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Oral Presentations |
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Poster Presentations |
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*Best Oral or Poster Presentation, Graduate
# Best Oral or Poster Presentation, Undergraduate |
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Using Routine PCR and qPCR Methods as Molecular Mapping Tools to Determine Bacterial Inputs into the Red Cedar River
Brian M Panzl and Joanna M Pope, Michigan State University, East Lansing, MI 48824
The Red Cedar River runs for 45 miles and the land use in its watershed ranges from urbanized to rural. Contamination of water has always been a concern with regards to the level of fecal indicator bacteria in water. These bacteria may originate from several sources of fecal contamination including humans, domestic animals and wildlife, as well as a combined sewer overflow (CSO) events. A discharge from a CSO occurs when capacity of the wastewater system is exceeded during a storm event and the partially treated storm water and sewage is discharged into the river. The objective of this research is to determine the sources of bacterial contamination present in the river. Water samples were collected starting in May 2010 at five sites on the river at approximately two-week intervals. Samples were filtered using EPA method 1600 to determine overall microbial water quality. Two indicator organisms were used for microbial source tracking, namely Bacteroides spp. and Enterococcus spp.. Routine polymerase chain reaction (PCR) coupled with agarose gel electrophoresis and quantitative PCR (qPCR) were used for microbial source tracking. Target genes for routine PCR were human and bovine in Bacteroides spp. (16S rDNA) and human esp in Enterococcus faecium. The human α-mannanase gene in Bacteroides thetaiotaomicron was the qPCR target gene. To date, the routine PCR data show that bovine contamination is not a factor; however in terms of the human Bacteroides spp. and esp genes they are sporadically positive for esp and inconclusive for the human Bacteroides spp. target. This shows that human contamination is a factor in the river, but perhaps not a substantial one. The qPCR data show that high concentrations of qPCR cell equivalent/100 mL are present throughout the river with the highest average per site occurring upstream of the CSO facility. These preliminary results are provided since sampling is continuing through the fall of 2010.The data thus far indicates that the qPCR method is a more effective tool in the search for environmental contamination. This conclusion is based on the fact that the data can be quantified as opposed to the qualitative routine PCR results.
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Pathogen TMDL Study of Evans Creek
Kandice L Karll and James Martin, Adrian College, Adrian, Michigan 49221
Evans creek watershed is a 33 square mile basin that includes portions of Washtenaw and Lenawee counties, in Southeast Michigan. The water quality in this stream not only affects the sub-basin it runs through, but also the River Raisin watershed downstream through to Monroe County and on to Lake Erie. Water quality monitoring for levels of the enteric bacteria Escherichia coli were conducted at six locations on Evans creek. Collection and testing procedures followed the Michigan Department of Natural Resources and Environment protocol for violations of the Total Maximum Daily Load (TMDL) for enteric pathogens as recommended by the USEPA. The standard membrane filtration method was used to enumerate the bacteria. USEPA approved Hach® Method 10029 was employed. Sampling and data collection were conducted on a monthly basis during a variety of weather conditions from October 2009 to April of 2010. During recreation season (May 1-September 30) samples were collected six times a month. Our data show that the amounts of E. coli present exceeded the legal allowable value at several of our testing sites, and through all weather conditions. Sample results from an urban location (downtown Tecumseh) are particularly interesting, showing E. coli colonies per deciliter routinely of 10 times and more the legal TMDL limit.
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Proteolytic Comparison of IdeS and IdeZ and Novel MWCO Filtration with ELISA Assay
Andrew M Howard and Jennifer L. Hess, Aquinas College, Grand Rapids, MI 49506
The immunoglobulin-degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). IdeS and a homologous cysteine protease, IdeZ, originating in Streptococcus zooepidemicus, have not been found to cleave any of the other known synthetic substrates that typical cysteine proteases hydrolyze. In this study, we have compared the proteolytic activity of catalytic site-directed mutant IdeS with homologous mutants from IdeZ to determine if there is any direct or indirect association between the cleavage activity of IgG-degrading enzymes. Using both qualitative protein immunoblotting and quantitative ELISA techniques, the IgG cleavage profiles of IdeS and IdeZ do not appear to be significantly different. Enzyme variants of IdeZ in which critical amino acids found in the active site of the enzyme were changed as a consequence of site-directed mutagenesis were found to be non-functional. The presence of these various bacterial cysteine proteases with such similar substrate preferences remains intriguing and merits further analysis, especially with respect to the expression pattern in infected hosts and the potential implications for clinical applications of research concerning these enzymes' activities.
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Role of methionine biosynthesis in the symbiosis between Sinorhizobium meliloti and the legume plant Medicago sativa
Aroha C. Rodrigues, Frans J. de Bruijn# and Silvia Rossbach, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008-5410 #Laboratoire des Interactions Plantes Micro-organismes, INRA/CNRS, 31326 Castanet Tolosan, France
Atmospheric nitrogen is in a form that is unavailable to plants. Some bacteria can convert nitrogen to ammonia and are found either free-living or in association with plants. This process is called biological nitrogen fixation. Bacteria that form a symbiotic relationship with plants are known as rhizobia and one example is Sinorhizobium meliloti, the nitrogen fixing symbiont of Medicago sativa (alfalfa). S. meliloti infects root hairs of alfalfa and induces the plant to form specialized organs called nodules. Bacteria residing within nodules fix atmospheric nitrogen using the enzyme nitrogenase. S. meliloti provides the plant with nitrogen and in turn the plant provides it with carbon sources. Some bacteria, known as auxotrophs, are incapable of synthesizing certain metabolites and can grow only when supplied with them. Past studies have shown a variation in the ability of different S. meliloti methionine auxotrophs to form nodules and fix nitrogen. The focus of our study is to characterize essential genes involved in the methionine pathway and to determine if the methionine auxotrophs of S. meliloti have the ability to form nodules and to fix nitrogen. The methionine biosynthetic pathway differs in various organisms. For example, in Escherichia coli methionine synthesis is a four step pathway starting with the conversion of homoserine into intermediates and then to methionine. Gene products involved are MetA, MetB, MetC, and MetE or MetH. In contrast, methionine biosynthesis is a three step pathway in Pseudomonas aeruginosa, involving MetW, MetX, followed by MetZ and MetH. We performed growth studies with S. meliloti mutants to confirm which genes, when mutated, result in methionine auxotrophy. Growth studies using intermediates of the methionine pathway were carried out resulting in the finding that the methionine biosynthetic pathway in S. meliloti is a combination of the pathways observed in E. coli and P. aeruginosa. The methionine auxotrophic mutants of S. meliloti were inoculated onto plants. The met mutants were found to form nodules and to fix nitrogen efficiently.
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Disinfection effectiveness of a household water treatment device that delivers bromine
Kyle S. Enger, Angela D.Coulliette, Matthew S. Field, Joan B. Rose, Michigan State University, East Lansing, MI 48824
Diarrheal illness kills approximately 1.8 million children per year in the developing world. Development workers promote many household water treatment (HWT) methods to mitigate diarrhea in poor communities. Such methods are less expensive than large drinking water treatment and distribution systems, but their effectiveness varies widely. We tested the antimicrobial effectiveness of the AquaPure™ device, which is commercially marketed in India. The device has an upper tank for untreated water, a central canister containing polymer beads (HaloPure®, HaloSource, Inc.) that release bromine, and a lower tank to collect treated water. Gravity moves water through the device. Methods:Cultures of Salmonella enterica Typhimurium, Vibrio cholerae, human adenovirus 2, and MS2 coliphage were added to 10 L of water entering the device. Some experiments also included 5% raw sewage. Since 10 L of water requires ~60 min. to flow through the device, specimens were collected as soon as disinfected water left the device, and at three 15-minute intervals thereafter. Log reductions were calculated from culture results. S. enterica, V. cholerae, and MS2 were enumerated by plate counts; a 3-tube MPN method was used for adenovirus, based on observation of cytopathic effect in cell culture.Results:S. enterica was reduced by 4 to 6.5 logs; V. cholerae by 5 to 7 logs; adenovirus by 0 to 4 logs; and MS2 by 0 to 2 logs. Specimens taken early in each run showed lower bromine residuals and disinfection effectiveness than later specimens. Further investigation of the effect of culture media on disinfection of MS2 suggested that small amounts of bacterial and cell culture media protected MS2 from disinfection, and that the cell culture media exerted a bromine demand.Conclusions:Although the device was highly effective against the bacteria tested, it was less effective against viruses. Effectiveness in clean water was probably underestimated due to the protective effect of culture media. It is unclear whether the culture media would affect disinfection in sewage-contaminated water; future experiments will test this. Acknowledgements:This work was carried out using funding from HaloSource, Inc. ADC is now affiliated with the Centers for Disease Control and Prevention. MSF is now a medical student in Indiana.
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Chromosomally Encoded Serum-Resistance in Salmonella typhimurium
Kristie C. Mitchell and James L. VandenBosch, Eastern Michigan University, Ypsilanti, MI 48197
Salmonella typhimurium is a leading cause of infectious gastroenteritis and results in systemic disease when it escapes into the bloodstream. One of the most important components of human serum in fighting bacterial infections is the complement system. Outside of cells, S. typhimurium exhibits resistance to killing by complement (serum-resistance), a trait conferred by several known virulence genes, primarily those encoding lipopolysaccharide length, but also including ancillary contributions from rck, pagC, and traT. This study investigates a potential complement-resistance gene located on the chromosome of S. typhimurium. S. typhimurium strain EM876 has a TnphoA insertion in the chromosome and also has reduced serum-resistance. Inverse PCR and subsequent DNA sequence analysis reveal the insertion to be in the gene glpQ. glpQ codes for a periplasmic enzyme involved in the glycerol degradation pathway. Complementation assays using a clone of glpQ on a multicopy plasmid are consistent with a role for glpQ in serum-resistance. Significantly, glpQ expression is temperature-dependent, preferentially expressed at 37°C rather than 30°C. This is similar to traT and suggests that multiple genes are regulated by temperature and, together, contribute to the overall serum-resistance of S. typhimurium. This poster was also recently presented at the 17th Annual Midwest Microbial Pathogenesis Conference.
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Inhibition of larval Aedes triseriatus (Diptera: Culicidae) growth associated with fungal blooms and maple leaf detritus
Rebecca J Morningstar, Michael G Kaufman, Edward D Walker, Michigan State University, East Lansing, MI 48824
Laboratory experiments in aquatic microcosms showed that growth of larvae of the eastern tree hole mosquito, Aedes triseriatus, which is a known vector of La Crosse Encephalitis, and a potential vector for West Nile Virus, was stalled in the presence of senescent sugar maple leaves and fungal blooms on the leaves and in the water column. The phenomenon, by contrast, was not observed when red oak leaves were provided and larval growth was not inhibited. 18S rDNA sequence analysis indicated that fungi in the water column were predominantly Aspergillus spp. along with other fungi in a consortium. ITS1/5.8S PCR also indicates the presence of fungus-like oomycetes. The observed dampening of larval growth could be due to: presence of toxic compounds, such as tannins, leached from the maple leaves; mechanical interference of feeding by the observed fungal bloom; toxic or growth inhibitory substances from the fungi; or interactions and combinations of these factors. The significance of these results to growth and production of mosquitoes from heterotrophic container habitats will be discussed.
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Evaluation of Recovery Efficiency and Survival of Bacteriophage P22 and Bacillus thuringiensis on Fomites
Amanda B. Herzog, Alok K. Pandey, Prianca Bhaduri, Charles P. Gerba 2, Joan B. Rose, and Syed A. Hashsham, Michigan State University, East Lansing MI, 48823; 2University of Arizona, Tucson AZ, 85721
Fomites (inanimate or nonliving objects) are known to play a role in the transmission of pathogenic bacteria and viruses. Quantitative analysis of the parameters that affect recovery at the lower limit of detection of organisms on fomites will aid in improving microbial risk assessment and infection control. In this study, the variability in recovery as a function of fomite type, fomite surface area, application medium, relative humidity, wetting agent, recovery materials and sampling time were evaluated. To quantify the recovery from fomites, bacteriophage P22 (a surrogate for influenza virus) and Bacillus thuringiensis (a surrogate for Bacillus anthracis) at concentrations of 102 PFU-CFU/ml were used. Nonporous fomites commonly found in indoor environments (acrylic, galvanized steel, and laminate) with surface areas of 100 and 1000 cm2 were sampled with pre-moistened wipe, KimwipeTM, and cotton swab. The parameters that had the most affect on the recovery of bacteriophage P22 was fomite surface area, time before sampling and relative humidity while for B. thuringiensis it was fomite surface area and time before sampling. An increase in size from 100 to 1000 cm2 decreased the recovery by 50% for bacteriophage P22 and 20% for B. thuringiensis. After the applied samples dried on the fomites, less than 2% of bacteriophage P22 and 20% of B. thuringiensis was recoverable. Under the conditions evaluated, the least significant parameters affecting the recovery of bacteriophage P22 and B. thuringiensis from fomites were the fomite type and recovery materials. Understanding these parameters impacting recovery, method limit of detection, can assist in the design of fomite sampling and use of the data in quantitative risk assessment models.
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Identification of HopM1 Targets using a Yeast Model System
Vanessa Revindran and John R. Geiser, Western Michigan University, Kalamazoo, MI.
We have created a model system to study the action of plant pathogen effectors in yeast (Saccharomyces cerevisiae). The inducible effector expression system utilizes the GAL1 promoter, contains a V5 epitope for microscopic visualization and 6xHIS tag for purification from yeast. Our expression constructs were created by PCR amplification followed by transferring into a Gateway SystemTM expression vector. The Pseudomonas syringae effector protein HopM1suppresses innate immunity causing disease. Upon expression in yeast, the strain containing HopM1 was lethal on solid media. The HopM1 strain in liquid has not duplicated the effect seen on solid media. In liquid media, colony forming units decrease to 73% at hour 24. At hour 48, only 38% of the initial cells are alive. The difference between liquid and solid media may suggest that the effect seen observed is a delay or arrest of growth phenotype and not an outright cytotoxic effect. Surprisingly, HopM1 is only lethal on solid media at 21°C, but not at 30°C or 37°C. To investigate this further, we have examined expression levels of the HopM1 protein at all three temperatures. At 21°C, HopM1 shows maximal expression at 24 hours and this corresponds to first evidence that HopM1 has an effect in strain growth in liquid culture. At 30°C maximal expression is at hour 3 and at 37°C maximal expression is at hour 24. These results suggest that the HopM1 protein structure may be unstable. In plants, the effects of the bacteria are seen at temperatures lower than 23°C, and at 31°C no effects are observed. To identify the cellular targets of HopM1, we have begun to isolate spontaneous suppressors that are capable of surviving the HopM1 imposed lethality on solid media. Currently, we have identified 14 suppressor strains. We are using immunofluorescent microscopy to visualize HopM1 localization and to determine if the suppressors have any effect on localization of HopM1. Further characterization of these strains are continuing and results will be presented to show growth rates and HopM1 protein expression levels in both wild type and suppressor strains.
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In vivo induced gene Hfq regulates virulence-associated phenotypes in Actinobacillus pleuropneumoniae
Sarguru Subashchandrabose1,2 and Martha H. Mulks1,2,3
1-Comparative Medicine and Integrative Biology Program, 2-Center for Microbial Pathogenesis, and 3-Department of Microbiology and Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease. Previous studies in our lab have shown that the gene encoding the RNA chaperone Hfq is up-regulated during A. pleuropneumoniae infection of porcine lungs. Hfq is a bacterial pleiotropic post-transcriptional regulator which facilitates interaction between small regulatory RNAs and their target mRNAs. A. pleuropneumoniae forms biofilm with poly N-acetyl glucosamine as the major matrix component. We hypothesized that Hfq would regulate biofilm production in A. pleuropneumoniae. An hfq deletion-disruption mutant was engineered. Biofilm production on polystyrene microtitre plates was assayed by crystal violet binding. WT and complemented mutant strains produced biofilm while the hfq mutant failed to produce biofilm. Q-PCR studies revealed that pgaC transcript encoding biofilm matrix biosynthetic enzyme was 15 fold lower in the hfq mutant compared to WT. SDS-PAGE analysis of outer membrane fraction of WT and the hfq mutant strains grown to stationary phase identified that cysteine synthase was expressed only in the presence of hfq. Since cysteine synthase is known to protect against oxidative stress and A. pleuropneumoniae is exposed to oxidative stress during infection, we probed the role of hfq in oxidative stress resistance with growth inhibition assays. We found that Hfq is involved in protection against superoxide stress and this effect is independent of its role in biofilm formation. In summary, we have identified that Hfq regulates biofilm formation by altering pgaC transcript levels and Hfq plays a role in regulating resistance to superoxide stress, at least in part by regulation of expression of cysteine synthase. This is the first report of regulation of poly N-acetyl glucosamine based biofilm by Hfq.
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Mycobacterium avium subspecies paratuberculosis infection prevents apoptosis of infected bovine primary macrophages
E. Kabara and P. M. Coussens, Departments of Animal Science, Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824
Johne’s disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), affects over 60% of Michigan dairy farms and is nationally responsible for as much as 1.5 billion dollars in losses annually. Clinical Johne’s disease presents as a massive infiltration of acid-fast staining macrophages in ileal tissues, combined with inflammation. The extent of mycobacterial infection may be explained by the ability of mycobacteria to bypass typical macrophage-mediated destruction, preventing phagosome maturation, coupled with an increase in cellular persistence of infected macrophages. This creates a large reservoir of the bacteria in the host. We hypothesize that MAP-infection prevents apoptosis of infected macrophages via inhibition of caspase activity to prevent clearance of infected macrophages and efferocytosis, essential for proper immune responses. To test this hypothesis, we labeled MAP bacteria with a fluorescent dye to enable studies on infected macrophages and compare them to uninfected or bystander macrophages in the same culture. We also employed Annexin V/ 7-AAD staining to study cell death as well as CaspaTag™ staining to study several caspase activities. We found significant differences in apoptosis rates between MAP-infected macrophages and control, uninfected macrophages while finding no significant differences between necrosis rates in these same cells. Furthermore, we have also observed a significant increase in apoptosis in bystander macrophages as compared to both the MAP-infected and control, uninfected macrophages. Control macrophages had the highest amount of caspase activity, bystander macrophages have an intermediate amount of activity, and MAP-infected macrophages displayed the lowest amount of caspase activity, regardless of the caspase studied (3/7, 8, or 9). Our results suggest that infection of macrophages with MAP dramatically reduces spontaneous and induced apoptosis of infected host cells and this may be caused by an inhibition of caspase activity. In infected animals, this process could be responsible for increased bacterial persistence and accumulation of infected cells.
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Ribosomal Database Project: Improved Tools and Services for rRNA Analysis
Benli Chai, Qiong Wang, Jordan A. Fish, Donna M, McGarrell, James M. Tiedje, and James R. Cole, Michigan State University, East Lansing, Michigan 48824
The Ribosomal Database Project (RDP) offers rRNA sequence data and analysis services through its website(rdp.cme.msu.edu). RDP's products are used for research and teaching in a broad range of fields such as molecular phylogeny and evolutionary biology, microbial ecology, bacterial identification, characterization of microbial populations, and understanding the diversity of life. Updated monthly, RDP maintains 1,418,497 aligned and annotated, quality-controlled public rRNA sequences as of October 2010 while researchers maintain over 2 million Sanger sequences in their myRDP accounts. To meet the analysis need for increasingly large numbers of next-generation sequences, RDP has made significant improvements to its rapid Pyrosequencing Pipeline. Since its release in 2008, the Pyrosequencing Pipeline has been used by over 800 researchers to process their next-generation rRNA sequence data. The Pipeline Initial Process, Aligner, and Clustering steps have been enhanced to allow users to visualize sequence quality and diversity via returned graphic summary files. A new RDP MultiClassifier is available as a command-line tool to help researchers carry out taxonomy-based analysis of large numbers of sequences in multiple samples. This new tool complements the command-line version of the RDP Classifier and both can be used as standalone tools or be integrated into researchers' local analysis workflow. RDP has also expanded the Pyrosequenceing Pipeline to support protein-coding genes by including tools for frameshift detection and correction. Other enhancements include a new distance matrix tool that generates distance matrices in two popular formats used by third-party tools such as Mothur. Tools in the Pipeline have been improved to accept compressed files in order to reduce the upload time of large amounts of sequence data.
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Relationships Between Landcover and the Parasitic Diseases Cryptosporidiosis and Giardiasis
Rebecca Ives and Joan Rose, Michigan State University, East Lansing, MI 48824
This study focuses on land cover and the occurrence of the parasitic diseases giardiasis and cryptosporidiosis in the human population in the Lower Peninsula of Michigan. The focus area includes the lower Grand River watershed in Kent County and Ottawa County and the River Raisin watershed in Hillsdale County and Lenawee County. The objective of this study is to compare disease occurrence in urban and rural zip codes, when urban and rural definitions are based on landcover elements. Research Question 1:Are there significantly more cases of cryptosporidiosis in rural zip codes? Research Question 2:Are there significantly more cases of giardiasis in urban zip codes? A database was constructed in ArcGIS (ArcGIS 9.2, ESRI) using census tract information, the 2001 landcover raster image of Michigan’s Lower Peninsula, ZIP code tabulation areas, and giardiasis and cryptosporidiosis case information over the time span of January 2000 to December 2008. When normalized by area, significantly more cases of cryptosporidiosis and giardiasis occurred in urban zip codes than in rural zip codes (P<0.05). When normalized by area, the number of giardiasis and cryptosporidiosis cases that occurred in urban zip codes are not significantly different (P>0.05). Significantly more cases of giardiasis than cryptosporidiosis occurred in rural zip codes (P<0.05). Case patterns of area normalized data suggest that transmission of Cryptosporidium from non-human hosts to human hosts in these areas may be less of a factor than human susceptibility and person to person transmission.
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Characterization of the phyllosphere community of immune-activated and untreated Arabidopsis thaliana
Mihir Boddupalli, James Kremer and Sheng Yang He, Michigan State University, East Lansing, MI 48824
In response to an infection, Arabidopsis thaliana will produce salicylic acid (SA). Release of SA will ultimately induce systemic acquired resistance (SAR), an important and highly conserved plant defense mechanism. SAR causes changes in phytohormone levels that leads to the production of antibiotic metabolites. Treating plants with benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH) stimulates SAR, resulting in the same phenotype as salicylic acid induced SAR. In agriculture, BTH has been proposed as an eco-friendly alternative to antibiotics for food crops, including strawberries and peaches. The phyllosphere, or the above-ground portion the plant, is inhabited by a highly diverse community of 2 to13 million diverse bacteria. These bacteria occupy a wide variety of ecological niches and typically include methanotrophs and nitrogen fixers. Prior to this novel study, the effects of induced defenses on the phyllosphere bacterial community as a whole had not been studied.Pyrosequencing the 16S rDNA of different phyllosphere extractions from BTH-treated and untreated Arabidopsis thaliana Col-0 revealed that the immune activated plants retain approximately the same degree of diversity. However, immune activated plants showed an increase in the abundance of a-proteobacteria and a decrease in b- and g-proteobacteria.
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Cryptosporidium and Giardia contamination in urban and rural watersheds in the midwestern US
Christopher Wendt, Rebecca Ives, Rachel McNinch, Shikha Singh, Joan Rose Michigan State University
The pathogens Cryptosporidium and Giardia have a history as etiologic agents in many recreational water outbreaks in the United States but the occurrence and risk associated with natural waters impacted by sewage or non-point sources has not been well explored. To evaluate the differences of parasite contamination in rural and urban watersheds, analyses were conducted at recreational areas and parks as well as in sources including combined stormwater / sewage discharges, and drains associated with agricultural activities throughout the Great Lakes region. Average concentrations of Giardia for the rural area represented by River Raisin watershed was 0.02137 per liter and 0.50576 per liter for Cryptosporidium. Infectious oocysts were detected in one sample at levels of 2301 per liter from an agricultural tile drain. Average concentrations at sites in the more urbanized Grand River watershed were 0.05095 per liter for Giardia and 0.03705 per liter for Cryptosporidium. Comparisons of parasite contamination evaluated in urban and rural waters using samples obtained from Chicago, Illinois and the River Raisin watershed in Michigan will be presented. Thus far, we have found that Giardia is of greater significance in urban watersheds influenced by sewage and Cryptosporidium is of greater concern in rural waters influenced by animal wastes.
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Fall 2010 Home |
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