1 ' LIBRARY $57 Michigan State University This is to certify that the thesis entitled DEVELOPMENT OF A RAPID METHOD FOR A HUMAN POLLUTION SOURCE TRACKING MARKER USING ENTEROCOCCUS SURFACE PROTEIN (ESP) IN E. faecium presented by Lekha Satheesh Kumar has been accepted towards fulfillment of the requirements for the Master of degree in Department of Fisheries and Science Wildlife \W «3%? Major Professor’ 5 Signature og\;ts’\c>’i Date MSU is an afinnative-acfion, equal-opportunity employer .—.—.-.--.- -u-l-O-Q-D-l-o-I-l-v-l--I-c-l-- — _..—.— -l-l-l- It - ._ __ —.—.——..—_ . _ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:/ClRC/DateDue.indd-p1 DEVELOPMENT OF A RAPID METHOD FOR A HUMAN POLLUTION SOURCE TRACKING MARKER USING ENTEROCOCCUS SURFACE PROTEIN (ESP) IN E. faecium By Lekha Satheesh Kumar A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 2007 ABSTRACT Development of a rapid method for a human pollution source tracking marker using enterococcus surface protein (esp) in E. faecium By Lekha Satheesh Kumar Enumeration of fecal indicator bacteria alone will not provide information on the sources of contamination in water, thus microbial source tracking is necessary to identify the source of pollution. Major limitations with microbial source tracking methods are time consumption and the cost of the technique. The objective of the first portion of the study was to develop a rapid method for source tracking human pollution using a host specific genetic marker “esp” gene. Methods (2 & 3) were compared to the conventional method (1) for the detection of the esp marker in E. faecium from raw sewage. The elimination of the DNA extraction process and the removal of the enrichment process from the conventional method were assessed. Ninety seven membranes from 10 different raw sewage samples were analyzed for comparing method 1 and method 2. The rapid method limit of sensitivity was 46i27 cfu/membrane compared to the 45d:28 cfu/membrane of the conventional method. In method 3 (without enrichment) the sensitivity was 26i40 cfu/membrane (n= 78). The second objective was to evaluate the marker in the sewage. The marker was present in all the raw sewage samples (n=23) and in 2/10 effluent samples. The marker was absent in all the non human samples (n=23) and was present in 2/25 environmental samples processed. In a blind study the marker correctly identified 6/7 USGS source samples from human and was absent in all the non human samples processed (n=7) from different geographical regions. ACKNOWLEDGEMENTS I would like to thank the National Oceanic and Atmospheric Administration (N OAA) for funding this research. I am very grateful to my advisor Dr. Joan B. Rose, for her guidance and support throughout my Master’s research. I would like thank my committee members, Dr. Darryl Warncke and Dr. Kelly Millenbah for their guidance during my research. Also, thank you to the other graduate students and employees in Dr. Rose’s lab for their support and help in my research. iii TABLE OF CONTENTS LIST OF TABLES ..... vi LIST OF FIGURES - vii CHAPTER 1 1 LITERATURE REVIEW -- - - _ - - - l 1.1 Water Pollution and Waterborne Disease Outbreaks ................................................ 1 1.2 Beach closure Days at US and Great Lakes Beaches ............................................... 3 1.3 Indicator Organisms .................................................................................................. 4 1.3.1 Michigan Water Quality Standards .................................................................... 5 1.4 Total Maximum Daily Load (TMDL) ...................................................................... 6 1.5 Microbial Source Tracking ....................................................................................... 8 1.5.1 Phenotypic Library Dependent .......................................................................... 9 1.5.2 Genotypic Library Dependent .......................................................................... 11 1.5.3 Genotypic Library Independent Methods. ....................................................... 14 1.5.4 MST using Viruses .......................................................................................... 15 1.5.5 Advantages and Disadvantages of Current Microbial Source Tracking Methods ..................................................................................................................... 15 1.6 Characteristics of Enterococci ................................................................................ 18 1.6.1 The Enterococci Surface Protein (ESP) .......................................................... 18 1.7 Objectives of this Study .......................................................................................... 21 CHAPTER 2 - -- - - - - -- - - - -22 MATERIALS AND METHODS. - - A- - - - - - - -- 22 2.1 Introduction ............................................................................................................. 22 2.2 Sample Collection ................................................................................................... 22 2.3 Positive and Negative Controls ............................................................................... 25 2.4 Concentration of Enterococci from Liquid Samples. ............................................. 25 2.5 Concentration of Enterococci from Solid Samples ................................................. 26 2.6 Method 1- Conventional Method ............................................................................ 26 2.7 Method 2 ................................................................................................................. 27 2.8 Method 3 ................................................................................................................. 29 2.9 PCR primers and reaction condition. ...................................................................... 29 2.10 Sequencing of Amplicons ..................................................................................... 30 iv 2.1 1 Statistical Analysis ................................................................................................ 31 CHAPTER 3 - -- - -- - - -- - 32 RESULTS - - - -- _ - - - - 32 3.1 Introduction..............................................‘ ............................................................... 32 3.2.1 Detection of esp gene in naturally occurring enterococci determining the range of the limit of detection in two methods. .................................................................. 32 3.2.2 Method Comparison using Environmental samples ........................................ 36 3.2.3 Sequencing of Amplicons ................................................................................ 37 3.2.4 Detection of esp in Naturally Occurring Enterococci using Method 3 ............ 38 3.2.5 Detection of esp Gene in Disinfected Effluent Samples .................................. 41 3.2.6 Persistence of the esp Marker in Sewage ......................................................... 42 3.2.7 Blind Analysis of the esp Gene ........................................................................ 43 3.2.8 Presence of esp Marker in Different Watersheds in US .................................. 46 3.2.9 Detection of esp Marker in Non-human Samples ............................................ 48 CHAPTER 4-- - - - 49 DISCUSSION---_-- - ............ - -_ -- - - -- .................. 49 4.1 Introduction ............................................................................................................. 49 4.2 Development of a Rapid Method ............................................................................ 49 4.2.1 Solid Samples ................................................................................................... 53 4.2.2 Presence or Absence of the Marker ................................................................. 53 4.2.3 Marker Testing ................................................................................................. 56 APPENDIX 1-- - _ - -_ -- - -- - -- - 59 APPENDIX 2- -- _- -- - - _ -- -- -- _ -- - - _ 71 REFERENCES - - -- - - - - - 72 LIST OF TABLES Table 1-1. Water quality standards/criteria for recreational water (US EPA -1986, MIDEQ- 2002) ................................................................................................... 5 Table 1- 2. Impairment of water segments due to pathogens in Michigan watersheds. (http://oaspub.epa.gov/waters/state_rept.control?p_state=MI)* ........................ 7 Table 1-3. Comparison of Microbial Source Tracking techniques (12, 29, 40, 42) ........ 17 Table 1-4. Human sewage pollution marker validation(3 8) ............................................ 20 Table 2-1. List of total samples assayed for esp detection in this study .......................... 24 Table 3-1. Conventional method versus Rapid using raw sewage showing percentage of agreement between the methods in each sample .............................................. 34 Table 3-2. Esp gene detection in Enterococci cultured from a variety of samples and the comparison of method one and method two. ................................................... 36 Table 3-3. Detection of esp gene from naturally occurring enterococci from untreated sewage using method 3. ................................................................................... 39 Table 3-4. Detection of esp gene in disinfected effluent samples .................................... 41 Table 3-5. Lowest esp positive in raw sewage using method 1, 2‘& 32 ........................... 42 Table 3-6. Enterococcus concentration and presence of E. faecium esp human pollution marker in USGS source samples (human). ...................................................... 45 Table 3-7. Enterococcus concentration and presence of E. faecium esp human pollution marker in USGS source samples (Non-human). .............................................. 45 Table 3-8. Detection of esp marker in non human samples‘ ............................................ 48 vi LIST OF FIGURES Figure 3-1. Comparison of two methods for detection of the esp gene in naturally occurring enterococci from sewage in the range of the limit of detection‘. (N = 61)*. ................................................................................................................. 35 Figure 3-2. PCR gel image showing amplification of positive control, raw sewage and Katrina sample. ................................................................................................ 37 Figure 3-3. Detection of esp gene in naturally occurring enterococci in the range of the limit of detection using method 3 (N= 75)*. .................................................... 40 Figure 3-4. Presence of esp marker in different watersheds in US .................................. 47 Images in this thesis are presented in color. vii CHAPTER 1 LITERATURE REVIEW 1.1 Water Pollution and Waterborne Disease Outbreaks Water quality at coastal beach areas are directly tied to the health of the people using these waters for recreational swimming activities. Prolonged contact with the contaminated water could seriously affect the health of the beachgoers (29). The major sources of contamination during disease outbreaks associated with use of recreational waters in the US during 1971-2000 were sewage discharges, wild or domestic animals and bathers themselves (8). Beach sand also harbors bacteria that can lead to poor water quality at beaches during rainfall or onshore winds (54). Rainfall has been associated with discharge of untreated sewage via Combined Sewer Overflow (CSO) systems. CSOs are overflows from older sewer systems designed to carry both domestic and storm water loads. A discharge from a C80 occurs in response to rainfall and/or snowmelt because the carrying capacity of the sewer system is exceeded. These discharges do not receive all the treatment that is available and utilized under ordinary dry weather conditions. Normally, during dry weather conditions, the wastewater is transported to a wastewater treatment facility where it receives appropriate treatment prior to discharge. In the US, CSOs serve an estimate of 772 communities containing about 40 million people. Most CSOs are located in the Northeast Great Lakes region, and the Pacific Northwest, and are considered a major contributor of untreated sewage to the waterways in Great Lakes region (www.in.gov). Unintentional ingestion of recreational water contaminated with pathogens from sewage can cause digestive tract illnesses. Pathogens are disease causing microorganisms present in high concentration in the polluted environment. Other than ingestion, full body contact in polluted waters can also cause ear, nose and throat infections, skin rashes and respiratory illness (52, 59). Pathogens that are abundant in the gastrointestinal tract are shed through the feces of warm blooded animals and humans. These are found in sewage as well as manures, animal feces, waste lagoons and wildlife (including birds). In addition to pathogens, enteric bacteria have been traditionally used as indicators of fecal pollution and potential waterborne disease risk. Water quality advisories or closures occur in beaches when levels of fecal indicator organisms exceed an individual state authority’s standards. Prior to 1970, waterborne disease outbreaks were collected by individual agencies. Since 1971 the Center For Disease Control (CDC) and the United States Environmental Protection Agency (USEPA) improved the surveillance system that keeps track of the national recreational waterborne disease outbreaks and the causative organisms (34, 58). Summary estimates based on published epidemiological studies have shown an increase in recreational waterborne disease outbreaks since 1993. There were 65 recreational waterborne outbreaks reported in US during 2001 to 2002 resulting in illness among an estimated 2,536 persons including eight fatalities. Thirty percent of the total outbreaks involved gastroenteritis and of these 60% were related to swimming or wading pools. All the eight fatalities were as result of swimming in lakes or rivers (30, 58). 1.2 Beach closure Days at US and Great Lakes Beaches In 2003, in United States at least 18,284 closing and swimming advisory days were posted at ocean and Great Lakes beaches, and of those, 46% reported sewage as a pollution source with 54% due to non—point sources. According to data released by EPA on July 27, 2004, the number of beaches monitored had tripled in 2004, compared with 1997, the first year when EPA began collecting beach-monitoring program data. Nationally the number jumped 9 % in 2004 compared to 2003. Of the 3,574 beaches that were monitored in 2004, 26 % had at least one advisory or closing during the 2004 season. Among the 4,025 beaches that were monitored in 2005, 28 % had at least one advisory or closing during the 2005 season due to high levels of certain bacteria that exceed standards (www.mdcorg). Monitoring data published by Natural Resources Defense Council (N RDC) on July 2006 indicated that 13% of the total monitored samples (n=18,309) from 530 Great Lakes beaches exceeded the beach water quality standards. The Beach Act suggests for E. coli a daily maximum standard of less than 235 colony forming units (cfu) per 100ml of fresh water. They also reported that 54 beaches situated in every Great Lakes states except Pennsylvania had exceeded the standard at least 25% of the time. The number of closing and/or advisory days increased 5% in 2005 compared to 2004. The increased monitoring of the beaches could be one reason for this. In 2005, the major reason for the beach advisory or closing was due to the elevated level of bacterial concentrations (87%). Unknown sources of pollution caused 79% of the total days closed, which was an increase of 403 closure days posted (some of the beaches closed more than one day per year) from 2004. Storm water run off, sewage spills and overflows and rain fall advisories from other sources contributed to the closures. 1.3 Indicator Organisms Monitoring of hundreds of pathogens on a routine basis in the environment is a costly and time-consuming process. Historically, Fecal coliform bacteria were used as indictor organisms but complications with their use and interpretation began to emerge (47). E. coli and enterococci, two indicators, used by States and recommended by EPA, are considered to have a higher degree of association with disease and have been recommended as the basis for bacterial water quality standards (6). Fecal coliforrns, E. coli and enterococci are present in abundance in the gastrointestinal tract of warm- blooded animals so that the presence of these bacteria is considered as a strong indication of fecal pollution. Water containing higher levels of indicator organisms is considered unsafe because it also indicates the possible presence of other waterborne pathogens(6). Recreational activities in these types of impaired waters are considered to pose serious health risks. The enterococci have been used as indicators of fecal pollution for many years and have been valuable in the marine environment and recreational waters as indicators of potential health risks and swimming related gastrointestinal and urinary tract illness (51). Studies demonstrated that there was a strong correlation between precipitation and the amount of time spent in water. Waterbome disease outbreaks, and particularly the presence of enterococci, was directly related to the increased risk of recreational related acquired gastrointestinal illness (4, 51, 52). 1.3.1 Michigan Water Quality Standards Michigan uses fecal coliform and E. coli bacterial concentrations for assessing the water quality standards for recreational waters. Michigan’s water quality standard specifies a geometric average of < 200 cfu per 100ml of fecal coliforrns and < 130 cfu per 100ml for E. coli (Table 1-1). For EPA, the recreational water criteria specify geometric average of < 200 cfu per 100ml for fecal coliforms, < 126 cfu per 100ml for E. coli and less than 33cfu per 100ml for enterococci. Table 1-1. Water quality standards/criteria for recreational water (US EPA -1986, MIDEQ- 2002) Indicator Geometric Means Michigan EPA Fecal Coliforms CF U <200cfu/ l 00ml <200cfu/100ml E. coli CFU <130cfu/100m1 <126cfu/100ml Enterococci CFU No standard <33cfu/100ml 1.4 Total Maximum Daily Load (TMDL) The Clean Water Act was passed in 1972, yet today many of the US water bodies do not meet the water quality standards, goals and/or designated uses. Each state has responsibility to identify highly polluted waterbodies that are not meeting water quality standards. These impaired waters must undergo an evaluation using the Total Maximum Daily Load (TMDL) approach according to the Clean Water Act. Microbial densities that a particular water body can receive can be addressed partially via the TMDL because a TMDL is defined as "the maximum pollutant load that a water body can receive and still meet water quality standards” (50). Exceeding the TMDL criteria for indicator bacteria generally focuses on the potential for illness in people who are in contact with such water bodies. Water quality improvements there by also reduce the risk of infectious diseases. As a part of this process, identifying the fecal pollution source could lead to better management and remediation. In 2004, Michigan Department of Environmental Quality (MDEQ) identified 580 water segments of impairment in Michigan watersheds. MDEQ specifies that 90 of the impairments in Michigan waters were due to pathogens, which is about 16% of the total (n= 580) impairment reported. In those 90 impairments, the majority (n= 84) were due to pathogens and untreated sewage discharges. Bacterial slimes, beach closures, E. coli and fecal material comprise the remainder (Table 1- 2) (MDEQ). Table 1- 2. Impairment of water segments due to pathogens in Michigan watersheds. (http://oaspub.epa.gov/waters/state_rept.control?p‘state=MI)* Cause of Impairment in MI Causes of Impairment (%) watersheds Bacterial Slimes 3% Beach closures 1% E. coli 1% Fecal 1% Pathogens 71% Untreated sewage discharges, 13% Pathogens Total numbers of segments 90 1.5 Microbial Source Tracking The TMDL attempts to calculate contaminant impairments and regulate the amount and type of pollution entering the water. To correct the problem, the source of pollution must be identified (50). Microbial Source Tracking (MST) is a developing field to help pinpoint the source of the microbial pollution in the aquatic environment (40). For example Hagedom et a1. (1999) were able to trace the fecal pollution in a Virginia watershed using a MST method and were able to implement Best Management Practices (BMP) for improving the water quality. The study was conducted for a 28 month period where the first 12 months concentrated on identifying the source pollution. They found that high concentration of fecal bacteria in stream areas during the summer were due to cattle (18). By restricting the cattle access to the river they were able to reduce the pollution in those stream areas. Traditional indicator bacteria have been used for many years for determining the sanitary quality of surface and recreational waters. Even though indicator organisms have been shown to relate to recreational health risks they cannot be used to address the sources of pollution. According to the Clean Water Act, recreational water should be protected from pathogenic microbial organisms. Thus there is a growing need for a science - based approach to determine the pollution source to abate the problem in a cost effective manner (38). Various methods have been developed and used for MST (40, 43). The basic premise of the source tracking methods is that bacteria demonstrate specific characteristics related to host specificity and adaptation to certain environmental conditions (29, 40, 42). The methods are classified into two main categories, Library-dependent or library- independent methods. Library dependent methods rely on the building of a library that is developed with bacterial isolates from known sources (40, 49). Most are culture based and often rely on genetic characterization. Reference libraries are built using isolates, collected directly from animals or collected directly afier excretion to ensure there is limited contamination. Library dependent methods are based on matching the reference libraries (phenotypic or genotypic profiles) of bacterial strains to isolates from the environmental sample (49). Library-independent methods rely on identification of a host- specific trait or gene. The following sections will describe some of the MST methods. 1.5.1 Phenotypic Library Dependent 1.5.1.1 Antibiotic Resistant Analysis (ARA) Antibiotic resistance profiling is the most widely used phenotypic method for MST. A library of antibiotic resistant genes in enterococci, fecal coliform or E. coli of known animal sources are used to identify the source of pollution (5, 18, 55). For this method, antibiotic resistant bacteria are isolated via inoculation onto agar plates containing different antibiotics. The growth of the bacteria in the antibiotic plates is compared to a control plate (without antibiotic) to develop profiles. This comparison is based on the assumption that bacteria from human, livestock and domestic animals are more resistant to different sets of antibiotics so that one can differentiate wildlife fecal pollution from domestic animals and from human waste, using this technique. This requires culturing and screening a large number of isolates against antibiotics of different concentrations to make a large library that represents a fair number of likely sources in a given watershed. So the accuracy of the results rely partly on the size of the library yet the heterogeneity of the bacteria contributes to a high false positive rate (21, 31). The fingerprints are compared to a reference database composed of bacterial isolates from known sources to find out the Average Rate of Correct Classification (ARCC). Results from various MST studies using antibiotic resistant genes summarized have reported ARCC ranging from 62 to 96% for this method, when classifying some subset of the library, however this rate goes down with new isolates (42). Geary et a1. (2003) were not able to identify any significant source of pollution when they assayed four different concentrations of four different antibiotics in a shell fish growing area in Australia (14). The reference library was composed of isolates of fecal streptococci from beef, dairy cattle, chickens and humans(14). Research has shown that isolates from point sources were more diverse than isolates from nonpoint sources and grouping is likely influenced by a strain's prior exposure to antibiotics (31). Another draw back of this method is that antibiotic resistance genes are carried by plasmids which may be lost upon cultivation. A study conducted in six Virginia watersheds by Wiggins et a1. (2003) concluded that antibiotic resistant plasmids may only be stable up to one year (55). 1.5.1.2 Carbon Source Utilization Carbon source utilization profile is another type of phenotypic MST method. This method is based on the BIOLOG system which has a 95 well microtitre plate that contains different carbon substrates. The microbial isolates from known sources are categorized in accordance with their utilization of carbon substrates. A study conducted by Hagedom et a1. (2003) used 365 enterococci isolates of known source from four different geographical regions using BIOLOG system. The ARCC ranged from 86.6- 92.7% for the human versus non-human two-way classification. But the ARCC was 10 lower for the three way classification system and a high percentage of false positives (51.5%) was also noted (17). Again new isolates were not classified with high accuracy. These phenotypic methods are not used very frequently because phenotypic characters are not always stable and most researchers are moving toward genotypic methods. In addition to that nutrient requirements of the microorganisms are based on environmental factors so the carbon source utilization may not be same in all environments (17, 42). 1.5.2 Genotypic Library Dependent Library based genotypic analyses use DNA finger prints and techniques such as rep- PCR, ribotyping, Amplified Fragment Length Polymorphism (AF LP), and Pulse Field Gel Electrophoresis (PFGE). For these methods DNA is extracted from bacterial cells and restriction enzymes are used to cut the DNA into different base pair lengths. Ladder- like patterns are visualized using gel electrophoresis and according to the size of the DNA fragments the isolates are grouped statistically. 1.5.2.1 Rep-PCR For these genetic methods, a specific repetitive portion of the microbial genome is amplified by Polymerase Chain Reaction (PCR) which is visualized by electrophoresis and pattern recognition by computer software. The three major repetitive elements in bacteria being used to generate bacterial finger prints are REP (Repetitive Extragenic Palindromic), ERIC (Enterobacterial Repetitive Intergenic Consenses) and BOX (154 basepare box element). A small study based on the BOX-PCR conducted by Dombeck et.al. ((200) used154 E. coli isolates obtained from six species of animals (cows, pigs, 11 sheep, chickens, geese and ducks) and humans and revealed that 100% of the chicken and cow isolates gave the correct classification. Between 19-29 isolates were used per source and the correct classification of other isolates was 78 to 90% (10). In this study they also suggested that BOX- PCR was better than REP-PCR for differentiating human from non human E. coli isolates. While in another study conducted by Johnson et a1. (2004) using E. coli strains from 12 different animal sources, the correct classification was only 60% despite the relatively large size of the known source data base(23). 1.5.2.2 Ribotyping Ribotyping includes multiple steps such as restriction enzyme digestion of genomic DNA, fragment separation by gel electrophoresis, transfer of the DNA fragments by Southern Transfer and hybridization using labeled rRNA probes to generate ribotype patterns that can be used to identify bacterial strains (9, 29, 37). The rRNA (ribosomal ribonucleic acids) is highly conserved and is an integral part of all living organisms. In a small study 287 E. coli strains from humans, cattle, pigs, horses, chickens, turkeys, migratory geese, and dogs were collected and riboprint patterns were generated (7). They found this method was useful for differentiating fecal bacteria of human and non- human origin but the Average Rate of Correct Classification (ARCC) was poor when pooled fecal sources were analyzed. This method also required extensive manipulation of DNA and use of labeled gene probes. However Jenkins et al. (2003) suggested that to make the ribotyping effective one should also consider the temporal variability (40). This observation was based on the study using wild deer and captive deer. Researchers found that the diet was influencing the ribotype diversity of E. coli between the wild and 12 domesticated deer (22). Another draw back of this method is the necessity of a large database that represents each watershed for the correct source identification. 1.5.2.2.1 Pulse Field Gel Electrophoresis (PFGE). Pulse Field Gel Electrophoresis (PFGE) is another technique used for ribotyping but it analyzes DNA rather than rRNA (ribosomal RNA). This was found to be more discriminatory than ribotyping with pure culture bacterial cells (29, 46). However Parveen et a1. (2001) were not able to distinguish between human versus non-human sources when they analyzed 32 E. coli isolates using PFGE technique (33). Another draw back of PFGE is electrophoreses time consumption (3 0-50 hours) and the specialized equipment for the process (42). 1.5.2.3 Amplified Fragment Length Polymorphism (AFLP) Guan et al. (2002) used 105 E. coli isolates from the feces of cattle, poultry, swine, deer, goose, moose, human sewage, and clinical samples that were genotyped by Amplified Fragment Length Polymorphism (AF LP)(16). These isolates were digested with restriction enzymes to generate a subset of DNA and PCR amplification was done on that subset. The success rate of this method solely depends on the availability of whole genomic sequence of the particular isolate. In this study they used a smaller number of isolates, since natural populations of E. coli comprise a large community with high genetic variation. So it is necessary to grow a large number of isolates and the method may take 10-12 days to complete the manual ribotyping, a large database is also needed for comparison (32, 35). 13 1.5.3 Genotypic Library Independent Methods. 1.5.3.1 Host specific molecular markers Host specific MST methods using genetic markers appear to have the ability to address human versus non-human sources with a much lower false positive rates compared to Library based methods (3, 21). Host specific methods are generally gene specific PCR methods that have been developed by targeting specific genes, found only in certain hosts. The prevalence of the gene determines the sensitivity and the false negative rates. 1.5.3.2 Terminal Fragment Length Polymorphism (T-RFLP) Terminal Fragment Length Polymorphism (T-RFLP) and length-heterogeneity polymerase chain reaction (LH-PCR) techniques use an automated sequencer for the detection of fluorescently labeled 16S rDNA. Bernhard and Field (2000) demonstrated that there are species composition differences between Bifidobacterium and the Bacteroides-Prevotella groups among human and ruminants. The specific primer sets for human and ruminant were designed (2, 3, 12). However, this method has only been tested against a limited number of animal fecal samples to date. Other potential sources also need to be included. Although this method was found to be helpful for MST, it is technically very demanding and expensive equipment is required for this host specific method for detection(42). The short term survivability of Bifidobacterium and its lower concentration in human feces needs to be considered before applying this for routine monitoring (41). 14 1.5.4 MST using Viruses Fecal viruses also have been used to trace sources of fecal contamination in environments. Human enteroviruses and bovine viruses were used to differentiate human versus bovine fecal contamination. The main advantage of using viruses as indicators includes the ability to monitor directly in water, without culturing(13). However large volumes of water need to be screened for virus detection compared to the traditional water quality indicator organisms. While screening larger volumes, PCR inhibitory substances also concentrate and that may interfere with the PCR results(44). Like other culture independent methods using viruses for source tracking will not always indicate a recent contamination because the detection method targets viable as well as non viable cells. 1.5.5 Advantages and Disadvantages of Current Microbial Source Tracking Methods. In summary different MST have their own advantages and disadvantages (Table 1-3). Most of these methods target was E. coli, however a few of them targeted fecal streptococci, enterococci and Bacteroides. The time for each technique and the cost are critical factors when considering the methods applicability. For instance culture dependent and library dependent phenotypic methods require culturing the organism and construction of large databases for comparison. At the same time Carbon Source Utilization is a simple process but is mostly applied to pure culture such as clinical isolates. Rep-PCR has high discriminatory power but this method also needs a reference library. Like Rep- PCR, ribotyping also has discriminatory power but this method is a 15 very complex and time consuming process. Despite the advantage of applicability for most of bacterial strains, AFLP and the PF GE methods are considered less demanding for MST because of the cost and the technical level needed. Unlike other methods RFLP does not need a library and it is a culture independent method. However the limitations of this method are also related to the expense and the complex processes. 16 Table 1-3. Comparison of Microbial Source Tracking techniques (12, 29, 40, 42) Method Description Advantage Disadvantage Microbial Target (ARA) Distinct ARA Useful for Time Fecal Antibiotic patterns used to smaller consuming. streptococci, Resistant identify the watersheds Antibiotic E. coli, analysis“ source resistant gene Enterococci pollution. may be lost during cultivation. Requires large database Carbon Substrate Applicable Carbon Source utilization clinical utilization can Utilization* differences microbiology. change and among bacteria Simple process depends on Enterococci indicate sources environmental factors. Requires large database Rep-PCR* PCR based High Culture amplification discrimination dependent. using Large database E. coli, palindromic required DNA sequences Ribotyping“ DNA digestion Discriminatory Time with restriction power and easy consuming enzymes, interpretation complex E. coli, hybridization procedure using labeled probe AF LP & DNA Applicable in Expensive and E. coli, PF GE* fingerprinting most strains, technically by digestion automatable demanding with restriction enzymes T-RFLP Used automated Culture Expensive sequencer for independent equipment the detection of Library needed and Bacteroides fluorescently independent technically labeled l6S demanding rDNA *Culture and Library based methods 1.6 Characteristics of Enterococci The US EPA in 1986 suggested the enteococci as pollution indicators for marine environments and both E. coli and enterococci as fecal indicators for fresh water environments. The taxonomic group Streptococci contains both Enterococcus and Streptococci. The enterococci sub group has the ability to grow at high and low temperatures (IO-45°C) and at high pH (9.6). Enterococci is a gram positive roughly spherical shaped bacteria found in the intestinal tract of warm blooded animals. The species of Enterococcus include opportunistic pathogens such as Efaecalis and E. faecium. Enterococci are well suited to survive in the intestine, vaginal tract and the oral cavity. These bacteria have a natural ability to readily acquire, accumulate, and share virulence traits (clustered on a large pathogenicity island) and/ or antibiotic resistance genes (24, 36, 38). According to EPA criteria, a single sample maximum concentration of Enterococci for marine water should be less than 61cfu/100ml. For fresh water it should be less than 104/ 100ml, while a geometric mean of 33 CFU/100ml was suggested. 1.6.1 The Enterococci Surface Protein (ESP) Wei et a1. (2001) suggested that PCR based detection of virulence factors would be a better indicator of the health significance and a tool for MST (53). The esp gene codes for the enterococcus surface proteins and studies have demonstrated that the esp gene in E. faecium is a virulence factor in this microorganism and plays a role in it’s pathogenesis (25, 36, 45). A study conducted using 120 isolates associated with hospital patients during outbreaks from USA, Europe and Australia reported that the esp gene in clinical isolates of E. faecium were genetically distinct from the non epidemic isolates (n=45) as 18 well as animal isolates (n=98), it was also suggested that its presence was a marker of increased virulence (56). Various E. faecium isolates from hospital outbreaks (11, 56) were used to clone and sequence the esp and these revealed the presence of PAI (pathogenicity island) related to epidemicity. Willems et a1. (2001) indicated that the esp in E. faecium is present in the chromosome of the bacteria and had enough evidence to believe that the presence of this gene was associated with nosocomial outbreaks of E. faecium (24). Nosocomial outbreaks are those that originate or occur in a hospital or hospital like settings. Primers specific for the E. faecium esp gene developed previously were used as a marker of human sewage (3 8). This target originated from a homologue that had been identified in isolates of E. faecium previously, and was used to develop the primers and the amplicon(56). A culture- PCR based method was evaluated to assess presence of the esp gene in Enterococci from livestock, birds, other animal sources and human sewage. A total of 167 wastewater and fecal samples were screened for the target gene and the gene was detected in primary sewage influent (40/40), secondary sewage effluent (10/10), filtered waste water (5/5) and septic tank effluent (8/ 10). The marker was never detected 0% (n=102) in animal waste water or fecal samples. Table 14 shown the esp marker validation using samples from a variety of sources. Survival studies showed that the marker was detectable in fresh water up to five days when the colony forming units ranged between 19 and 122 and in simulated seawater up to seven days when the total number of culturable eenterococci was more than 70 CFU (3 8). 19 Table 1-4. Human sewage pollution marker validation(3 8) Source* No of samples positives/No of samples tested Primary sewage influent 40/40 Secondary sewage effluent 10/10 Filtered waste water 5/5 Septic tank 14/ 16 Waste lagoons- Poultry 0/8 Waste lagoons-Swine 0/ 12 Waste lagoons -Cattle 0/35 Feces-Canada Geese 0/ 12 Feces-Seagull 0/28 Feces-Pelican 0/7 Feces-Wild Birds 0/10 *Culture based, library independent host specific assay based on populations of bacteria not individual isolates 20 1.7 Objectives of this Study Based on the initial study by Scott et al. (2005) further evaluation of the esp marker in enterococci was warranted .The overall objective of this study was to further examine the utilization of the esp gene as human sewage marker and streamline the potential method, allowing for more sensitive results. Specific objectives included: 1) Comparison of a rapid PCR (Direct PCR) to a conventional (DNA extraction) method. 2) Further validation of the presence or absence of the marker in various sources. 3) Persistence of the marker in raw sewage and the sensitivity of the method and prevalence of the marker. 21 CHAPTER 2 MATERIALS AND METHODS 2.1 Introduction Effective assays for source tracking are available, but these are often expensive, take time and are complex (Table 1-3). Based on epidemiological studies the most serious threat to human health comes from human and not from animal sources (52). The elimination of human source pollution is important for managing water quality and thus protecting public health. The source tracking method used by Scott et al. (2005) for enterococci esp gene included cultivation followed by a three hour enrichment step, DNA extraction procedure and PCR (3 8). One of the specific goals of this study was to minimize the processing time without affecting the test results. Thus, procedures to eliminate the extraction and enrichment steps were evaluated and these were termed rapid PCR approaches. 2.2 Sample Collection Domestic raw sewage and secondary disinfected wastewater effluents were collected from a waste water treatment plant in East Lansing, MI from August 2005 to July 2006. Twenty three raw sewage samples were collected in 50mL sterile centrifuge tubes and 10 secondary effluent samples were collected in one liter sterile bottles with sodium thio sulphate to neutralize any chlorine residual. Because of the high concentrations of the bacteria in sewage and fecal samples, numerous replicates were prepared from each sample. In addition to waste water samples, 13 water samples from New Orleans after 22 Katrina were collected and shipped overnight to the Michigan State University and tested for indicator bacteria and the esp gene. Four samples from animal waste water, six samples from ditches of a near by farm and nine bird samples were also collected. A one day beach and bird survey was conducted on July 9, 2006 at North Beach, along Lake Michigan in Grand Haven, Michigan. The goals of this research project were to examine bird behavior and determine the extent that bird feces impacts beach sand and water quality at a recreational beach. Sand samples were collected that day along the beaches and eight of the sand samples were also analyzed for esp marker. All samples were processed within 24 hours from the time of collection. Samples were also collected by the USGS throughout the US from human and animal pollution sources, and duplicates of these were shipped to the MSU Environmental Quality Laboratory. These were not identified as to the source at the time of processing and were treated as blind samples. Samples were collected from Colorado, Texas, Iowa, New Jersey, Minnesota, Virginia, New York, Idaho and Ohio. All liquid samples were collected in one liter sterile bottles and the solid samples were collected in sterile 4 ounce specimen containers. All samples were collected using aseptic techniques and were transported to the laboratory on ice before being processed. Table 2-1 shown the total number of samples assayed for the esp marker detection. 23 Table 2-1. List of total samples assayed for esp detection in this study Total no. of Source Sample Type samples collected 23 WWTP* Raw sewage 10 WWTP Secondary effluent 13 Katrina (New Flood water Orleans, LA) 4 MSU (Farm) Animal waste water 9 MSU (Farm) Drainage water 9 Bird Feces 8 Beach Sand 18 USGS” Human 9 USGS Non-human *waste water treatment plant “US Geological Survey 24 2.3 Positive and Negative Controls Efaecalis (ATCC# 19443) was used as negative control. E. faecium C68 strain containing the esp gene was used as positive control (provided by Dr. Louis B. Rice of the Cleveland Veterans Medical Center in Cleveland Ohio). The PCR products of the E. faecium C68 were purified using QIAquick PCR Purification Kit (Qiagen, Inc.). A TOPOTA Cloning® Kit (Invitrogen, Co.) was used to clone the PCR products and this plasmid was used as a positive control for all conventional and rapid methods. 2.4 Concentration of Enterococci from Liquid Samples. Enterococci were enumerated in liquid samples by membrane filtration. Serial (ten fold) dilutions were used and lmL of the diluted solution was filtered through a 0.45 pm pore-size 47mm nitrocellulose-mixed ester membrane filter (GE Osmonics). The first step in making a serial dilution is to take a known volume (usually lml) of the concentrated sample and place it into a known volume of buffer (usually 9ml). This produces 10ml of a diluted solution. The dilute solution has lml of extract /10ml. This is a 10 fold dilution. This process was repeated to make successive ten fold dilutions. Three ten fold (10", 10-2, 10-3) dilutions were made from each of the samples and replicates of each dilution were filtered as described above. The filters were then placed on mEnterococcus (mE) agar (Difco) supplemented with indoxyl substrate (mEI) (Sigma Inc.) according to the methodology outlined in EPA (48). Filters and media were incubated at 413: 05°C for 24h. After 24 hour incubation, the blue halo colonies were counted as enterococci. Filters were stored at 4°C until further analysis for the esp gene. 25 2.5 Concentration of Enterococci from Solid Samples Three grams of each of the solid fecal sample were weighed and placed into a sterile 50ml centrifuge tube. Thirty milliliters of the 1X Phosphate Buffer Water (1 .25ml of 0.5M KH2PO4, 5mL of 0.44M MgCl2; pH 7) (PBW) were added to the tube, which was then vortexed to suspend the sample. Serial dilutions were performed using PBW and appropriate dilutions were filtered through 47mm nitrocellulose-mixed ester membrane filter (GE Osmonics) placed on mEI media and incubated at 41d: 05°C for 24 hour incubation. After 24 hour incubation Colony Forming Units (CFU) were counted and the membrane filters on the agar plates were stored at 4° C until further analysis. 2.6 Method 1- Conventional Method In the conventional method the E. faecium esp analysis and DNA extraction were performed as follows: Membranes that were positive for enterococci colonies were further evaluated to determine the presence of the target gene esp (3 8). Membrane filters containing zero to too numerous to count (TNTC) colony forming units (CFUs) were lifted, suspended in a lSmL centrifuge tube containing ten mL of Tryptic Soy Broth TSB (Difco), vortexed vigorously for 30 seconds and incubated at 41°C for three hours. The centrifuge tubes were removed from the incubator and vortexed again. One milliliter of the resulting culture was placed into a microcentrifuge tube and the tube was centrifuged (NapCo 2002 centrifuger) at maximum speed for five minutes. The supernatant was removed and 180uL of lysis buffer (20mg/ml lysozyme; 20mM Tris-HCL, pH8.0; 2mM EDTA; 1.2% Triton® was added to the pellet. The pellet was resuspended and placed into a 35°C water bath for 30 minutes. Then 20uL of Proteinase K (Qiagen, Inc) and 200uL of lysis 26 solution (Qiagen,Inc) was added to the tube and vortexed. The tube was placed into a 45°C water bath for 30 minutes. The temperature of the water bath was increased to 95°C for 15 minutes. The microcentrifuge tube was removed from the bath and kept at room temperature. DNA extraction was performed using the QIAamp DNA Mini Kit according to the manufacturer’s instructions (Qiagen, Inc.). Then lul of the DNA was used as the template for the PCR reactions from the total (400 pl) extracted DNA. The total time consumption for the DNA extraction step was 3.5 hours. 2.7 Method 2 The main difference between method 2 and method 1 was in the preparation of the template for rapid PCR reaction where by no extraction was used (Figure 2-1). For the rapid PCR lml of the TSB was transferred after the two hour incubation to a 1.5m] centrifuge tube. 27 Figure 2-1. Schematic diagram showing the sample analysis for detecting enterococci and the esp gene using three methods Sample Collection Membrane Filtration Incubation Enumeration (cfu) V I Method 1 Method 2 Method 3 Enrichment (TSB) Enrichment Cell concentration from the (3 hours) (TSB) (2 hours) membrane filter (30 minutes) DNA Extraction Cell concentration (3.5 hours) from TSB ' PCR 28 The cells were spun down by centrifuging five minute at maximum (14000 rpm) speed (N apCo 2002 centrifiiger). The supernatant was removed without disturbing the pellet using a micropipette. This step was repeated one more time. Finally the pellet was resuspended with 500ul of molecular grade water (DNase/RNase free water) and placed into a -20°C freezer until analyzed by PCR. Two micro liters of the cell suspension was placed into PCR reaction tube as the template for the marker detection. 2.8 Method 3 In method 3 the preparation of the template was done without the enrichment step (Figure 2-1). After 24 hours of incubation the blue halo colonies were counted and the cells were washed off the filters with lml of molecular grade water (Sterile water) using a sterilized plastic cell spreader. The cell suspension was spun down by centrifuging five minutes at the maximum speed (14000 rpm) (NapCo 2002 centrifuger). The pellet was resuspended with 500 pl of molecular grade water and washed via centrifugation. Finally the cell suspension was placed into a -20°C freezer until analyzed by PCR. Two micro liters of the cell suspension were placed into a PCR reaction tube as the template for esp detection. 2.9 PCR primers and reaction condition. The primers specific for the esp gene in Efaecium previously developed and examined for specificity to human fecal pollution were used for detecting the esp marker (28). The forward primer: (5’-TAT GAA AGC AAC AGC ACA AGT-3’) and the conserved reverse primer (5’ —ACG TCG AAA GTT CGA TTT CC-3’), were used for all reactions. 29 PCR reactions were performed in ZOuL reaction mixtures containing 1X PCR buffer, 1.5mM MgClz, ZOOuM of each of the four deoxyribonucleotides, 0.3 uM of each primer 0.5Unit of HotStarTaq DNA polymerase (Qiagen) and lul of template DNA. Amplification was performed in Eppendorf Mastercycler gradient (Eppendorf scientific inc. Westbury, New York) with an initial step at 95°C for 15 minutes (to activate Taq polymerase) , followed by 35 cycles of 94 °C for 1 min,58 °C for 1 min, and 72 °C for 1 min. The PCR products were separated on a 1.5% agarose gel stained with Gelstar nucleic acid stain (Cambrex Bio Science, Inc.) and viewed under ultraviolet (UV) light. The PCR product is 680 base pairs in length. 2.10 Sequencing of Amplicons The amplified products were isolated in a 1.75% low melting temperature agarose gel (Sea Plaque GTG; Cambrex Bio Science Baltimore Inc. Baltimore, MD) and the product was purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA). The same primers for esp gene were used for dye terminator cycle sequencing of each amplicon. Sequencing was carried out with an ABI 377 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA) at the Michigan State University (MSU) sequencing facility. The multiple sequence alignment of the Enterococcus esp gene was executed using the pileup program of genetics Computer group (Madison, Wis.) Squeb software. The sequence analysis was also performed by the blast program on the National Center for Biotechnology Information. 30 2.11 Statistical Analysis A paired t-test was performed at the average cfu/membrane limit of detection on a log transformed data to address the statistical difference between method 1 and method 2 and a chi-square test was done to test the significant association between method 1 and method 2 in the relative presence versus absence of the marker per membrane. All statistical analysis (Chi-squared, t-tests) were performed using SPSS for windows, Rel.12.0.0.2003.Chicago: SPSS Inc. 31 CHAPTER 3 RESULTS 3.1 Introduction Microbial source tacking (MST) is becoming an accepted field of study because of its applicability to better define the relationship between environmental quality, source of pollution and human health (40, 42, 51). However the MST field needs to be improved with regard to affordability to the communities using them and accuracy of the results. This research focuses on improving a host specific test and addressing the accuracy for identification of improperly treated sewage. 3.2.1 Detection of esp gene in naturally occurring enterococci determining the range of the limit of detection in two methods. Ninety seven membranes from ten different samples of raw sewage were analyzed by method 1 and 2. Method Us a conventional method which includes membrane filtration, incubation on mEI media, recovery of all colonies from membrane, enrichment and DNA extraction followed by PCR for esp detection where as the method 2 excludes the DNA extraction and is considered rapid. Membranes from zero to TNTC were assayed for the comparison. Results are shown in Table 3-1. All four membranes assayed from sample 1 with cfu ranging between 29 and TNTC were esp positive in method 1 and in method 2. Sample 2 had cfu between zero and 48 per membrane and of the six membranes all were negative by both methods. In samples 3, 4, and 5, 13 membranes with cfu from 4 to 560 were compared and six were positive and seven were negative by both methods. There was 100% agreement between the two methods in these samples. Forty seven membranes 32 from sample 6 were assayed and comparative results in 40/47 were obtained using the two methods. There were five esp positive membranes from the conventional method and two positives from the rapid method that did not agree each other (85% agreement). Figure 3-1 shows the results graphically for each membrane. Agreement in sample 6, 7, 8 was 88% to 95 %. In all other samples (sample 9 & 10) the results by the two methods were in 100% agreement. Overall the two methods agreed 91% of the time (88/97). The sensitivity of method 1 was 45128 cfu/membrane and the sensitivity of the method 2 was 46i27 cfu/membrane. The sensitivity was calculated by taking the geometric average of all counts cfu/membrane with esp negative results. Positive esp membranes had cfu ranging from 21 to TNTC in method 1 and method 2 where as the negative esp membranes had cfu range from zero to 117 in method 1 and and zero to 106 in method 2. The paired t-test performed showed that there was no difference between the two methods ((p = 0.53) chosen level of p = 0.05). Hence the paired t-test failed to reject the null hypothesis at the 95% confidence limit. 33 Table 3-1. Conventional method versus Rapid using raw sewage showing percentage of agreement between the methods in each sample No cfu No.0f Method 1 Mehod 2 Method 1 Method 2 Agreement in (Range)* membranes esp +ve esp +ve esp -ve esp -ve both methods assayed (%) 1 29-TNTC 4 4 4 0 0 100 2 0-48 6 O O 6 6 100 3 19-560** 4 3 3 l 1 100 4 12-500" 4 1 1 3 3 100 5 4-280 5 3 3 2 2 100 6 45-120 47 23 21 24 26 85 7 36-65 14 4 3 10 ll 93 8 39-100 5 4 5 l O 80 9 58-140 4 4 4 0 0 100 10 44-160 4 l 1 3 3 100 Positive Negative Total Method 1*“ 47 50 97 Method 2 45 52 97 *cfu/membrane cultured on mEl ** Higher values extrapolated from plates in countable range. ***Chi-square test showed no significant difference in the results obtained by method 1 versus method 2. (p< 0.001) 34 Figure 3-1. Comparison of two methods for detection of the esp gene in naturally occurring enterococci from sewage in the range of the limit of detection'. (N= 61)*. . ti“ :17 In :21 I nnnl 010 DIIIJCIZII ESP No 000:. ‘0 [[0 ‘0I0 0!:10020 0 00 CI] 0 0 0 I ESP -vo CFU I Membrane ‘Sensitivity was 45:1:28 cfu/membrane in method 1 & 463:27 cfu/membrane in method 2 ‘No. of membranes in this figure is 61. 35 3.2.2 Method Comparison using Environmental samples A total of 36 membranes from 23 different environmental samples were processed and compared using method 1 and method 2. All of the results between the two methods were in 100% agreement. Results are shown in Table 3-2. The majority of the samples were from the New Orleans Katrina affected area, but the cfu range was generally below the detection limit of the methods (30). Only one sample came up as positive for the esp marker in Katrina where the membrane had 121 cfu. The rest of the samples (n= 12) were esp negative by both methods. Nine water samples from ditches near the MSU farm were assayed for the esp marker. One sample was esp positive by method one and two. It was speculated that a leaky sewage line or an older septic field could have contributed to the finding of the human marker. The membranes processed for the esp marker from the MSU samples had a cfu range between 10 and TNTC where the total membranes assayed was 14. A ground water sample was also processed from a private house well which had a cfu ranging between 67 and 189 per membrane. All three membranes assayed for the esp marker were esp negative by both methods in this sample. Table 3-2. Esp gene detection in Enterococci cultured from a variety of samples and the comparison of method one and method two. Membranes No. of Source Esp + Esp - Agreement assayed samples (%) 14 9 MSU Farm 1 8 100 3 1 House Well 0 1 100 19 13 New Orleans 1 12 100 36 3.2.3 Sequencing of Amplicons The PCR product from the positive control, one sample from raw sewage and one from the Katrina affected area which were positive for the esp marker in method 2 previously, were sequenced to examine the specificity of the esp marker in the samples (Figure 3-2). The lanes were loaded with Marker (lkb), positive control, negative control, raw sewage, and Katrina sample respectively except lane five which was empty. The remaining lanes were loaded alternatively with samples that had been previously negative for esp. The lanes between the samples were empty. The three amplified PCR products were used for the sequencing. Figure 3-2. PCR gel image showing amplification of positive control, raw sewage and Katrina sample. Marker Positive control Raw sewage - :0 I! G ""'—‘i —¥'#‘ -_,_ Katrina sample 37 3.2.3.1 Sequencing Results Multiple sequence alignment of the three selected isolates was undertaken. A total of 612 base pairs (bp) of the esp marker was assessed (appendix 1). Alignment of Esp gene from the two environmental samples (isolates from Katrina samples and raw sewage) matched the positive control with 100% sequence similarity and without a single mismatch of nucleotides. This genetic evidence supports that the human marker esp gene present in raw sewage and in Katrina samples were the same as that of the positive control. 3.2.4 Detection of esp in Naturally Occurring Enterococci using Method 3 In an attempt to reduce the time taken for the esp analysis, the next step was to eliminate the enrichment step. A total of 78 membranes from 12 different samples were processed by method 3 for the detection of esp marker. All samples were raw sewage collected from February 2005 to July 2006 from East Lansing waste water treatment plant. Of the total 78 membranes processed, 38 were esp positive with cfu ranging between 1 and TNTC per membrane. Results are shown in Table 3-3 and in Figure 3-3. Positive esp membranes had cfu ranging fiom 2 to TNTC while negative esp membranes had cfu ranging from 1 to 140 in method 3. Over all method 3 was found to be more sensitive but had greater variability than method 1 and method 2. The sensitivity of method 3 was 26i40 cfu/membrane at the 95% confidence limit. Unlike method 1 and method 2, the positive esp was detected on membranes with lower cfu (< 10). 38 Table 3-3. Detection of esp gene from naturally occurring enterococci from untreated sewage using method 3. No No. of cfu range/membrane Esp+ Esp- Total membranes esp+ analyzed (%))‘k 1 14 15-53 4 10 27 2 6 51-1 15 6 0 100 3 5 63-150 3 2 60 4 5 47- 1 98 4 l 80 5 3 125-140 1 2 33 6 7 1-36 4 3 57 7 5 5-70 4 1 80 8 4 . 13-64 2 2 50 9 8 10-1 13 1 7 12 10 5 4-48 3 2 60 1 1 6 4-142 3 3 50 12 5 4-149 2 3 40 13 5 4-TNTC 5 0 100 *percentage of membranes positive for esp 39 Figure 3-3. Detection of esp gene in naturally occurring enterococci in the range of the limit of detection using method 3 (N= 75)‘. N E§P we .5 ,ESP -v.e CFU I Membrane *Number of membranes in this figure is 75 4o 3.2.5 Detection of esp Gene in Disinfected Effluent Samples Ten disinfected effluent samples were collected from the waste water treatment plant in East Lansing. The first three samples were processed by method 1 and 2 where as the remaining samples were processed by method 3. The colony forming units were below the detection limit of the esp marker for majority of the samples due to the disinfection. Results are shown in Table 3-4. Less volume was filtered through the membrane for samples one two and three, hence the cfu range was below the detection limit (30). For samples four thru ten the volume filtered through the membrane was increased to one liter. Samples six and eight were esp positive with a cfu range between 68 and TNTC when assayed from a total of 10 membranes. Sample seven was esp negative despite higher enterococci counts where as samples nine and ten were esp negative with cfu range below the detection limit (30). Table 3-4. Detection of esp gene in disinfected effluent samples. Method used Membranes cfu range Results Esp + assayed (%)* 1& 2 5 0-17 - 0 1&2 6 10-16 - 0 3 9 1-21 - 0 3 4 68-100 + 100 3 4 . 54-139 - 0 3 6 >100 + 67 *percentage of membranes positive for esp 41 3.2.6 Persistence of the esp Marker in Sewage Twenty three samples were assayed for the detection of the esp marker over a year from August 2005 to March 2006 using method 1 and 2. Raw sewage samples were collected twice a month. The marker was detected in 100% of the samples processed (22/22) when the cfu range was above the detection limit (45:1:28). This indicates that the marker was persistent over the time. All the four membranes were esp negative in only one sample. The lowest positive membrane fell within the limit of detection for all methods previously described (Table 3-5). Table 3-5. Lowest esp positive in raw sewage using method 1, 2‘& 32 Method Month Lowest positive Range (cm/membrane) (cfu/membrane) 1&2 Aug-05 23 23-TNTC 1&2 Sept-05 21 0-560 1&2 Nov-05 144 4-500 1&2 Dec-05 1 12 1 12-260 1&2 Jan-06 45 45-120 1&2 Feb-06 37 37-100 3 Feb-06 19 17-1 15 1&2 Mar-06 58 44-160 3 Mar-06 47 63-198 3 Apr-06 2 2-140 3 May-06 4 4-1 1 3 3 June-06 4 4-142 3 J uly-06 4 4-TNTC ‘Number of membranes tested in method 1&2 was 97 2 Number of membranes tested in method 3 was 78 42 3.2.7 Blind Analysis of the esp Gene 3.2.7.1 Introduction Blind wastewater and fecal samples were used to evaluate the predictive ability of the host specific esp marker in conjunction with a 2004 project run by the United States Geological Survey (USGS) on source sampling for pharmaceuticals. The 2004 USGS source sampling was conducted to provide a refined estimate on the relative potential environmental contributions of Organic Wastewater Contaminants (OWC’s) from a variety of urban and agricultural waste sources. This study was part of the USGS Toxic Substances Hydrology Program - Source Characterization (2004). The MSU Environmental Quality Laboratory assisted in the study by analyzing the samples collected from various animal and human sources by the USGS for the abundance of Enterococci and for the presence of an E. faecium esp marker (30). The sources were revealed after the analysis. 3.2.7.2 Results Method 1 was used to analyze for the esp marker in the USGS Blind samples except for one sample. Results are shown in Table 3-6 and Table 3-7. Enterococcus concentrations ranged from <3 to >103 cfu/ lOOmL. The E. faecium esp human marker was found in 6/7 of the human sourced samples with high enterococci counts and 0/6 animal source samples examined. The human sourced samples were from septic tanks of an elementary school, restaurant, multi-family home, convenience store, single family household and from a waste water treatment plant. The non-human sources were from a veterinary 43 hospital, antibiotic-free swine operation, antibiotic intense swine operation, cottage cheese factory, cattle operation (ranch) and antibiotic free poultry operations. All of the six septic tank effluent samples analyzed were esp positive (100%).The solid form of sample was esp negative in 4/6 septic tank samples and 2/2 municipal pond samples due to the low concentrations of enterococci. Less volume was filtered for the solid samples and this may be one reason for the low cfu count and hence the absence of the esp marker. One solid sample with high enterococci counts was also esp negative. Only one septic tank sample was esp positive for both solid and liquid portions with TNTC enterococci using method 3. Enterococcus concentrations in three samples from animal sources were also found below the detection limit (45i28) for the esp marker and the remaining six samples were negative for the esp marker. The presence of antibiotics may account for the low microbial numbers in these samples. 44 Table 3-6. Enterococcus concentration and presence of E. faecium esp human pollution marker in USGS source samples (human). Source State No. of Esp Esp samples + - Septic tank CO 5 5 0 Septic tank NJ 1 1‘ 0 Municipal pond CO 1* NA NA Municipal pond MN 1* NA NA Municipal pond IA 1* NA NA Biosolid NY 1 0 1 ‘Analyesd in method 3 * Not analyzed as number of cfu/membrane was below detection limit (45i28). Table 3-7. Enterococcus concentration and presence of E. faecium esp human pollution marker in USGS source samples (Non-human). Source State No. of Esp Esp samples + - Hog lagoon IA 4 O 4 Cheese factory ID 1* NA NA Poultry litter VA 1 0 1 Poultry litter OH 2* NA NA Biosolid (Ranch) TX 1 0 1 *Not analyzed as number of cfu/membrane was below detection limit (45:1:28). 45 3.2.8 Presence of esp Marker in Different Watersheds in US The marker was tested for in samples collected from different watersheds in US during the USGS source sample analysis. The marker was present in human samples from Colorado and New Jersey and the marker was absent in non-human samples from Texas, Virginia, and Iowa. One sample from Florida, (Katrina area) was esp positive by PCR and in the sequencing confirmed the presence of the marker. Despite high enterococci count the marker was absent in the Waste Water Treatment Plant (WWTP) solid sample from New York. Figure 3-4 shows the different watersheds evaluated and the results. 46 Figure 3-4. Presence of esp marker in different watersheds in US + States in which samples were positive for esp . States in which samples were negative for esp. 47 3.2.9 Detection of esp Marker in Non-human Samples The esp marker was tested using 29 membranes from numerous non-human sources. Three manure and one swine slurry samples were collected from animal waste points at the MSU farm. All four samples were negative. Nine bird samples assayed from parks and lakes were also negative. The bird samples included 2 gulls, 1 goose and 6 duck feces. All samples were assayed from numerous membranes and some membranes with cfu well above the detection limit. Eight sand samples collected from the North Beach, along Lake Michigan in Grand Haven, M1, were processed and were esp negative. These came from the same area where bird samples were also negative for the esp marker. Results are shown in Table 3-8. Table 3-8. Detection of esp marker in non human samples‘ No of samples Method Source cfu Range Esp+/- analyzed 3 1&2 Manure 250-TNTC - 1 1&2 Swine slurry 156 - 2 1&2 Sea gulls 28-TNTC - 6 3 Duck 12-TNTC - 1 3 goose 16-90 - 8 3 Beach sand* 6-125 - ‘ 29 membranes tested for esp marker. * Sand collected from beaches. High enterococci count possibly due to fecal pollution from birds or wild life. 48 CHAPTER 4 DISCUSSION 4.1 Introduction Microbial Source Tracking is becoming widely used but factors like library size, cost and complexity of the procedures are limiting application in environmental samples (49). Understanding the potential of each method before use is also challenging. The recently published MST guide document by EPA (2005) addresses these difficulties (49).The literature addressed all source tracking methods that are currently available and compared the methods with respect to their applicability, methodology and cost. According to the EPA an ideal MST method should be able to differentiate all the sources of pollution using a single approach, but none of the current methods meet that criterion (5, 18, 42, 55). So EPA has suggested that approaches involving multiple techniques are suitable for source tracking, particularly host specific methods that target genes which are highly specific, having the greatest potential in the future compared to the library based methods. The EPA guide document can be used as a reference for community managers to learn about MST methods for addressing the pollution issues, prioritization and remediation. However because this field is developing rapidly this EPA guide may be out of date quickly. 4.2 Development of a Rapid Method The first portion of this research addressed a rapid method for detecting a human pollution source tracking marker using the enterococcus surface protein (esp). The 49 method that was previously developed by Scott et a1. (2005) included a two day incubation at 41°C and an enrichment step lasting three hours followed by a 3.5 hour DNA extraction method and then the PCR step for the esp detection (3 8). The time from sample collection to result was about 3.5 days. The rapid method that was developed in this study compared to the conventional method obtained results in 1.5 days. By applying this method modification in the future, labor costs and the cost for manufactured DNA extraction kits would be reduced. Developing a rapid method was also seen as important for identifying the sources in a timely manner, hence community managers could take corrective actions to protect human health (51). Presence of a sewage marker in a particular water body is a strong indication of sewage discharges or leaking septic systems in the surrounding area (26, 28). If sewage is the likely source of pollution this suggests a more serious health, alert should be given to the people engaged in recreational activities in those waters, particularly young children. The method may be useful for indicating when to close a beach as opposed to just posting advisories and may be useful for indicating when to reopen a beach. In addition, by routinely monitoring suspected areas using rapid and affordable methods, one could address future water quality degradation and prioritize beachsheds for remediation. The enterococci is currently being used as an indicator organism and the method for detecting the esp gene allows the laboratories to implement this method easily complementing what they are already doing (48). 50 There were 97 membranes assayed for the comparison of method 1 and 2 taken from 12 different raw sewage samples. The cfu ranged from zero to TNTC. When the results were compared there were slightly more positive results from the conventional method (method 1) compared to the rapid method (method 2). The rapid method (method 2) used whole bacterial cells as a template for PCR, which is simpler and faster than using extracted DNA. However, DNA extraction is seen as a critical step for getting a high quality DNA by removing inhibitory substances like humic acids (42). Thus the negative esp by the rapid method may be a result of interferences from the material in the mEI media or from the increased number of cells in the PCR reaction. Some of the esp positives in the rapid method also showed faint bands indicating that the enzyme concentration (Taq polymerase) may need to be increased per reaction (9). While the marker is a clear indication of human fecal contamination, it is also clear that the marker containing strain is not always excreted by healthy human beings (24, 56, 57). Thus more membranes or larger volumes of water samples from the suspected areas may need to be processed with mixtures of enterococci. This study was mainly based on the raw sewage sample, hence the bacterial concentrations and the presence of the marker in the natural aquatic environment should be lower because of dilution (54). Thus as with many environmental monitoring programs proving the negative is difficult. Molecular sequencing of positive samples allowed for greater confidence that the PCR reaction was specific and that human sewage was present. This was done with the raw sewage and the positive environmental sample from the Katrina affected area. A similar 51 approach will be important proof for other samples in the future for further identification of the specificity of the marker. Some of the host specific markers that have been used for MST methods previously have shown geographic and host- impacted diet variations (20, 22, 40). The esp has been detected from numerous areas across the US. In future sequence analysis in a national survey would be beneficial to examine the distribution in different geographic regions. Method 3 concentrated on removing the enrichment step by taking viable cells from the membrane and using this as a template for the PCR. This variation in the method increased the sensitivity, and detection of the esp marker was below the detection limits (26i40) of methods 1 and 2. The marker was detected from membranes containing as few as 2 cfu. However this method failed to detect the marker in one membrane from sewage with enterococci counts of 140. One reason for this result could be the PCR inhibitors which may have prevented the amplification. Removing PCR inhibitors from environmental samples is challenging without DNA extraction. By selecting optimizing enzyme concentrations inhibitors may overcome this to some extent (1). Faster methods for MST targeting specific genes include Quantitative PCR (QPCR) and do not require live cells for cultivation. The advantage of this approach is that this method could detect past contamination in the environment (51) and possibly provide results within several hours. A study conducted by Lleo et a1. (2005) pointed out that 50 % of the ground water samples tested (n=30) contained nonculturable cells for E. faecalis (27). Thus molecular tests are able to detect the nonculturable cells along with the viable cells. In this study the method targeted the human pollution in viable cells only because 52 the objective of the research was to identify recent pollution and address adequately treated and disinfected sewage. Wastewater that was adequately treated contained low enterococci counts and no esp marker was found using the methods in this study unless the numbers of cfu increased. 4.2.1 Solid Samples The majority of the solid samples tested were esp negative. Most of the time the concentration of the enterococci was below detection limits for the esp. The only solid sample that was esp positive was a sample from a septic tank (Table 2-7) from the USGS source. That sample was tested using method 3. While processing sediment/solid type samples, the presence of a dark background due to the solids in the membrane may prevent the correct enumeration of CFU. This can be avoided by dilution and filtering smaller volumes for the enumeration and filtering larger volumes in replicate for testing the sample for the esp marker. The survival of the bacteria containing the marker has been evaluated in only one experiment to date in fresh and marine water. Further studies are also needed to better understand the persistence of the sub-population of enteroccci containing the marker particularly in solids and sediments (3 8). 4.2.2 Presence or Absence of the Marker This study reconfirmed the absence of the marker in animal wastes. However, interpretation of the esp result in the environment should be done with the concept that a negative result does not guarantee a true negative, where as a positive indicates that at least some of the pollution is from humans. The reasons for obtaining negative results 53 may be a low concentration of enterococci in that particular sample or the absence of marker in the sample. Absence of the marker may indicate that there are other pollution sources other than from humans in the area, so further investigation needs to be done to pinpoint the sources. There are no methods that warrant complete resolution for tracking all the sources of contamination. Forensic approaches which match exactly isolates would mean thousands of samples and isolates using methods like QPCR, AF LP, PFGE, T- RF LP which is not affordable for most of the communities(49). Many samples and more than one test need to be included in any source tracking study. A “toolbox approach “(EPA 2005) has been recommended. The success of the application of library dependent and library independent source tracking methods depends on the geographic stability of the marker and the size of the library. Several studies were able to pinpoint the source of pollution in smaller watersheds, where as the accuracy of the method was lower when compared to broad geographic regions(19, 20, 39). Thus more studies are needed to quantify the relative numbers of esp positive samples to esp negative in various types and sizes of water bodies. A combination of different and inexpensive methods is approachable for the community managers when they deal with environmental quality issues and it is concluded by this study that the Rapid esp method can be included as one of those. Unlike contamination from other sources, human fecal contamination in the environment has a direct and adverse impact on human health(15). Human fecal contamination and sewage are generally considered to be of a higher risk because it is possible that the wastes contain more bacteria, viruses and parasitic pathogens. However, 54 wastewater treatment can be implemented, changed and optimized, correcting the problem effectively compared to control of birds or wildlife fecal contamination. The EPA source tracking guide compared 8 field applications ((1) Targeted sampling and Enterococcus speciation- Georgia, (2) ARA with fecal colifonns, ribotyping with E. coli, and human pathogenic enterovirus detection-Tampa, Forida.(3),Rep-PCR with E. coli,- Minnesota, (4) ARA and PFGE with enterococci- Maryland,(5) Two-enzyme ribotyping with E. coli.- Virginia, (6) Host-specific Bacteroides/Prevotella markers and human- pathogenic enterovirus detection- California, (7) ARA with E. coli- Virginia, (8) F+ RNA coliphage genotyping- Florida) using MST methods, but follow up studies and remediation steps were undertaken only with three studies, where the major contributor of the pollution was human and cattle(49). In other case studies the major contributors of the pollution were birds and wildlife, so no implementation was taken in any of these watersheds. One major implementation based on the Virginia study (case no.5) was fencing along the river side where the main source of pollution was found to come from cattle. A corrective measurement of slip-lining the sewer lines that run along the beach were taken based on the Host-specific Bacteroides/Prevotella markers and human- pathogenic enterovirus detection study in Avalon Bay, California. In the Virginia study (case no.7) human sources were the dominant cause of the pollution; hence a septic system maintenance project was undertaken because all of the homes used septic systems and wells in the area where the study was conducted. These case studies demonstrate the usefulness of the MST tool box. 55 4.2.3 Marker Testing The lack of a single genetic marker to differentiate all possible sources of pollution, and the geographic variability of many markers are some of the limitations when considering MST. The esp marker has been tested in samples from different geographical regions. All liquid wastewater samples from humans thus far tested from Colorado, Florida and Michigan were also esp positive. The marker was absent in the non human sources from Idaho, Ohio, Virginia and Iowa. McDonald et al. (2006) were able to detect the esp marker in environmental samples from Georgia watershed (28). More samples from other geographical regions need to be evaluated. This study focused on a rapid method for the evaluation of raw sewage. More environmental samples need to be tested for the esp marker using methods 2 and 3. When dealing with samples of low concentrations of enterococci, method 3 may be the most appropriate because methods 1 and 2 failed to detect positives from membranes with <20 cfu. More membranes with higher cfu (>100) need to be evaluated in method 3 to explore the role of inhibitors in preventing the amplification. The esp marker should be evaluated in more geographical regions. More sediment samples also need to be processed and the stability of the marker in the environment including ponds, lakes and sludge need to be further investigated. Sewage discharges including CSOs are still one of the leading causes of human pathogens in recreational waters and adversely affecting the people’s health who engage activities in those waters. This research targeted development of a method which is 56 comparatively faster than a conventional method developed for detecting the human pollution marker esp (3 8). The research concentrated on the esp marker present in sewage to better define the prevalence of this gene in the human population and in community sewage. Additional research needs to be undertaken to examine how the esp marker behaves in the environment. The esp positive environmental sample from New Orleans was collected two months after Hurricane Katrina and demonstrated that the marker can persist up to two months in the environment. The absence of the marker in all non-human sources and the geographic stability of the marker are promising. A more comprehensive study with targeted sampling in problem areas would increase the understanding of the survival of the marker in the environment and the effectiveness of the rapid test. Case studies before and after remediation should be undertaken. Routine monitoring and targeted sampling are ways to identify a water pollution problem before affecting public health. The MST methods have been found to be very helpful for finding the root cause of the pollution. Enterococci is already being used as a water quality indicator by EPA and by many States. By adding the esp detection test after assessing the water quality one can begin to address more definitively the role of sewage as a source of waterbody impairment. Based on this study, the rapid method is as effective as the conventional method, is cheaper and less time consuming. Using the traditional indicator enterococci for water quality and then testing membranes with high enterococci counts (:20) for the esp marker using the rapid method is a simple approach for tracking a human source of contamination. Public health agencies that monitor beaches, as well as water utilities that are concerned with drinking water supplies should 57 find this method useful. In addition such as the case in New Orleans, the recovery and identification of hot spots of areas where sewage contamination remained risky could be monitored for. 58 Table A-1. Method Comparison using raw sewage APPENDIX 1 Sample Filtered esp No ID Date Volume cfu‘ +/- Method 1 RW1-1 8/10/2005 10p| 23 + 1 2 RW 1-1 8/10/2005 10pl 23 + 2 3 RW 1-2 8/10/2005 50pl 136 + 1 4 RW 1-2 8/10/2005 50pl 136 + 2 5 RW 1-3 8/10/2005 100111 270 + 1 6 RW 1-3 8/10/2005 100p| 270 + 2 7 RW 14 8/10/2005 1 ml TNTC + 1 8 RW 1-4 8/10/2005 1 ml TNTC + 2 9 RW 2-1 9/7/2005 10111 38 - 1 10 RW 2-1 9/7/2005 10111 38 - 2 11 RW 2-2 9/7/2005 10p| 44 - 1 12 RW 2-2 9/7/2005 10111 44 - 2 Dilution2 1 3 RW 2-3 9/7/2005 103 9 - 1 14 RW 2-3 9/7/2005 10'3 9 - 2 15 RW 2-4 9/7/2005 10'3 6 - 1 16 RW 2-4 9/7/2005 10'3 6 - 2 17 RW 2-5 9/7/2005 10'4 0 - 1 18 RW 2-5 9/7/2005 10'4 0 - 2 19 RW 2-6 9/7/2005 10'5 1 - 1 20 RW 2-6 9/7/2005 10'5 1 - 2 Volume 21 RW 3-1 9/21/2005 5011' 560 + 1 22 RW 3-1 9/21/2005 50111 560 + 2 23 RW 3-2 9/21/2005 10pI 121 + 1 24 RW 3-2 9/21/2005 1Cyl 121 + 2 Dilution 25 RW 3-3 9/21/2005 10'3 19 - 1 26 RW 3-3 9/21/2005 10'3 19 - 2 27 RW 3-4 9/21/2005 103 21 + 1 28 RW 3-4 9/21/2005 10'3 21 + 2 Volume 29 RW 4-1 1 1/2/2005 50111 500 + 1 30 RW 4-1 1 1/2/2005 50p| 500 + 2 31 RW 4-2 1 1/2/2005 10p| 98 - 1 32 RW 4-2 1 1/2/2005 10p| 98 — 2 Dilution 33 RW 4-3 1 1/2/2005 10'3 22 - 1 34 RW 4-3 1 1/2/2005 10'3 22 - 2 35 RW 4-4 11/2/2005 10'4 12 - 1 36 RW 4-4 1 1/2/2005 10'4 12 - 2 37 RW 5-1 11/15/2005 10p| 280 + 1 59 Table A-1 continued 38 RW 5-1 11/15/2005 10p| 280 + 2 39 RW 5-2 1 1/15/2005 10pI 280 + 1 40 RW 5-2 1 1/15/2005 10p| 280 + 2 41 RW 5-3 11/15/2005 10'2 144 + 1 42 RW 5-3 1 1/15/2005 10"2 144 + 2 43 RW 54 1 1/15/2005 10'3 20 - 1 44 RW 5-4 1 1/15/2005 10'3 20 - 2 45 RW 5-5 1 1/15/2005 10'4 4 - 1 46 RW 5-5 1 1/15/2005 10'4 4 - 2 47 RW 10-1 1/18/2006 10'2 50 - 1 48 RW 10-1 1/18/2006 10'2 50 - 2 49 RW 10-2 1/18/2006 10'2 51 - 1 50 RW 10-2 1/18/2006 10'2 51 - 2 51 RW 10-3 1/18/2006 10'2 52 - 1 52 RW 1 0-3 1/18/2006 10'2 52 - 2 53 RW 10-4 1/18/2006 10'2 49 + 1 54 RW 10-4 1/18/2006 102 49 + 2 55 RW 10-5 1/1 8/2006 10'2 60 + 1 56 RW 1 0-5 1l18/2006 102 60 + 2 57 RW 10-6 1/18/2006 10'2 57 - 1 58 RW 10-6 1/18/2006 10'2 57 - 2 59 RW 10-7 1/18/2006 10'2 54 + 1 60 RW 10-7 1/1 8/2006 10'2 54 + 2 61 RW 10-8 1/18/2006 10'2 58 - 1 62 RW 10-8 1 I 1 8/2006 10'2 58 - 2 63 RW 1 0-9 1/1 8/2006 102 56 - 1 64 RW 1 0-9 1/1 8/2006 102 56 - 2 65 RW 10—1 0 1l18/2006 10'2 45 - 1 66 RW 10-10 1/18/2006 10'2 45 + 2 67 RW 10-1 1 1/18/2006 10'2 46 + 1 68 RW 10-1 1 1/18/2006 10'2 46 + 2 69 RW 10-12 1l18/2006 10'2 59 + 1 70 RW 10-12 1/18/2006 10'2 59 + 2 71 RW 10-13 1l18/2006 10’2 69 - 1 72 RW 10-13 1/18/2006 10'2 69 - 2 73 RW 10-14 1l18/2006 102 64 - 1 74 RW 10-14 1/18/2006 102 64 - 2 75 RW 10-15 1/18/2006 10'2 62 - 1 76 RW 10-15 1/18/2006 10'2 62 - 2 77 RW 10-16 1/18/2006 10'2 63 + 1 78 RW 10-16 1/18/2006 10'2 63 - 2 79 RW 10-17 1/18/2006 102 65 - 1 80 RW 10-17 1l18/2006 10'2 65 - 2 60 Table A-1 continued 81 RW10-18 1/18/2006 10'2 68 + 1 82 RW 10-18 1/18/2006 10’2 68 + 2 83 RW 10-19 1/18/2006 10-2 67 - 1 84 RW 10-19 1/18/2006 10"2 67 - 2 85 RW 10-20 1/18/2006 10'2 66 + 1 86 RW 10-20 1/1 8/2006 10-2 66 + 2 87 RW 10-21 1/18/2006 10'2 75 + 1 88 RW 10-21 1/18/2006 10'2 75 - 2 89 RW 10-22 1/18/2006 10'2 72 - 1 90 RW 10-22 1/18/2006 102 72 - 2 91 RW 10-23 1/18/2006 10'2 71 + 1 92 RW 10-23 1l18/2006 10’2 71 - 2 93 RW 10-24 1/18/2006 10'2 70 + 1 94 RW 10-24 1/1 8/2006 10'2 70 + 2 95 RW 10-25 1/18/2006 10'2 74 + 1 96 RW 10-25 1/18/2006 102 74 + 2 97 RW 10-26 1/1 8/2006 10'2 78 + 1 98 RW 10-26 1/18/2006 10'2 78 + 2 99 RW 10-27 1/18/2006 10'2 77 - 1 100 RW 10-27 1/18/2006 102 77 - 2 101 RW 10-28 1/18/2006 10"2 73 - 1 102 RW 10-28 1/18/2006 10"2 73 - 2 103 RW 10-29 1/18/2006 10'2 84 + 1 104 RW 1029 1/18/2006 10'2(2ml) 84 + 2 105 RW 10-30 1/18/2006 10'2(2ml) 85 + 1 106 RW 10-30 1l18/2006 10'2(2ml) 85 + 2 107 RW 10-31 1/18/2006 10'2(2mI) 83 - 1 108 RW 10-31 1/18/2006 10'2(2m|) 83 - 2 109 RW 1 0-32 1/18/2006 10'2(2m|) 88 + 1 1 10 RW 10-32 1l18/2006 10'2(2m|) 88 + 2 1 11 RW 10-33 1/18/2006 10'2(2m|) 81 - 1 112 RW 10—33 1/18/2006 10'2(2ml) 81 - 2 113 RW 10-34 1/18/2006 10'2(2ml) 89 - 1 1 14 RW 10-34 1/18/2006 1072(2ml) 89 - 2 1 15 RW 10-35 1/18/2006 10'2(2mI) 86 + 1 1 16 RW 10-35 1/18/2006 10'2(2m|) 86 + 2 117 RW 10-36 1/18/2006 10'2(2ml) 82 + 1 1 18 RW 10-36 1/18/2006 10'2(2m|) 82 + 2 119 RW 10-37 1/18/2006 10'2(2m|) 96 + 1 120 RW 10-37 1/18/2006 10'2(2m|) 96 - 2 121 RW 10-38 1/18/2006 10'2(2ml) 91 - 1 122 RW 10-38 1/18/2006 10'2(2ml) 91 - 2 123 RW 10-39 1/18/2006 10'2(2ml) 90 + 1 124 RW 10-39 1/18/2006 10‘2(2ml) 90 - 2 61 Table A-1 continued 125 RW 1040 1/18/2006 10'2(3m|) 93 + 1 126 RW 1040 1718/2006 10'2(3ml) 93 + 2 127 RW 1041 1/18/2006 10‘2(3ml) 92 - 1 128 RW 1041 1/18/2006 10'2(3m|) 92 + 2 129 RW 1042 1718/2006 10'2(4m|) 97 - 1 130 RW 1042 171872006 101ml) 97 - 2 131 RW 1043 171872006 10‘2(5ml) 106 - 1 132 RW 1043 171872006 10'2(5m|) 106 - 2 133 RW 1044 1/18/2006 10'2(5ml) 110 + 1 134 RW 1044 171872006 10'2(5m1) 110 + 2 135 RW 1045 1/18/2006 10'2(5ml) 101 — 1 136 RW 1045 1/18/2006 10'2(5ml) 101 - 2 137 RW 1046 171872006 10’2(5ml) 117 - 1 138 RW 1046 171872006 10’2(5m|) 117 + 2 139 RW 1047 171872005 10'2(5ml) 120 + 1 140 RW 1047 1/18/2006 10'2(5ml) 120 + 2 141 RW 11-1 271372006 102 41 - 1 142 RW 114 271372006 102 41 - 2 143 RW 11.2 271372006 102 52 - 1 144 RW 11.2 271372006 102 52 - 2 145 RW 11-3 271372006 102 65 - 1 146 RW 11-3 271372006 102 65 - 2 147 RW 114 271372006 102 54 + 1 148 RW 114 271372006 102 54 + 2 149 RW 1 15 271372006 102 53 + 1 150 RW 115 271372006 102 53 - 2 151 RW 11-6 271372006 102 48 - 1 152 RW 115 271372006 102 48 - 2 153 RW 11-7 271372006 102 58 - 1 154 RW 1 1-7 271372006 102 58 - 2 155 RW 11-8 271372006 102 51 - 1 156 RW 11-8 271372006 102 51 - 2 157 RW 11-9 271372006 102 37 + 1 158 RW11-9 271372006 102 37 + 2 159 RW 1 1-10 271372006 102 42 - 1 160 RW 1 1-10 271372006 102 42 - 2 161 RW 1141 271372006 102 44 - 1 162 RW 1 1-1 1 271372006 102 44 - 2 163 RW 11-12 271372006 102 38 + 1 164 RW 11-12 271372006 102 38 + 2 165 RW 11-13 271372006 102 43 - 1 166 RW 1143 2/13/2006 102 43 - 2 167 RW 11-14 2/13/2006 102 36 - 1 168 RW 1 144 271372006 102 36 - 2 62 Table A-1 continued 169 RW12-1 2/24/2006 10'2(1.7mD 100 + 1 170 RW12-1 2/24/2006 10'2(1.7m|) 100 + 2 171 RW12-2 2/24/2006 10'2 60 + 1 172 RW12-2 2/24/2006 10'2 60 + 2 173 RW12-3 2/24/2006 10'2 48 - 1 174 RW12-3 2/24/2006 102 48 + 2 175 RW12-4 2/24/2006 10'2 39 + 1 176 RW12-4 2/24/2006 10'2 39 + 2 177 RW12-5 2/24/2006 10'2 70 + 1 178 RW12-5 2/24/2006 10'2 70 + 2 179 RW13-1 3/7/2006 10'2(2ml) 126 + 1 180 RW13-1 3/7/2006 10'2(2m|) 126 + 2 181 RW13-2 3/7/2006 10'242ml) 140 + 1 182 RW13-2 3/7/2006 10'2(2ml) 140 + 2 183 RW13-3 3/7/2006 10'2 73 + 1 184 RW1 3-3 3/7/2006 10"2 73 + 2 185 RW13-4 3/7/2006 10'2 58 + 1 186 RW13-4 3/7/2006 10'2 58 + 2 187 RW14-1 373172006 10'2(2ml) 44 - 1 188 RW14-1 3/31/2006 10'2le) 44 - 2 189 RW14-2 3/31/2006 10'2(2m|) 75 - 1 190 RW14-2 3/31/2006 10'2le) 75 - 2 191 RW14-3 3/31/2006 10'2(2m|) 92 - 1 192 RW14-3 3/31/2006 10'2(2ml) 92 - 2 193 RW14-4 3/31/2006 10'1(2m|) 160 + 1 194 RW14-4 3/31/2006 10'1(2m|) 160 + 2 ‘cfu/membrane 2 In all diluted samples lmL of the volume was filtered unless noted otherwise 63 Table A-2. Method 2 only Sample Filtered No ID Date Volume cfu‘ Esp+/- Method 1 RW 6-1 11/30/2005 101.11 117 + 2 2 RW 6-2 11/30/2005 10111 109 + 2 Dilution2 3 RW 6-3 11/30/2005 103 17 - 2 4 RW 6-4 1 1/30/2005 103 26 - 2 5 RW 7-1 12/21/2005 101 260 + 2 6 RW 7-2 12/21/2005 10'2 151 + 2 7 RW 7-3 12/21/2005 102 1 12 + 2 8 RW 8-1 1/3/2006 10'2 124 + 2 9 RW 8-2 1/3/2006 10'3 34 - 2 1O RW 8-3 1/3/2006 10'3 19 - 2 11 RW 9-1 1/12/2006 10'2 29 + 2 12 RW 9-2 1/12/2006 10'2 30 - 2 Volume 13 RW 9-3 1/12/2006 50111 31 + 2 14 RW 9-4 1/12/2006 50111 34 - 2 Dilution 15 RW 9-5 1/12/2006 10'1 77 - 2 16 RW 9-6 1/12/2006 10'1 90 + 2 ‘cfu/membrane 2 In all diluted samples lmL of the volume was filtered unless noted otherwise 64 Table A-3. Method Comparison using environmental samples Filtered No Sample ID Date Volume cfu‘ Method esp+/- 1 MSU Farm 12 9/27/2005 50ml TNTC 1 + 2 MSU Farm 12 9/27/2005 50ml TNTC 2 + MSU Farm 3 12(duplicate) 9/27/2005 50ml TNTC 1 + MSU Farm 4 12(duplicate) 972772005 50ml TNTC 2 + 5 MSU Farm 15 9/27/2005 50ml TNTC 1 -- 6 MSU Farm 15 9/27/2005 50ml TNTC 2 - 7 MSU Farm 1 9/29/2005 60ml 33 1 -- 8 MSU Farm 1 9/29/2005 60ml 33 2 -- 9 MSU Farm WSP 15 11/9/2005 10ml 84 1 - 10 MSU Farm WSP 15 11/9/2005 10ml 84 2 - 11 MSU Farm WSP 1 11/9/2005 50ml 115 1 - 12 MSU Farm WSP 1 11/9/2005 50ml 115 2 - 13 MSU Farm WSP 15 11/17/2005 10ml >250 1 -- 14 MSU Farm WSP 15 11/17/2005 10ml >250 2 - 15 MSU Farm WSP 1 11/17/2005 10ml 58 1 - 16 MSU Farm WSP 1 11/17/2005 10ml 58 2 - 17 MSU Farm WSP 12 A 01/12/06 150ml 15 1 -- 18 MSU Farm WSP 12 A 01/12/06 150ml 15 2 - 19 MSU Farm WSP 12 A 01/12/06 100ml 11 1 - 20 MSU Farm WSP 12 A 01/12/06 100ml 11 2 -- 21 MSU Farm WSP 12 A 01/12/06 100ml 10 1 — 22 MSU Farm WSP 12 A 01/12/06 100ml 10 2 - 23 MSU Farm WSP 12 B 01/12/06 10ml 48 1 - 24 MSU Farm WSP 12 B 01/12/06 10ml 48 2 - 25 MSU Farm WSP 12 3 01/12/06 50ml 150 1 - 26 MSU Farm WSP 12 3 01/12/06 50ml 150 2 - 27 MSU Farm WSP 12 8 01/12/06 100ml TNTC 1 - 28 MSU Farm WSP 12 8 01/12/06 100ml TNTC 2 - 29 MSU Farm WSP 12 3 01/12/06 150ml TNTC 1 -- 30 MSU Farm WSP 12 3 01/12/06 150ml TNTC 2 - 31 Randall House 9/26/2005 350ml 189 1 - 32 Randall House 9/26/2005 350ml 189 2 - 33 Randall House 9/26/2005 250ml 131 1 - 34 Randall House 9/26/2005 250ml 131 2 - 35 Randall House 9/26/2005 100ml 67 1 -- 36 Randall House 9/26/2005 100ml 67 2 -- ‘cfu/membrane 65 Table A-4. Method Comparison using Environmental samples Filtered No Sample ID Date Volume cfu‘ Method esp+/- 1 Gulf sample 9/30/2005 160ml 37 1 -— 2 Gulf sample 9/30/2005 160ml 37 2 - 3 NO#1 Water 10l19/2005 100ml 44 1 - 4 NO#1 Water 10/19/2005 100ml 44 2 - 5 NO#2 Water 10/19/2005 100ml 222 1 - 6 NO#2 Water 10/19/2005 100ml 222 2 - 7 NO#3 Water 10/19/2005 100ml 11 1 - 8 NO#3 Water 10/19/2005 100ml 11 2 -- 9 NO#4 Water 10/19/2005 100ml 22 1 - 1O NO#4 Water 10/19/2005 100ml 22 2 -- 11 NO#5 Water 10l19/2005 100ml 16 1 - 12 NO#5 Water 10/19/2005 100ml 16 2 -- 13 NO#8 Water 10l19/2005 100ml 29 1 -- 14 NO#B Water 10/19/2005 100ml 29 2 - 15 NO#9 Water 10/19/2005 100ml 289 1 - 16 NO#9 Water 10/19/2005 100ml 289 2 - 17 NO#1O Water 10/19/2005 100ml 121 1 + 18 NO#10 Water 10/19/2005 100ml 121 2 + NO#1O Water 19 (duplicate) 10/19/2005 100ml 121 1 + NO#1O Water 20 (duplicate) 10/19/2005 100ml 121 2 + 21 NO#11 Water 10/19/2005 100ml 245 1 - 22 NO#11 Water 10/19/2005 100ml 245 2 -- 23 LPOO4Sediments 10/19/2005 100ml 42 1 - 24 LPOO4Sediments 10/19/2005 10ml 42 2 - 25 LP005 NIA 10ml 1 1 -- 26 LP005 NIA 10ml 1 2 -- ‘cfu/membrane 66 Table A-5. esp validation on non human samples No Source Date cfu‘ Method esp+/- MSU Farm 1 swine slurry 10/27/2005 1 56 1 -- 2 MSU Farm swine slurry 10/27/2005 156 2 — 3 MSU Farm Manure 10/27/2005 250 1 - 4 MSU Farm Manure 10/27/2005 250 2 -- 5 Manure S P 11/3/2005 TNTC 1 - 6 Manure S P 11/3/2005 TNTC 2 - 7 Manure C P 11/3/2005 TNTC 1 - 8 Manure C P 11/3/2005 TNTC 2 - 9 008 sea gull feces 12/16/05 38 2 - 10 008 sea gull feces 12/15/05 45 2 - 11 008 sea Mfeces 12/ 15105 TNTC 2 - 12 005 sea gull feces 12/15/05 28 2 - 13 005 sea gull feces 12/16/05 41 2 - 14 005 sea gull feces 12/ 16105 TNTC 2 - 1 5 Goose feces 3/1 5/06 90 3 - 16 Goose feces 3/1 5/06 82 3 - 17 Goose feces 3/ 1 5/06 16 3 - 18 F/D/002 Duck feces 3/3/2006 133 2 - 19 FID/002 Duck feces 3I3/2006 143 1 - 20 F/D/002 Duck feces 3/3/2006 143 2 -- 21 F/D/002 Duck feces 3/3/2006 12 1 — 22 F/D/002 Duck feces 3/3/2006 12 2 - 23 F/D/006 Duck feces 3/3/2006 135 3 - 24 F/D/008 Duck feces 3/3/2006 103 3 -- 25 F/D/007 Duck feces 3/3/2006 44 3 - 26 F/D/010 Duck feces 3/3/2006 100-200 3 - 26 F/D/004 Duck feces 3/3/2006 TNTC 3 -- 27 SU-U2-4 Sand 7/10/2006 45 3 - 28 SU-L-12 Sand 7l10/2006 125 3 - 29 SU-U2-12 Sand 7/10/2006 35 3 -- 30 SU-B1-12 Sand 7/10/2006 25 3 - 31 SU-L-8 Sand 7/10/2006 6 3 - 32 SU-B1-12 Sand 7/10/2006 25 3 - 33 SU-U2-4 Sand 7/10/2006 47 3 -- 34 SU-B2-12 Sand 7l10/2006 20 3 - ‘cfu/membrane 67 Table A-6. Preliminary data without enrichment step Filtered No Sample ID Date Volume cfu‘ esp +/- Method 1 RW11-15 2/14/2006 250111 6+5+9 - 3 2 RW 1 1 -16 2/14/2006 400111 15+6 + 3 3 RW 11-17 2/14/2006 250g 8+4+3 - 3 4 RW 11-18 2/14/2006 500111 17 - 3 5 RW 11 -1 9 2/14/2006 400111 19 + 3 6 RW 11-20 2/14/2006 400111 18 - 3 7 RW 11-21 2/14/2006 750111 35 - 3 8 RW 1 1-22 2/14/2006 750111 32 - 3 9 RW 11-23 2/14/2006 7501.11 22 - 3 10 RW 11-24 2/14/2006 750111 22 - 3 11 RW 11-25 2/14/2006 750111 28 - 3 12 RW 11-26 2/14/2006 750111 31 - 3 Dilution2 13 RW 11-27 2/14/2006 10'2 53 + 3 14 RW 11-28 2/14/2006 10'2 38 + 3 15 RW12-1 2/24/2006 10'2 73 + 3 16 RW12-2 2/24/2006 10'2 57 + 3 17 RW12-3 2/24/2006 10'2 51 + 3 18 RW12-4 2/24/2006 10'2 63 + 3 19 RW12-5 2/24/2006 10'2(1.7m1) 110 + 3 20 RW12-6 2/24/2006 10'2(1.7m|) 115 + 3 21 RW13-1 3/7/2006 10'2 63 + 3 22 RW13-2 3/7/2006 10'2 72 - 3 23 RW13-3 3/7/2006 10'2 80 - 3 24 RW13-4 37772006 10'2(2m1) 150 + 3 25 RW13-5 3/7/2006 10'2(2m|) 161 + 3 26 RW14-1 3/31/2006 10’2(2ml) 47 + 3 27 RW14-2 3/31/2006 10'2(2m|) 71 + 3 28 RW14-3 3/31/2006 10'1(2ml) 198 + 3 29 RW14-4 3/31/2006 10'1(2ml) 180 + 3 30 RW14-5 3/31/2006 10‘2(2ml) 67 - 3 31 RW15-1 4/18/2006 10'2(25011|) 125 + 3 32 RW15-3 4/18/2006 10'2(25011|) 126 - 3 33 RW15-4 4/18/2006 10'2(25011|) 140 - 3 34 RW16-1 4/19/2006 10'21500111) 36 - 3 35 RW16-2 4/19/2006 10'3 3 + 3 36 RW16-3 4/19/2006 10'3 9 + 3 37 RW16-4 4/19/2006 10'2(50011|) 28 + 3 38 RW16-5 4/19/2006 103 2 - 3 39 RW16-6 4/19/2006 10'3 1 - 3 40 RW16-7 4/19/2006 10'3 6 + 3 41 RW17-1 4/20/2006 10'A3ml) 70 + 3 68 Table A-6 continued 42 RW17-2 4/20/2006 10'2 55 + 3 43 RW17-3 4/20/2006 10'3 2 + 3 44 RW17-4 4/20/2006 10'2 43 + 3 45 RW17-5 4/20/2006 103 5 - 3 46 RW18-1 4/21/2006 10'3(2ml) 13 - 3 47 RW18-2 4/21/2006 10'2 64 + 3 48 RW18-3 4/21/2006 10'3(2m|) 14 - 3 49 RW18-4 4/21/2006 1 O'2(50011|) 39 + 3 50 RW19-1 5/25/2006 10'3(2m|) 1 1 - 3 51 RW19-2 5/25/2006 1073(3ml) 25 - 3 52 RW19-3 5/25/2006 102 69 - 3 53 RW19-4 5/25/2006 10'3 5 - 3 54 RW19-5 5/25/2006 10'3(3ml) 22 - 3 55 RW19—6 5/25/2006 10’2(2ml) 113 - 3 56 RW19-7 5/25/2006 10'2(2m1) 89 + 3 57 RW19-8 5/25/2006 10'3(2m|) 10 - 3 58 RW 20-1 5/31/2006 10'2(2m1) 42 - 3 59 RW 20-2 5/31/2006 10’3fiml) 7 - 3 60 RW 20-3 5/31/2006 10'2(2ml) 48 + 3 61 RW 20-4 5/31/2006 10'3(3ml) 4 + 3 62 RW 20-5 5/31/2006 10'2(2m|) 19 + 3 63 RW 21 -1 6/8/2006 10'3(1 ml) 4 + 3 64 RW 21-2 6/8/2006 101(500119 120 - 3 65 RW 21 -3 6/8/2 006 10'2 43 + 3 66 RW 21 -4 6/8/2006 10'1(500111) 142 + 3 67 RW 21 -5 6/8/2 006 1072 49 - 3 68 RW 21 -6 6/8/2006 10'3(2m|) 19 — 3 69 RW 22-1 6/1 312006 10'1 (500111) 106 - 3 70 RW 22-2 6/13/2006 10'2 45 - 3 71 RW 22-3 6/13/2006 102 29 + 3 72 RW 22-4 6/13/2006 10'3(1 ml) 4 + 3 73 RW 22-5 6/13/2006 10'1 (500111) 1 18 - 3 74 RW 23-1 7/25/06 10'2 101 + 3 75 RW 23-1 7/25/06 10'2 102 + 3 76 RW 23-1 7/25/06 10'1 TNTC + 3 77 RW 23-1 7/25/06 1m| TNTC + 3 78 RW 23-1 7/25/06 10'4 4 + 3 ‘cfu/membrane 2 In all diluted samples lmL of the volume was filtered unless noted otherwise 69 Table A-7. Detection of esp gene in disinfected Effluent samples. Sample Filtered Date cfu/100mL esp Method No volume 1A 50ml 8/11/05 4 - 1&2 lB 20ml 8/1 1/05 1 - 1&2 2A 100ml 09/08/05 0 - 1&2 ZB 200ml 09/08/05 0 - 1&2 3A 100ml 09/30/05 4 - 1&2 3B 200ml 09/30/05 9 - 1&2 3C 200ml 09/30/05 17 - 1&2 4A 1L 09/07/06 17 - 2 48 IL 09/07/06 14 - 2 4C 1L 09/07/06 19 - 2 4D 1L 09/07/06 15 - 2 4E 1L 09/07/06 13 - 2 4F 1L 09/07/06 21 - 2 5A 1L 09/08/06 10 - 2 5B 1L 09/08/06 14 - 2 5C 1L 09/08/06 21 - 2 5D 1L 09/08/06 1 1 - 2 5E 1L 09/08/06 16 - 2 SF 1L 09/08/06 10 - 2 6A 2L 09/09/06 TNTC + 2 6B 2L 09/09/06 TNTC + 2 6C 2L 09/09/06 TNTC + 2 6D 1.5L 09/09/06 TNTC + 2 6B 1 .5L 09/09/06 TNTC - 2 6B 1 .5L 09/09/06 TNTC - 2 7A 1L 09/25/06 1 - 2 8A 1L 09/26/06 8 - 2 88 IL 09/26/06 16 - 2 9A 1L 10/03/06 83 + 2 9B 1L 10/03/06 78 + 2 9C 1L 10/03/06 68 + 2 9D 1L 10/03/06 100 + 2 10A 1.5L 10/04/06 139 - 2 108 1L 10/04/06 56 - 2 10C 1L 10/04/06 65 - 2 10D 1L 10/04/06 54 - 2 70 APPENDIX 2 Esp gene sequence of positive control, Katrina sample and raw sewage. EspATCC EspKatrtna EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatr1na EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawSewage EspATCC EspKatrina EspRawaewage EspATCC EspKatrtna EspRawSewage 1 Cttfgattct crr:gar:cc Ctttgatfc: 51 atggaacagg atggaacagg atggaacagg 101 gacgataaag gacgaraaag gacgaraaag 151 attagaagaa attagaagaa attagaagaa 201 'ccacgagtt tccacgagft accacgagit 251 ggtacrgtat ggractgzat ggtacrgtat 301 :ccaatttca tccaarttca iccaattzca 351 aa*'aatcgc aattdatcgc aattaatcgc 401 garaaaccag gazaaaccag gataaaccag 451 tgataaacaa :gaiaaacaa tgataaacaa 501 rtcggaaraa ttcggaaraa ttcggaazaa 551 gagcctgaia gagcc:gaza gagcc:gata 601 raagatcgga taaga"cgga raagarcgga rgg'tgtcgg tggttgtcgg tggitgtcgg t' '.7 81' 'Ta'. ’Q’.a1.'_.at [Laltat 1:.a Eta L a o I aagagagcgg aagagagcgg aaQaQaQCQQ gtarargarg gtaratgaig gtata*9aig agcgggaaca agcgggaaca agcgggaaca tagcagaaaa tagcagaaaa ragcagaaaa gatactgtag gatactgtag gazacrgrag gaaagargaa gdaagatgaa gaaagargaa aagaaggt'c aagaagg'rc aagdaggitc accgtggtag accgzggtag accgaggtag taaaaaC'ta taaaaac:fia :aaaaaczta Ct‘C"aacgfi c:;c;aacgt CZ’CZaacg: 612 atacgaaiaa atacgaataa atacgaataa tfgcaaga'a rtgcaagata ttgcaagata agacacgaat agacacgaat agacacgaat ttgacacaac ttgacacaac tcgacacaac ggtcacaaag gg'cacaaag ggzcacaaag aacratcgct aacra'cgc1 aac:atcgcr aatttgaat: aatttgaatt aattigaat: ‘C T. aaaaat CC L 1. 61616161111"- CC t 7. aaaaat' CC aaccgtrart aaccgiraflt aaccgiaazr ttggatrtaa riggaittaa ttggarttaa CCtgaagatg ccrgaagatg eczgaagazg tactgacagt Lac:garag: tac;gatagt 71 816867. T. aat C aaaat taarc 8.651137. F 6161' C rigatgg'ga r39131991113 ttgatggzga ccaiararcg ccatatarcg ccazatatcg agtraagggg agitaagggc agitaagggg cccaaCt'gt cccaacrrgr cccaaCZtgt ccaaatgaaa ccaaa'gaaa ccaaatgaaa racgggtgta racgggtgta tacgggtgta aaatcgirtc addLCQLLLC aaatcgtttc aaggaarcac aaggaaicac aaggaaicac accagatgcr accagatgc: accagatgcc cagagtaffc cagag:a::c cagagtaiic acgaaaggfia acgaaagg:a acgaaaggta 50 cagaacactt cagaacaCtt cagaacacrt 100 zggaaaccc: tggaaaccct tggaaaCCCt 150 ggaaaCC1ga ggaaacc:ga ggaaaccrga 200 aaagtattca aaag'attca aaagtattca 250 tgaraaagaa tgataaagaa agazaaagaa 300 aagatggggc aagarggggc aagatggggc 350 gattcaagta gattcaagta gattcaagta 400 tccaggtttt Lccaggnztt tccaggtttt 450 caaaagcggt caaaagcggt caaaagcggt 500 aaagaafcaa aaagaaicaa aaagaatcaa 550 atggaaaaca arggaaaaca atggaaaaca 600 ttgtaactg: t:g:aactgt t:gzaac:g: 10. REFERENCES Abu Al-Soud, W., and P. Radstrom. 1998. 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