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Sample Collection

Collected 1 liter of stream water from each

site.

FilterSamples were concentrated using a .45 µg

membrane filter.

CultureSmear of filter plated on McConkey Agar

with MUG.

VerificationIsolated colonies were verified as E. coli

using citrate, indole, Voges-Proskauer, and

methly red IMViC tests.

RibotypingIsolated E. coli colonies were ribotyped

using a Dupont Riboprinter©. ECORI

restriction enzyme fragments DNA, and

electrophoresis is conducted. DNA is

removed from gel via Southern blotting and

the membrane is washed by a 16s rDNA,

RNA probe.

Cluster Analysis Unweighted pair-grouping (UPGMA)

Euclidian distance method was used to

create a dendogram using Bionummerics©

software package.

IdentificationThe unknown stream samples were

compared to the known human and livestock isolates of E. coli. The highest similarity value of 92 or more indicated a match. The identification

was compared to the cluster analysis for confidence and consistency.

•Phosphorous is a key nutrient essential to life. Too much however can

cause problems in an aquatic system. These problems can include an

overload of nutrients that cause turbidity and algae blooms (1)

Phosphorous loading on a waterway is caused from a variety of factors,

and these factors are in large part due to land use .

•The three different land types concentrated on in this study were

agricultural, impervious ( urban ), and forested. The stream sections were

classified based on the predominant surrounding and upstream land uses.

•Nineteen stream sections from tributaries in the Lamoille River drainage

basin were examined in the Summer of 2008 for amounts of total

phosphorus and fecal coliform bacterial contamination.

•Total P concentrations in stream water were generally three times higher

during a period of heavy precipitation in late July than during base flow

in the middle of June. Some agricultural and impervious sites had total P

concentrations comparable to reference forested sites, but impacted

agricultural and impervious sites could have total P concentrations two to

twenty-fold higher than forested sites during base flow and storm events.

•The overall trends of the phosphorous discharge point to Agricultural sites producing the highest

amounts, followed by the impervious sites, while the forested sites show the lowest amount of daily

discharge

•The source of E. coli in agricultural areas was predominantly from livestock.

• The source of E. coli in impervious sites was also largely from livestock, but the contribution from

human sources increased to one-fourth of the isolates.

•The source of most isolates of E. coli in forested sites was unable to be determined, possibly because

wildlife were not included in the genetic library, but livestock were still the source for one-fourth of the

E. coli isolated.

• Total Phosphorus was measured on seven occasions from June 13

through August 5 for a total of 400 samples.

• Phosphorus samples were analyzed by the University of Vermont

Vermont Streams group

• Coliform and E. coli enumerated utilizing Hach coliblue24© method

• Library and unknown stream E. coli isolates were processed by

ribotyping (see below)

1.Lake Champlain Basin Program, date n/a, http://www.lcbp.org/PHOSPSUM.HTM. Retrieved on 11/1/08

2.Lamoille Valley watershed map taken from

www.lcbp.org/Atlas/MapJPEG/nat_lamoille.jpg. Retrieved on 8/5/08.

• The discharge trends illustrates clearly the areas where the state could use

more prominent buffer zones, in particularly, the agricultural sites and a

number of impervious sites.

•The frequent occurrence of E. coli from livestock (predominantly cows,

goats, horse, and sheep) reflects the pastoral nature of the landscape in

Lamoille County. As would be expected, human sources were predominantly

associated with human habitation (impervious) sites.

•Library classification of wild life, and a more extensive human E.coli library

would be beneficial, and may happen in the near future in order to help

identify the unknown samples acquired this summer and for future studies.

ABSTRACT

METHODS

RESULTS

CONCLUSIONS

BIBLIOGRAPHY

Comparison of total phosphorus and microbial source tracking by land use type in the Lamoille

River watershedGreggory A Perry, Timothy R Thurston, and Robert Genter PhD

Johnson State College, Johnson VT

RIBOTYPING RESULTS

PHOSPHOROUS DISCHARGE

ACKNOWLEDGMENTS

For funding of our project we thank the NSF and VT

EPSCoR. KathiJo Jankowski for an amazing job coordinating the streams project, and the

laboratory team at UVM, who were instrumental in processing our phosphorous samples.

Jim Ryan whom is part of the Vermont Department of Natural Resources played a vital role

in helping us choose our sites. We also thank Farley Ann Brown, Declan MaCabe, and

Carlos Pinkham for advice and inspiration.

**Values of zero notate either values too small to

record, or incomplete processing of samples.

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