes lost the ability to accumulate FUM gene transcripts. List any significant activities that support special target populations. None. Progress report. To determine if production of polyketide compounds in Fusarium verticillioides contributes to the ability of this fungus to cause corn ear rot, gene disruption vectors were constructed for each of the 15 polyketide synthase genes that have been identified in this fungus. To date, 8 polyketide synthase genes have been disrupted. We determined that one of these genes is required for production of the polyketide mycotoxin fusarin, and we have begun corn ear rot tests in the field to determine if any strains with a disrupted polyketide synthase gene is less able to cause ear rot than the wild-type strain from which it was derived. To examine the possibility of using fumonisin-nonproducing mutants as biological control agents to reduce fumonisin contamination in corn, the ability of fumonisin-producing and nonproducing strains to colonize corn seedlings and to compete with each other was examined. Both producing and nonproducing strains were able to colonize corn seedling tissue. In addition, a nonproducing strain was able to exclude a producing strain when applied prior to the producing strain. To determine the potential impact of two cryptic species of Fusarium subglutinans on US agriculture, twenty strains of each cryptic species were examined for their ability to produce moniliformin and to cause corn ear rot in the field. Individual strains varied in their ability to produce moniliformin, but all strains caused high levels of corn ear rot. A paper on sensitivity of corn to fumonisins was submitted for publication and a paper on the F. verticillioides EST database was submitted and published. Back to top 3620-42000-026-00D Project Title: Control of Fusarium Graminearum Mycotoxins in wheat, barley and corn Project No: 3620-42000-026-00D SY(s): Alexander, Nancy; McCormick, Susan; Proctor, Robert; Kendra, David; Desjardins, Anne; Busman, Mark Location: National Center for Agricultural Utilization Research Mycotoxin Research What was the single most significant accomplishment this past year? Butenolide biosynthesis: To further define the role of other Fusarium mycotoxins in pathogenesis, we, along with our colleagues at Ag Canada, characterized a gene that is highly expressed in a genomic library of wheat infected with Fusarium. This gene is involved in the biosynthesis of butenolide, a mycotoxin. However, it does not appear to play a role in virulence of Fusarium on wheat. This was accomplished by knocking out the function of a gene involved in the biosynthesis of butenolide and then analyzing the mutants for virulence on wheat in greenhouse assays. This result is valuable for developing tools for the analysis of genes found by genomic research. List other significant accomplishments, if any. Heterologous expression of mycotoxin genes: In order to fully characterize genes in the biosynthetic pathway of Fusarium mycotoxins, we developed a unique transgenic expression system. We cloned two specific genes involved with mycotoxin production and, individually, placed them behind a promoter from another Fusarium species that does not produce trichothecenes, and then analyzed the resulting mutants. We clarified the function of these specific genes. This transgenic expression system will be helpful in analyzing the many genes that are being identified in genomic projects. Maize ear rot testing system: We developed a greenhouse virulence assay for maize ear rot using the cultivar, Gaspe flint. We determined the best conditions for growth and flowering of this maize cultivar in the greenhouse and tested for corn ear rot infection using known lines of Fusarium. This set the groundwork for tests to be done in FY06 to determine virulence in mycotoxin-producing lines of Fusarium. Greenhouse testing minimizes the risk to the environment of testing lines of Fusarium. Progress report. We have continued to define the trichothecene biosynthetic pathway by manipulating expression of a gene responsible for negative regulation and by measuring expression of genes that are responsible for critical biosynthetic steps. We are using fungal genomics approaches to continue identification and characterization of Fusarium graminearum genes expressed during the plant-fungal interaction. Back to top 3620-42000-027-00D Project Title: Develop and Evaluate rapid and sensitive methods to detect mycotoxins using novel sensors Project No: 3620-42000-027-00D SY(s): Marago, Chris; Dombrink Kurtzman, Mary Ann; Kendra, David; Appell, Michael Location: National Center for Agricultural Utilization Research Mycotoxin Research What was the single most significant accomplishment this past year? Moniliformin is a mycotoxin produced by several fungi that are pathogenic to cereal grains. Moniliformin is acutely toxic to both animals and plants, and is found in maize, wheat, rye, rice, and oats worldwide. A sensitive, reproducible, and reliable analytical method to separate and quantify moniliformin was developed and applied to samples of field-inoculated maize. The method developed involves the separation of the moniliformin using a strong electrical field (capillary electrophoresis). The method is more rapid than traditional liquid chromatographic methods, and may find use in measurement of this mycotoxin in maize. List other significant accomplishments, if any. Conditions for production of patulin, specifically Penicillium strains and composition of culture media to use were identified. This will facilitate the production and isolation of patulin and studies of patulin biosynthesis. List any significant activities that support special target populations. None. Progress report. Molecularly Imprinted Polymers (MIPs) for moniliformin were synthesized, and their binding efficacies were evaluated. Patulin MIPs and control polymers synthesized using ultraviolet (UV) photo-initiated polymerization at low temperatures in the Department of Chemistry, University of California, Irvine, were processed and evaluated for binding of patulin at NCAUR. When evaluated using equilibrium binding assays there were no differences between the patulin-imprinted and control polymers. Partial sequences of the isoepoxydon dehydrogenase (idh) gene and the ITS1, 5.8S gene, ITS2 and 28S gene (nuclear large rDNA subunit) were identified for 8 strains of Penicillium that produce patulin. Back to top 3620-42000-030-00D Project Title: Critical Control Points in corn Resistance/Susceptibility to Aspergillus Flavus and Aflatoxin Project No: 3620-42000-030-00D SY(s): Wicklow, Donald; Brown, Daren; Kendra, David; Vacant; Vacant Location: National Center for Agricultural Utilization Research Mycotoxin Research What was the single most significant accomplishment this past year? None. List other significant accomplishments, if any. Mycoparasitic fungi are proving to be rich sources of antifungal protein/genes that can be expressed in crops, conferring resistance against fungal pathogens. Several mycoparasites isolated from A. flavus sclerotia, or as colonists of fungus-infected corn kernels at harvest, were shown to produce extracellular proteins with potent antifungal activity against A. flavus and F. verticillioides. Gliocladium catenulatum, a soil fungus known to be a mycoparasite of Aspergillus flavus sclerotia displayed potent extracellular chitinase activity in a liquid minimal medium supplemented with colloidal chitin. Yields of extracellular chitinase were substantially increased by optimizing selected growth parameters. Microbial endophytes of cereals represent under explored sources of antifungal proteins and metabolites that can suppress fungal growth or silence genes critical to mycotoxin synthesis while also being adapted to function in planta. We recently reported that the protective corn endophyte Acremonium zeae produces pyrrocidines A and B, antibiotics that display significant antifungal activity in assays against A. flavus and F. verticillioides. Further evaluation of the antimicrobial activity of pyrrocidine A revealed potent antibiotic activity against two major stalk and ear rot pathogens of maize Fusarium graminearum and Stenocarpella maydis (syn. Diplodia maydis), in addition to the ear and kernel rotting pathogens Nigrospora oryzae, Penicillium oxalicum, and Cladosporium cladosporioides. It was also shown that pyrrocidines A and B exhibit potent antibiotic activity against Clavibacter michiganense subsp. Nebraskense, an important bacterial pathogen of maize, as well as Bacillus mojaviense and Pseudomonas fluorescens, bacterial endophytes of maize used as biocontrol agents. In 2005 we developed a scalable method for the isolation and purification (crystallization) of pyrrocidines A and B. There is an urgent need for new sources of antifungal agents and fungi that parasitize and kill other fungi offer a potential source of novel antifungal agents useful to agriculture and medicine. Using the two most important mycotoxin producers A. flavus and F. verticillioides as targets, a university scientist in Iowa City, IA, in collaboration with an ARS scientist in Peoria, IL, have isolated and identified numerous antifungal metabolites present in culture extracts of fungi that grow on larger fungi. These ongoing studies are contributing to a growing data base that will be useful in interpreting structure function relationships and potential cellular targets in Aspergillus and Fusarium. This research is supported, in part, with funds provided by the National Science Foundation. In 2005, our library of pure compounds and crude extracts has produced multiple "priority hits" with pharmaceutical targets including the microbial panel, HIV panel, 60 tumor cell line panel, angiogenesis inhibitor activity, retroviral RNase H inhibitor activity, and diffuse large B cell lymphoma activity, in tests performed through the "NIH Inter-Institute Program for the Development of AIDS-Related Therapeutics" (National Cancer Institute & National Institute for Allergy and Infectious Diseases, Frederick, Maryland). List any significant activities that support special target populations. None. Progress report. Current research, in summary, has resulted in the successful classification of individual white corn kernels showing visible symptoms of fungal infection, with special emphasis given to kernels infested with F. verticillioides and contaminated with fumonisins. A target gene list of putative F. verticillioides regulatory genes has been produced that appear to be differentially expressed under growth conditions that include a maize plant component. Developed a scalable method for the isolation and purification (crystallization) of pyrrocidines A and B from the protective maize endophyte Acremonium zeae. Several mycoparasites isolated from A. flavus sclerotia, or as colonists of fungus-infected corn kernels at harvest, were shown to produce chitinase and other extracellular proteins with potent antifungal activity against A. flavus and F. verticillioides. Discovered several new antifungal antibiotics, produced by mycoparasitic fungi, with potent activity against the mycotoxin fungi Aspergillus flavus and Fusarium verticillioides. Back to top 3620-22000-007-00D Project Title: Insect-Plant-Microorganism interations affecting corn quality and safety Project No: 3620-22000-007-00D SY(s): Bartelt, Robert; Petroski, Richard; Cosse, Allard Location: National Center for Agricultural Utilization Research Crop Bioprotection Research What was the single most significant accomplishment this past year? See report for 3620-22000-008-00D. Back to top 3620-22000-008-00D Project Title: Identification and Practical use of semiochemicals for the management of Agriculturally important insects Project No: 3620-22000-008-00D SY(s): Bartelt, Robert; Cosse, Allard; Petroski, Richard Location: National Center for Agricultural Utilization Research Crop Bioprotection Research What was the single most significant accomplishment this past year? Chemical identification was completed for the male-produced aggregation pheromone of Galerucella calmariensis (a beetle species that is a biological control agent of purple loosestrife), the compound was synthesized, and its attractiveness was verified under field conditions. This accomplishment contributes significantly to the basic understanding of the chemistry and biology of chrysomelid beetle pheromones. A small (17-microgram) sample of the compound was accumulated during a 2-year period at NCAUR from beetles obtained from the Illinois Natural History Survey (in collaboration with Robert Wiedenmann and Susan Post). A key analytical step was nuclear magnetic resonance (NMR) spectroscopy using a specially configured, highly sensitive instrument (at Pfizer Global Manufacturing, Kalamazoo, MI, in collaboration with Stephen Grode). The compound, a novel dimethylfuran lactone, was subsequently synthesized at NCAUR and successfully field tested in Northeastern Illinois by personnel from NCAUR and the Illinois Natural History Survey. The pheromone has promise to become a practical tool for monitoring/manipulating populations of this important biocontrol agent. List other significant accomplishments, if any. The major component of the male-produced aggregation pheromone of the eggplant flea beetle, Epitrix fuscula, was chemically identified as a novel nine-carbon aldehyde with three double bonds, synthesized, and successfully field tested for attractiveness. This accomplishment adds to the understanding of the pheromone chemistry and biology of flea beetles, which include serious crop pests as well as important biological control agents. The compound was collected after being emitted from feeding male beetles and was analyzed and synthesized at NCAUR. Field tests at a local organic farm showed good attraction to the synthetic compound. The pheromone has potential as a pest management tool. List any significant activities that support special target populations. None. Progress report. Good progress has been made on an improved (5-step instead of the original 7-step) synthesis of nitidulid beetle (Carpophilus spp.) pheromones. While the pheromones are already commercially available (from Great Lakes IPM, Vestaburg, MI) and are in use in the U.S. and Australia, the new synthesis has the potential to lower the cost of production and improve commercial viability. Back to top 3620-42000-028-00D Project Title: Insect Management for Reduction of Mycotoxigenic Fungi in Midwest Corn Project No: 3620-42000-028-00D SY(s): Dowd, Patrick; Slinger, Patricia; Johnson, Eric Location: National Center for Agricultural Utilization Research Crop Bioprotection Research What was the single most significant accomplishment this past year? Insect damage and associated ear mold toxins cause hundreds of millions of dollars in losses each year. ARS scientists evaluated corn plants that expressed a novel plant-derived gene that produces a protein type we previously demonstrated would kill insects, for enhanced insect resistance. Percent mortality of the representative caterpillar pest was significantly correlated with expression of the gene as detected histochemically. Further validation and optimization of expression and form of the protein should allow for a useful new insect resistance mechanism. Incorporation of these gene types into corn through transgenic means should result in reduced levels of mycotoxins, thereby improving the health of animals and people, and increasing the exportability of U.S. corn. List other significant accomplishments, if any. 1. Insect damage and associated ear mold toxins cause hundreds of millions of dollars of losses each year. ARS scientists evaluated hybrid transgenic plants in a model plant system that expressed two plant-derived genes they previously showed could independently promote enhanced resistance to insects. The plants that expressed both the proteins had significantly less damage by caterpillar and beetle pests, and in some cases significantly enhanced mortality, compared to corresponding plants that did not produce the proteins at the same level. Incorporation of these genes into corn through multigenic transgenic means should result in reduced levels of mycotoxins, thereby improving the health of animals and people, and increasing the exportability of U.S. corn. 2. Insect damage and associated ear mold toxins cause hundreds of millions of dollars of losses each year. ARS scientists, working with farmers, examined different management strategies of potential use in reducing mycotoxins in corn in the field. Bt versions of preferred hybrids, early planting to escape caterpillar damage, scouting of damaging insects including lures and traps identified in project research, fungal monitoring using leaf axil material, and a predictive computer program indicating when mycotoxin-producing fungi are present, and specific mycotoxin levels that may occur without intervention by the farmer, could be rationally combined into a management plan. Application of the management plan should result in reduced levels of mycotoxins, thereby improving the health of animals and people, and increasing the exportability of U.S. corn. List any significant activities that support special target populations. A predictive computer program used as part of an ear mold toxin (mycotoxin) management program underway with representative farmers from a 200 farmer organization again provided useful predictions of fumonisins and aflatoxins in 2004, predicting values close to those actually encountered. A compiled, Windows version was received from cooperators at a local college has been revised into a version that is nearly ready to be made public. Once made widely available, this computer program should allow farmers to more economically produce larger amounts of healthier, high-quality corn with reduced levels of mycotoxins. This higher quality product should help the farmers to increase sales, and help livestock productivity of farmers using corn for on farm animal feed. Progress report. Plant pigments produced in food plants that contribute to insect resistance have been identified. A health-related food plant secondary metabolite was found to be significantly active against insects. A gene construct potentially useful in increasing the site specificity of transgene insertion was found to be active in a corn system. A corn-derived gene potentially useful as a selectable marker was cloned, sequenced, and introduced into a model system where it conferred resistance to a selecting agent. Back to top 3620-42000-031-00D Project Title: Molecular Genomics and rapid Diagnostics of plant pathogens and food safety microorganisms Project No: 3620-42000-031-00D SY(s): Kurtzman, Cletus; Ward, Todd; Peterson, Stephen; ODonnell, Kerry; Rooney, Alejandro Location: National Center for Agricultural Utilization Research Microbial Genomics and Bioprocessing Research What was the single most significant accomplishment this past year? A system for rapid identification of microorganisms was developed. This system is based on the Luminex flow cytometer (Luminex Corporation, Austin, Texas), in which species-specific DNA probes are attached to small plastic spheres and reacted with the DNA of species to be identified. The technology allows up to 100 species-specific probes to be used in combination, which can be placed in a single well of a 96-well plate. Identification time, starting with a microbial culture, is 8 hours. The procedure has been successfully used to identify up to 30 different clinical yeasts (to be published in the September 2005 issue of the Journal of Clinical Microbiology), and is now being applied to detection of lineages of the foodborne pathogen Listeria monocytogenes, species of the plant pathogenic and mycotoxigenic genus Fusarium and species in the aflatoxin producing Aspergillus flavus complex. The successful application of this system is based on the large database of gene sequences determined by the staff of the Microbial Genomics and Bioprocessing Research Unit (MGB). List other significant accomplishments, if any. A single-well multilocus genotyping (MLGT) assay was developed for L. monocytogenes subtype identification based on nucleotide variation identified in 22 genes (23 Kilobase (Kb) of DNA sequence from 65 isolates) from seven regions of the Listeria chromosome. The current assay utilizes 59 SNP probes to distinguish among the closely related strains within lineage 1 of L. monocytogenes, which are responsible for the majority of human listeriosis outbreaks. Application of the assay to 175 additional lineage 1 isolates and comparison of MLGT results with strain histories demonstrated that the assay was able to uniquely identify isolates from each of the eight different listeriosis outbreaks examined, and was able to differentiate epidemiologically relevant groups of strains (serotypes 4b and 1/2b, and epidemic clones 1, 1a and 2). These results indicate that MLGT represents a significant new tool for use in epidemiological investigations as well as studies of the ecology, evolution, and population dynamics of L. monocytogenes. The goal of the research on Clostridium perfringens was to define distinct genetic lineages within this species on the basis of phylogenetic clustering patterns reconstructed from gene sequence data. Sequence data has been collected from seven genes (five housekeeping, two virulence) covering of a total of 6 Kb from each of 265 food, veterinary and human clinical isolates. Currently, the genes that we have sequenced (plc, colA, perfringolysin O regulator S (pfoS), histidine Kinase (hisK), gyrase subunit A (gyrA), rpoB, and ribosomal binding protien L7/L12 (rplL)) represent two distinct regions of the genome. The first region surrounds the origin of replication and extends from rpoB to colA and contains five of the seven genes that we have sequenced. The second region is on the opposite end of the genome (approximately 1.2 Megabase (Mb) from the other region). We plan to expand the number of genes from this region to 5 in order to obtain a total of 10 Kb of sequence data (approximately 5 Kb from each region). These additional data will be used to test for phylogenetic incongruence between genes within each genomic region and to test for incongruence between entire genomic regions. Over 100 strains representing food and beverage spoilage yeasts in the genus Zygosaccharomyces were identified to species from their unique gene sequences. Additional genes are being sequenced for each of the strains to determine population structure and genetic markers that may signal specificity for spoilage of particular food and beverage products. Three new food spoilage yeasts were described and placed in the genera Saturnispora, Tetrapisispora and Kregervanrija, the latter genus also newly described. Genetic boundaries of OA producing species in Aspergillus section Circumdati were determined through multi-locus DNA sequence analysis. Adding 200 new isolates from section Circumdati to the basic species set has revealed the presence of additional species that have been described by colleagues in Europe. OA is regulated in many countries, and American fruits and coffee are potential targets for the OA producing molds. OA is the putative cause of kidney failure of human populations chronically exposed to this mycotoxin. We have used multilocus DNA sequence data and multiplex polymerase chain reaction (PCR) assays to establish a baseline of FHB species and trichothecene toxin chemotype diversity globally. These studies have shown for the first time that the primary etiological agent of FHB, Fusarium graminearum, comprises at least 12 phylogenetically distinct and biogeographically structured species worldwide. Although F. graminearum accounts for over 99% of FHB within North America, three additional Fg clade species, including two recently introduced foreign FHB pathogens, were detected in our survey of FHB within the U.S. To facilitate communication among scientists within the FHB community and quarantine specialists, eight unnamed species within the Fusarium graminearum (Fg) clade resolved by our multilocus phylogeny were formally described (O’Donnell et al. 2004). Development of robust PCR multiplex and SNP-based molecular tools for Fg clade species identification and chemotype determination have significantly improved global monitoring efforts and disease surveillance, thereby making available for the first time detailed information on the host and geographic distributions of FHB pathogens and their trichothecene chemotypes. Such knowledge is critical for enhancing our knowledge of the ecology, epidemiology and population biology of these mycotoxigenic cereal pathogens. Our studies are the first to alert plant breeders charged with developing wheat and barley cultivars with broad-based resistance to FHB that the morphospecies Fusarium graminearum comprises at least 12 phylogenetically distinct and biogeographically structured species and that half of these species are still segregating for trichothecene toxin chemotype. Furthermore, our studies alert plant disease specialists and quarantine officials that only a fraction of the FHB pathogen and toxin chemotype diversity is currently represented within North America. Providing names for the newly discovered FHB pathogens should greatly facilitate communication among scientists within the FHB community, including quarantine specialists. Moreover, the unique multilocus DNA sequence database we have developed is currently being interrogated to develop the first SNP microsphere array for the high-throughput identification of all known B-trichothecene toxin-producing FHB species and their toxin chemotypes in order to improve disease surveillance efforts and to facilitate a greater understanding of the ecology, epidemiology and population dynamics of these FHB pathogens. This research has been funded in part by competitive research grants with the U.S. Wheat and Barley Scab Initiative (USWBSI) grant and the U.S. Department of Agriculture (USDA), Cooperative State Research Education Extension Service (CSREES), National Research Initiative (NRI), Competitive Grants Program and with the aid of an administer post-doc position. Back to top 3625-32000-056-00D Project Title: Swine Manure Management Effects on Zoonotic Pathogens in the Environment Project No: 3625-32000-056-00D SY(s): Ziemer, Cherie; Kerr, Brian Location: Ames, Iowa National Soil Tilth Reseearch Laboratory Swine Odor and Manure Management Research What was the single most significant accomplishment this past year? Development of group specific DGGE assays: Denaturing gradient gel electrophoresis (DGGE) was recently adapted to examine two of the predominant clostridial groups in swine intestinal, fecal, and manure samples. Data from this research demonstrates that this method allows for a higher resolution comparison of the dynamics of targeted bacterial groups in swine samples. Consequently, this data can be utilized by other scientists to assist in developing techniques or methods to reduce the prevalence of this bacteria in swine production facilities. List other significant accomplishments, if any. Assessments of quantitative PCR assays, although the two primer sets assessed were not suitable for our samples we are closer to having this operational in our lab. Back to top 3625-32000-069-00D Project Title: Charcterization and Enhancement of Immune Responses of Calves Project No: 3625-32000-069-00D SY(s): Nonnecke, Brian; Goff, Jesse Location: Ames, Iowa National Animal Disese Center Periparturient Diseases of Cattle What was the single most significant accomplishment this past year? Characterized the effects of increased dietary protein and energy on adaptive (i.e., antigen-specific) immune responses in milk replacer-fed dairy calves: Increased dietary protein and energy affected the composition of blood-borne immune cell populations, specifically the percentages of CD4**+ and gammadelta-TCR**+ T cells, and monocytes. Antigen-induced interferon-gamma or nitric oxide secretion by in vitro and in vivo reactivity to antigen (i.e., cutaneous delayed-type hypersensitivity reaction); however, were not affected by plane of nutrition, suggesting differences in protein and energy between the two treatments were not sufficient to influence antigen-specific recall responses. General reactivity (i.e., mitogen-induced IFN-gamma and NO secretion and expression of markers of activation (i.e., CD25, CD44, and CD62L cell surface molecules]) of blood leukocyte populations was affected by increased protein and energy. Additional research is necessary to determine if these nutrition dependent changes in immune function influence the susceptibility of the calf to infectious disease. Infectious diseases of calves, especially diarrheal and respiratory diseases are a major economic concern for the U.S. cattle industry. They also impact human health and food safety through pre-harvest contamination, and zoonotic transmission. More than 40% of neonatal calves on U.S. dairy farms are either clinically ill or die during the neonatal period. Morbidity and mortality of preweaned calves cost the industry between $90-$180 million annually. Customers of the research include dairy cattle producers, supporting industries, scientists, and consumers wanting a safer food supply. List other significant accomplishments, if any. Demonstrated adult-like cell-mediated immune responses in neonatal calves vaccinated during the first week of life: Research results indicate that early vaccination of the neonatal calf produces strong and sustained antigen specific, cell-mediated immune responses comparable to responses of vaccinated adult dairy cattle. In vivo and in vitro antibody responses of the vaccinated calf, however, were weaker than corresponding responses of adults suggesting the neonatal calf may be limited in its capacity to mount a humoral (i.e., antibody-based) immune response. To determine if the weak or nonexistent humoral responses of the calf were specific to the sensitization/challenge model, newborn calves were vaccinated with ovalbumin, an antigen not present in the natural environment of the dairy cow. Results indicated that the calf is capable of robust humoral (i.e., primary and secondary OVA-specific antibody) responses to vaccination. Conceivably, maternal antibody acquired through the ingestion of colostrum may inhibit humoral responses to natural antigens. Infectious diseases of calves, especially diarrheal and respiratory diseases are a major economic concern for the U.S. cattle industry. They also impact human health and food safety through pre-harvest contamination, and zoonotic transmission. More than 40% of neonatal calves on U.S. dairy farms are either clinically ill or die during the neonatal period. Morbidity and mortality of preweaned calves cost the industry between $90-$180 million annually. Customers of the research include dairy cattle producers, supporting industries, scientists, and consumers wanting a safer food supply. Back to top 3625-31320-001-00D Project Title: Microbial Factors/Pathogensis of Subacute Rumen acidosis(SARA) in cattle to assure food safety Project No: 3625-31320-001-00D SY(s): Rasmussen, Mark Location: Ames, Iowa National Animal Disease Center Pre-Harvest Food Safety and Eenteric Diseases What was the single most significant accomplishment this past year? Antiprotozoal screening assay: We developed an in vitro assay using live/dead fluorescent dyes to screen test compounds for their ability to kill rumen protozoa. Since engulfment of bacterial pathogens by rumen protozoa increases their virulence, i.e. S. typhinurium DT104, strategic defaunation will be advantageous to the dairy and cattle industry in terms of animal productivity and health. In our search for rumen defaunation compounds, we have found that some natural plant extracts (Yucca schidigera and Rosmarinus officinalis) will effectively control protozoa in the rumen without harming the general fermentation. List other significant accomplishments, if any. Chlorate for defaunation: We have also demonstrated that rumen protozoa are inhibited and killed by sodium chlorate. In vitro studies have shown that sodium chlorate in conjunction with Yucca schidigera extract effectively kill rumen protozoa within two hours. Previous work has shown the efficacy of sodium chlorate treatment to reduce the numbers of food borne bacterial pathogens in the rumen. A combination of these two treatments when strategically administered to cattle could greatly reduce the incidence and shedding of salmonella, food-borne pathogens, as well as other pathogens at critical time points in the production cycle of both beef and dairy cattle. Gene transfer studies: In collaboration with Steve Carlson, USDA-ARS, NADC, we have demonstrated that some strains of salmonella, after in vitro passage through rumen protozoa, display greater virulence in neonatal calves. We have demonstrated this ability in co-incubation studies where different species of bacteria can exchange antibiotic-resistance genes within rumen protozoa. These aspects of the project deserve further study. PCR for Prevotella: We have developed a real-time PCR method for the rumen probiotic bacteria, Prevotella bryantii. In upcoming work, we will be using this method to monitor establishment of populations of Prevotella introduced into the rumen of acidosis-stressed cattle. Back to top 3625-32000-051-00D Project Title: Prevention of Losses From Colibacillosis and E. Coli O157:H7 in Cattle and Swine Project No: 3625-32000-051-00D SY(s): Casey, Thomas; Sharma, ViJay; Nystrom, Evelyn; Rasmussen, Mark Location: Ames, Iowa National Animal Disease Center Pre-Harvest Food Safety and Eenteric Diseases What was the single most significant accomplishment this past year? Regulation of O157:H7 virulence factors: Understanding the mechanism(s) governing expression of genes that facilitate colonization, persistence, and shedding of E. coli O157:H7 may lead to novel strategies to reduce O157:H7 in the gastrointestinal tract of cattle. Adherence of E. coli O157:H7 to intestinal epithelial cells depends on the secretion of specific factors (e.g., Esps and Tir). Ongoing studies in Canada indicate that cattle immunized with these secreted factors have fewer O157:H7 bacteria in their feces than non-vaccinated cattle. Deletion of the E. coli O157:H7 gene called "hha" dramatically increases expression of a number of genes that promote bacterial colonization in animals. Scientists at NADC used in vitro tissue culture assays to demonstrate that an hha deletion mutant of E. coli O157:H7 exhibited increased adherence to cultured epithelial cells compared to the strain carrying hha and showed that the increased adherence was due to a 10- to 100-fold enhanced expression of Esps and Tir. These results suggest that cell-free supernatants obtained from the hha mutant strain could serve as an important source of large quantities of Esps and Tir for immunizing cattle against E. coli O157:H7. We have also constructed a hha-sepB double mutant strain of E. coli O157:H7 that accumulates increased intracellular quantities of Esps and Tir. This double mutant could potentially be converted into a bacterin for immunizing cattle to reduce E. coli O157:H7 colonization and shedding. List other significant accomplishments, if any. O157:H7 binding sites in cattle: Determining sites where E. coli O157:H7 bacteria colonize and persist in cattle is critical for designing and testing interventions aimed at reducing colonization and shedding. Lymphoid follicle-dense mucosa at the terminal rectum has been identified as an important site of O157:H7 colonization in cattle. Scientists at the NADC and a visiting scientist from the School of Veterinary Medicine, Hannover, Germany, studied E. coli O157:H7 binding in tissues from experimentally infected cattle and observed O157:H7 bacteria tightly attached to squamous epithelia cells of the rectal anal junction. This is the first evidence that O157:H7 bind to squamous epithelial cells at this site. Our findings confirm that the rectal anal junction is a prominent site of O157:H7 colonization in cattle and extend previous studies by showing that O157:H7 bind to squamous epithelial cells at this site and cause lesions which are similar to the attaching and effacing lesion produced by O157:H7 on enterocytes. Shiga toxin-induced kidney damage in piglets: The hemolytic uremic syndrome and renal failure are serious sequelae of Shiga toxin-producing E. coli infections in humans. Shiga toxin-induced kidney damage has been demonstrated in several different animal models, including piglets, but the sequence of events from ingestion of Shiga toxigenic E. coli to development of the hemolytic uremic syndrome is not understood. Scientists at the NADC and a visiting scientist from the School of Veterinary Medicine, Hannover, Germany, retrospectively examined kidney tissues obtained from neonatal piglets from previously reported and ongoing STEC infection experiments. Kidney lesions, similar to those in humans with the hemolytic uremic syndrome, were observed histologically in piglets inoculated intragastrically with different strains of STEC bacteria, but not in control piglets. To our knowledge, this was the first report of tubular epithelial cell necrosis in pigs infected with Shiga toxin-producing E. coli. The early presence of both vascular and epithelial damage (as early as 24 hours after inoculation) suggests that tubular necrosis is not dependent on prior glomerular capillary damage, as previously proposed. Immunohistochemical identification of Shiga toxin binding sites and globotriaosylceramide (Gb3, receptors for Shiga toxin) in the sites where renal lesions were seen in piglets inoculated with Shiga toxin-producing E. coli extends the evidence that Gb3 are receptors for Shiga toxin. These findings identify the neonatal piglet STEC infection model as an excellent model for studying the pathogenesis of the life-threatening complications of Shiga toxin-producing E. coli infections. Back to top 3625-32000-052-00D Project Title: Salmonella-Host Interactions Project No: 3625-32000-052-00D SY(s): Bearson, Shawn; Rasmussen, Mark; Vacant; Carlson, Steven Location: Ames, Iowa National Animal Disease Center Pre-Harvest Food Safety and Eenteric Diseases What was the single most significant accomplishment this past year? Up-regulated swine genes during Salmonella infection map to quantitative trait loci for infection and stress: The contamination of uninfected pigs on the farm, during transport or in the slaughter plant environment by animals shedding pathogenic bacteria such as Salmonella is a major food safety obstacle; thus, identifying swine genes that respond to Salmonella infection is important to determine the mechanisms involved in swine resistance (and susceptibility) to Salmonella. In a collaborative research project with Dr. Chris Tuggle at Iowa State University, genetic mapping and physical linkage studies of swine genes up-regulated during Salmonella infection revealed that two of the differentially expressed genes mapped to quantitative trait loci (QTL) sites for controlling mitogen-induced proliferation of whole blood cells (SDCBP) and the number of circulating lymphocytes following mixing and transport of pigs (CXCL10). Therefore, investigating gene expression alterations during Salmonella infection will be useful in developing markers and screening for DNA polymorphisms associated disease response, potentially improving pig genetics. List other significant accomplishments, if any. In vitro exposure of Salmonella Typhimurium to norepinephrine alters in vivo tissue prevalence in swine: An increase in the shedding of Salmonella and other infectious bacteria in swine have been found to coincide with the transportation of animals and other stressful events. Recent research has suggested the catecholamine norepinephrine (NE) as a potential host signal during stress. Therefore, the prevalence of Salmonella enterica serovar Typhimurium in swine following inoculation with S. Typhimurium exposed to NE in vitro was investigated in collaboration with Dr. Don Lay, Jr., at the USDA-ARS-Livestock Behavior Research Unit, West Lafayette, Indiana. Bacterial quantification revealed an increase in the number of Salmonella associated with various swine tissues following experimental inoculation with NE-treated S. Typhimurium compared to the Salmonella that did not receive NE exposure. Furthermore, upon observing an increase in the number of Salmonella associated with the stomach wall tissues at 3 h p.i. for the NE-treated culture, an experiment was conducted using an ex vivo stomach contents assay to determine the effect of NE exposure on the ability of the organism to survive the conditions of the porcine stomach; NE treatment enhanced the survival of S. Typhimurium more than 2 logs (p < 0.007). A possible scenario could be envisioned with a Salmonella-infected pig being stressed during transportation/mixing, resulting in the shedding of NE-stimulated Salmonella and exposure of naive, stress-compromised penmates with a "primed" microorganism. Back to top 3625-32000-053-00D Project Title: Ecology and Epidemiology of Salmonella and other Foodborne Pathogens in livestock, primarily swine Project No: 3625-32000-053-00D SY(s): Wesley, Irene; Rasmussen, Mark; Vacant; Scuphan, Alexandra Location: Ames, Iowa National Animal Disease Center Pre-Harvest Food Safety and Eenteric Diseases What was the single most significant accomplishment this past year? Distribution of Campylobacter associated with perimarketing events in turkeys: Viscera of birds (crops, duodenum, jejunum, ileum, colon, ceca) from six commercial flocks in the Midwest were examined for Campylobacter before (n = 30/flock) and after (n = 30/flock) transport to the abattoir in collaboration with scientists at the USDA-ARS-National Animal Disease Center (NADC), Ames, IA, and Iowa State University (D. Trampel), and partially funded by the Iowa Turkey Federation. Overall, when data for the six farms are combined, no difference was seen in Campylobacter spp. recovered from ceca, duodenum, ileum, spleen, and large intestine, whereas after transport, recovery was significantly higher from the gall bladder and crop when compared to on-farm levels (P < 0.05). Overall, C. coli was isolated more often from the crop and cecum, whereas C. jejuni predominates in the intestine (duodenum, ileum, colon) resulting in its more frequent isolation from cloacal swabs. Ability to differentiate C. jejuni from C. coli is fundamental to understanding dynamics of Campylobacter in the avian gut and in evaluating on-farm pathogen reduction interventions. List other significant accomplishments, if any. Salmonella populations are not altered during perimarketing events: We compared the prevalence of Salmonella enterica in turkeys on six commercial flocks pre- (30 birds/flock) and post- (30 birds/flock) transport in collaboration with scientists at the Iowa State University (D. Trampel), and partially funded by the Iowa Turkey Federation. No statistical difference was found between the overall prevalence found on-farm and at slaughter, based on any sample type analyzed (crop, ceca, liver/gall bladder, and spleen) with cecal contents having the highest relative sensitivity. This demonstrates that the perimarketing practices of feed withdrawal, catching, loading, transportation, and pre-slaughter holding do not significantly affect the prevalence of S. enterica in market-age turkeys in contrast to what we have previously reported for hogs. Therefore, it may be possible to estimate on-farm prevalence based on samples collected at slaughter. Vitamin E does not facilitate survival of L. monocytogenes in irradiated turkey meat: We monitored survival of Listeria monocytogenes (LM) following irradiation in ground meat from turkeys fed diets supplemented with vitamin E. These collaborative efforts between scientists at the USDA-ARS-NADC and Iowa State University were partially funded by CSREES-Special Program 406. Dietary vitamin E, which we have previously shown augments the immune response of turkeys, did not diminish the survival of LM in meat following irradiation. This suggests that vitamin E while boosting the immune response of birds does not compromise the bactericidal effects of irradiation on turkey meat. Culture methods bias the recovery of Salmonella from swine feces: Four culture methods (A, B, C, and D) were evaluated for their ability to recover Salmonella enterica from pooled swine fecal samples (n = 100). None of the methods was able to isolate the Salmonella at the same frequency and of the same serotype from all positive samples. We concluded that culture methods differ in efficiency of recovering S. enterica serotypes from naturally contaminated swine fecal samples. Therefore, depending on the objective(s) of investigations on the ecology and epidemiology of S. enterica populations in swine, a method(s) or a combination should be considered for more reliable results. Comparison of DNA extraction protocols: We performed preliminary experiments for analysis of the microbial ecology of the turkey cecum, including comparison of 14 DNA extraction methods. Electrophoretic and spectrophotometric analysis showed a wide range of DNA yield, shearing and contamination resulted from the methods. In addition, bacterial ribosomal small subunit quantification of the extraction products indicated a wide range of PCR inhibition resulting from template contaminants. This indicates that the DNA extraction protocol must be carefully selected prior to use in analysis of microbial communities. Seasonal distribution of Arcobacter: The prevalence of Arcobacter detected by cloacal swab (6/298) and cecal contents (3/145) suggests that Arcobacter colonizes the intestinal tract at very low levels. The overall prevalence of Arcobacter in the drinker water decreased from 63.04% (29/46) in the summer of 2003 to 24.66% (18/73) in the spring of 2004 with a concommitant shift in the species of Arcobacter during this interval. The prevalence of Arcobacter in the water appears to be related to the chlorination level present in the drinker water. Antibiotic resistance profiles of Campylobacter isolated from swine: Human Campylobacter infections are commonly treated with quinolone or macrolide antibiotics, such as erythromycin. The observed rise in the number of Campylobacter resistant to quinolone and macrolide antibiotics may be related to the use of growth promotants such as the macarolide tylosin in animal agriculture. C. coli strains (n = 152) isolated from swine farms in the Midwest were resistant to ciprofloxacin (7.9), to doxycycline (57.8%), erythromycin (65.1%), but not to gentamicin (0.0%), and minimally to meropenem (1.3%). Together these findings suggest that the swine C. coli isolates are highly resistant to erythromycin, which may be due to the use of macrolides as growth promoters during swine production. Back to top Project Title: Antibiotic Resistance of Enteric Bacteria Project No: 3625-32000-054-00D SY(s): Stanton, Thaddeus; Carlson, Steven Location: Ames, Iowa National Animal Disease Center Pre-Harvest Food Safety and Eenteric Diseases What was the single most significant accomplishment this past year? Identified "collateral effects" of subinhibitory levels of commonly used antimicrobial for swine: Factors contributing to persistence of bacterial resistance to an antibiotic in the absence of direct selection by that antibiotic are ill defined. We discovered that the quinoxaline antibiotic, carbadox, stimulates production of a gene transfer mechanism (VSH-1, a bacteriophage-like gene transfer agent) of the spirochete B. hyodysenteriae, the etiologic agent of swine dysentery. The gene transfer mechanism was shown by us to transfer resistance to the macrolide antibiotic tylosin. The carbadox concentrations that stimulate gene transfer are lower than those inhibiting bacterial growth and are equivalent to subtherapeutic in vivo levels. Carbadox is a common antimicrobial fed to swine for growth promotion and disease prophylaxis. We hypothesize, for future research, that carbadox can stimulate the transfer of tylosin resistance between B. hyodysenteriae strains in the absence of tylosin selection. We further hypothesize that the "carbadox effect" is not limited to spirochete bacteria and that carbadox can stimulate bacteriophage induction and promote generalized transduction of genes and stimulate transfer of other genetic elements among other intestinal bacteria species. List other significant accomplishments, if any. Increased virulence in antibiotic resistant Salmonella: Multiresistant Salmonella (i.e., DT104) have been purported to be more pathogenic that antibiotic-sensitive cohorts. For other bacterial pathogens, enhanced pathogenicity has been observed following growth of the pathogen within protozoa. In research performed at the National Animal Disease Center (NADC) in Ames, Iowa, we set out to evaluate the possibility that multiresistant Salmonella (specifically DT104) are more pathogenic after surviving within rumen protozoa. After surviving within rumen protozoa, we found that DT104 was more invasive than antibiotic-sensitive Salmonella. A specific DT104 gene was identified as the major contributor to this phenomenon. Additionally, rumen defaunation (elimination of protozoa) was identified as an intervention strategy for disruption. The results are important since they identify an intervention point (protozoa) for eliminating a reservoir for Salmonella, for disrupting a "training ground" for Salmonella pathogenicity, and direct future research focus on a specific gene important for protozoa-Salmonella interactions. Protozoa as sites of antibiotic resistance gene transfer: Plasmids are circular pieces of DNA that often carry antibiotic resistance genes. Bacteria can exchange these plasmid via a process termed conjugation. In research performed at the National Animal Disease Center (NADC) in Ames, Iowa, we set out to evaluate the possibility that bacterial conjugation can occur within rumen protozoa. Specifically, we found that Salmonella can acquire plasmids from other bacteria while existing within rumen protozoa. This phenomenon was also perturbed by rumen defaunation. These results reveal a bacterial gene exchange locus that can be disrupted. Back to top Table of Contents Next

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