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STUDENT POSTERS (eligible for judging at the meeting)
CLARK, E.S. and ALM, E.W. Biology Department, Central Michigan University, Mt. Pleasant, MI, 48858 Migratory waterfowl infected with Avian Influenza Virus (AIV) shed high titers of virus in their feces, and AIV shed into the environment can persist for substantial periods of time under certain conditions. Human use of recreational areas contaminated by infected birds raises concerns of bird-human transmission, therefore monitoring of potentially impacted sites should be considered and methods for rapid assessment of AIV in environmental samples developed. We have been optimizing protocols for collection, processing, and extraction of viral RNA from environmental samples so that they are amenable to real time PCR. Droppings from waterfowl were collected at migratory stopover sites and human recreational sites along the west coast of Lake Huron. Viral RNA was extracted from the samples and then real-time reverse transcriptase PCR with primers specific to a universal matrix protein of Influenza type A viruses was used to determine the presence of AIV in these fecal samples. Preliminary processing of fecal samples has shown 12 out of 69 samples to be positive for type A Influenza. Recent improvements in the extraction process indicate that this is likely an underestimate of the actual prevalence of AIV positive bird droppings. [received 9/02/07] * A poster on this research will also be presented at the Great Lakes Beach Association meeting in October 2007. Screening and Characterization of Soil Microbes Capable of Degrading Cellulose from Switchgrass (Panicum virgatum) Sarah Plecha and Danielle Hall,
The
Linking Microbial Diversity and Geochemistry of Uranium-Contaminated Groundwater Danielle Hall and Sarah Plecha,
The Faculty Advisor: Sonia M. Tiquia, Department of Natural Sciences, The
Mentor: Christopher W. Schadt, Environmental Sciences Division,
Development of Real Time Assay for the Detection of Three Pseudomonas species from Contaminated Metalworking Fluids
Metalworking fluids (MWFs) are lubricating oils used in different machining operations and are highly prone to microbial contamination that leads to degradation and biofouling. Pseudomonas oleovorans, P. pseudoalcaligenes, and a newly described species P. lubricans, all belonging to the mendocina sublineage, are the most dominant bacteria found in contaminated MWFs. The actual distribution of these bacteria in MWFs is difficult to study using standard culturing techniques as P. lubricans, in particular, will not grown on the commonly used Pseudomonas Isolation Agar. Real Time (RT)-PCR is a powerful technique for the identification and enumeration of these bacteria from MWFs, being highly sensitive and providing results within 2hrs. This study involved the development of a RT-PCR assay for the detection of the mendocina sublineage of Pseudomonas species from contaminated MWFs. The TaqMan primer-probe pair developed from the gyrB sequence of P. lubricans was highly sensitive and specific for these three species. Whole cell RT-PCR was performed and the sensitivity of the primer-probe was 101 CFU/ml and 13.7 fg with genomic DNA. Same results were obtained with spiked bacterial cells in 5% diluted MWF in phosphate buffer. The primer-probe pair did not give any amplification with P. aeruginosa, P. fluorescens, P. alcaliphila, Escherichia coli, Brevidimonas dimunita and Staphylococcus aureus, indicating the specificity of the assay. On the basis of the observation from the RT-PCR assay the designed TaqMan primer-probe pair can be effectively used to study the distribution of the three Pseudomonas species in contaminated MWFs. [received 9/12/07]
Effects of Oxidative Damage on Mitochondria and Yeast Spore Development TRICIA BROWN* AND STEVEN W. GORSICH Stress Tolerance Using Bakers Yeast as a Producer of Bio-ethanol SANDRA ALLEN*, WILLIAM CLARK, AND STEVEN W.
GORSICH Mutated Immunoglobulin-degrading Enzymes of Streptococcus Species and Their Effects on Human IgG
Immunoglobulin-degrading enzymes can cause an increase in virulence in certain pathogenic bacteria. These enzymes are specifically known for cleaving IgG to create fragments similar to those created by papain digestion. These enzymes are known as IdeS and IdeZ and are originally found in two different Streptococcus species. Though both enzymes have different primary protein sequences, the amino acids coding for the active sites are identical. In this research, IdeS and IdeZ plasmid DNA was obtained from recombinant E. coli, which could then be mutated using specific PCR primers. Mutations were focused around the active site, in order to determine amino acids that influenced the structure of the enzymes and their ability to cleave human IgG. In IdeZ, a serine residue that is positioned between two amino acids directly involved in the active site of the enzyme was changed to an alanine residue. This mutation eliminated IdeZ function as determined by western blot analysis. Characterizing IdeS and IdeZ IgG-cleavage functions could have medical and veterinary applications. [Received 10/05/07]
The objective of this study was to
assess the background microbial concentrations in air and fomites at two campuses of a hospital
before installation of the UVGI-HVAC. Air samples were
collected using an impingement method. Approximately
1500 L (4 impinges x 30 min vacuuming with the flow rate of 12.5 L/min) of air was
collected from
each of the four rooms (two waiting rooms and two patient areas). In each room, five commonly touched and five
untouched surfaces
were also
collected using electrostatic wipes. Samples
were assayed with culture methods using selective
agars, i.e. Tryptic(ase) Soy Agar (TSA), R2S, Mannitol
Salt Agar (MSA) and Malt Extract Agar (MEA). The results suggested that concentrations of
bacteria ranged from 12 to 66 cfu/m3 in the
bioaerosols and 0.1
to 64 cfu/cm2 on the fomites. In the indoor air bacteria concentrations were
strongly correlated with each other (r = 0.774 to 0.980) while the fungi concentrations
showed low correlations with the bacterial levels. Microorganisms detected from untouched fomites were
comparatively higher than the commonly touched surfaces. It can be hypothesized that microorganisms can survive on
fomites for a long period of time and
common surfaces in the hospital are most often
cleaned by staff
or microbials are transferred between different people touching the surfaces
frequently. Untouched surfaces represent accumulation
over time from the air. The effectiveness of the system will be assessed by
comparing the results before and after installation.
Impairment
of Water Quality at Silver Lake Sand Dunes Recreational Area, Michigan
ESTABLISHMENT
OF A PLANT PATHOGEN EFFECTOR MODEL SYSTEM
Our aim is to create a
model system to study the action of plant pathogens when they infect plant cells in
furtherance of our desire to find ways to stop bacterial pathogen infections. We have begun screening effector proteins of Pseudomonas syringae to determine which ones are
lethal in Saccharomyces cerevisiae. We have
initially picked 20 Pseudomonas syringae
pathovar tomato effectors to screen, as
homologues exist in common with Pseudomonas
syringae pathovar phaseolicola.
We have established an
inducible expression system in Saccharomyces
cerevisiae to identify which effectors are lethal. The expression vector contains an
inducible GAL1 promoter, a V5 epitope that will
be used for visualization of the effector protein and 6xHIS tag for purification. At present, we have PCR
amplified and cloned 13 of the 20 effectors into the pCR-Blunt II-TOPO vector. Each construct has been sequenced and verified
correct. Nine effectors have been moved to an intermediate donor vector using the Gateway
System. We have successfully transferred 1 of the effectors, HopM1, to the yeast
expression plasmid and have transformed it into S.
cerevisiae for examination of phenotypes. HopM1 when expressed in
yeast is lethal on solid media. Examination of the lethal effect using a titer assay has
not duplicated the effect seen on solid media, suggesting that the effect seen is a delay
or arrest of growth but not an outright cytotoxic effect. Results will be presented to
show the effect of the growth rates and comparison of death, compared to WT strains
containing HopM1.
Murine dendritic cells produce
IL-12 and induce Th1 polarization in response to Campylobacter jejuni infection
Campylobacter
jejuni is a frequent cause of food-borne
gastroenteritis. Dendritic cells (DCs) are central to initiating immune responses to
pathogens. Interaction of C. jejuni with murine DCs and its impact on T-cell
responses are unknown. We hypothesized that C. jejuni activates murine bone
marrow-derived DCs (BM-DCs) to undergo maturation, secrete T-helper cell (Th)-polarizing
signal(s) and initiate Th1-type responses. To test this hypothesis, immature BM-DCs were
infected with C. jejuni and various responses were assessed. BM-DCs were efficient
at C. jejuni uptake and killing; they readily internalized C. jejuni
by 2 h post-infection (p.i). Viable intracellular bacteria were reduced rapidly and no
live intracellular bacteria were recovered by 8 h p.i. Following infection, BM-DCs had
significant up-regulation of surface MHC-II, CD40 and costimulatory molecules (CD80 and
CD86), thus demonstrating a mature phenotype. C. jejuni-infected BM-DCs secreted
tumor necrosis factor-alpha, interleukin (IL)-6 and IL-12p70, which peaked at 24 h p.i.
Formalin-killed C. jejuni also induced maturation of and cytokine production by
BM-DCs but at a lower magnitude than responses elicited by live bacteria. Various strains
of C. jejuni elicited differential cytokine production from BM-DCs. In a DC-CD4+
T cell co-culture system, C. jejuni-infected BM-DCs induced high-level interferon-?
production from CD4+ T cells indicating Th1 polarization. In conclusion, we
demonstrate that murine DCs infected with C. jejuni undergo maturation, secrete
proinflammatory cytokines, and induce Th1 polarization, thus playing an important role in
mounting anti-C. jejuni immunity in C57BL/6 mice. (Funded by NIH
NO1-AI-30058) [Received
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