Non Technical Summary
In recent years, we have experienced a substantial increase in demands from our stakeholders to assist in identifying the sources of Escherichia coli (E. coli) - the sources of fecal pollution - in both surface and groundwater. These demands have been driven by the needs for taking effective steps towards restoring water quality. To meet the needs of our stakeholders, we will enhance our capacities via this project through both research and extension activities. The research aims to develop new methods, eliminating the limitations of current methods, for accurately detecting E. coli originating from agricultural sources. The research team at Lincoln University (LU) have already achieved success in developing two methods for identifying E. coli of human origin. Building on this progress and collaborating with the Virginia State University (VSU), this project aims to further extend the research by developing approaches to detect E. coli of livestock origin. At the same time, the outreach activities are designed to enhance awareness and understanding among stakeholders about E. coli and fecal pollution. This endeavor will identify and clarify common public misconceptions about E. coli and fecal pollution through social media and summer workshops. This project is the partnership and collaborative efforts of two 1890 land-grant universities, multiple federal, state, and local agencies, and a network of volunteers in Missouri. The successful execution of this project will enhance the capabilities of all partner institutions in fecal source tracking, and thereby contributing to the overall improvement of water quality and public health.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0210	1040	40%
133	0210	1100	40%
133	0210	2080	20%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0210 - Water resources;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 2080 - Mathematics and computer sciences;

Goals / Objectives
The goal of this project is to reduce instances of human illness resulting from direct bodily contact (such as in recreational water activitiesor the ingestion of water, produce, and food contaminated by fecal matter. The goal is to be fulfilled through both research and extension activities:1. Research objective: To develop new methods for accurate detection of E. coli originating from agricultural sources, with a specific focus on those of cattle, pigs, and poultry. The objective will be achieved through comprehensive approaches of bioinformatics, molecular biology, and environmental microbiology, further validated by practical field tests.2. Extension objective: To enhance awareness and understanding among stakeholders about E. coli and fecal pollution. The objective will be realized through comprehensive outreach and education initiatives.

Project Methods
Primary methods to be used for research activities:1.in-silicon searching for potential genetic markersThis proposed project will capitalize on the extensive repository of whole-genome information for E. coli isolated from diverse countries and host species. This vast dataset is readily accessible through public databases like EnterBase and GenBank. Employing bioinformatics strategies such as comprehensive whole-genome comparisons and data mining, we will strive to identify potential host-specific DNA sequences (i.e. genetic markers) among the E. coli genomes.A minimum of 100 E. coli genomes (contigs or complete assemblies) each from human, cattle, pigs, chicken, turkey, and other livestock animals, if available, will be downloaded and subsequently subjected to gene recognition using GeneMarkS-2. This process will yield the gene composition for each genome. The resulting gene compositions will then undergo whole-genome comparisons to identify genes exclusive to a single host species, using BLAST or other suitable digital platforms. These unique genes are known as host-specific genetic markers.2.To develop polymerase chain reaction (PCR) assays for the detection of E. coli associated with livestock sourcesThe putative genetic markers identified in silico will be utilized to design PCR primers, and through PCR assays, we will determine thespecificities and sensitivities of the potential genetic markers in differentiating different sources ofE. colistrains isolated from at least 10 fecal samples each of at least 10 host species includingat least humans (from wastewater treatment plant), livestock (cattle, chicken, pig, and turkey), wild animals (deer and raccoon), and waterfowl (duck and goose). Thefecal sampleswill becollected from the states of Missouri and Virginia, and thePCR testswill be conducted intwo laboratories, at Lincoln Univesity and Viginia State University, toidentify reliable genetic markersthat are not geographically limited.Primary methods to be used for extension activities:We will strike toenhance awareness and understanding among stakeholders about fecal pollution to make informed decisions in allocations of funds and resourcesTo achieve theextension goal, the following methodologies will be used.1.A survey will be designed and then conducted among the stakeholders to identify any misconceptions about E. coli and fecal pollution and to understand the needs of the stakeholders.Although the public is generally aware of the health risk associated with fecal pollution, there are several misconceptions that are prevalent in the public perception. We will strivetoenhance awareness and understanding among stakeholders about fecal pollution to make informed decisions in allocations of funds and resources. To achieve theextension goal, the following methodologies will be used.1.Todesignand conductamong the stakeholders to identify any misconceptions aboutE. coliand fecal pollution and to understand the needs of the stakeholders.The survey will include the following issues, but not limited to: a. AllE. colistrains are harmful: This is one of the most common misconceptions. While some strains ofE. colican cause illness, many others are harmless and even beneficial. b. Lack of personal impact: Individuals may believe that fecal pollution is not relevant to their lives, thinking it only affects remote or rural areas. c. Clean appearance equals clean water: The misconception that clear water is always safe water can lead to underestimating the presence of fecal contamination in seemingly clean bodies of water. d. Blame on visible pollution: Visible waste or debris might be wrongly attributed as the main source of contamination, while invisible or dissolved pollutants are overlooked. e. Assumption of natural purity: Some individuals might believe that natural water bodies are inherently pure and immune to fecal contamination. f. Inadequate understanding of sources: Misidentifying sources of contamination, such as assuming that all animal waste originates from domestic pets rather than considering wildlife contributions.2. To develop research-based extension education materials and disseminate them to the stakeholders Based on the result of the survey mentioned above and existing knowledge ofE. coliand fecal pollution, research-based extension education materials will be developed and disseminated to thestakeholders. Three extension education modules will be developed to meet the needs of different stakeholders:Module 1: Clarification of common misconceptions ofE. coliand fecal pollution.Module 2:E. coli-based fecal source tracking methods, and their applications, advantages, and limitations.Module 3: Non-E. coli-based fecal source tracking methods and their applications, advantages, and limitations.