Citation
Selection and characterization of bacteriophage for use in adsorption and transport studies

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Title:
Selection and characterization of bacteriophage for use in adsorption and transport studies
Creator:
Boice, Cheryl M., 1972-
Publication Date:
Language:
English
Physical Description:
xii, 146 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Dissertations, Academic -- Microbiology and Cell Science -- UF ( lcsh )
Microbiology and Cell Science thesis, Ph.D ( lcsh )
Bacteriophages ( jstor )
Adsorption ( jstor )
pH ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Cheryl M. Boice.

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78017518 ( OCLC )

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Full Text










SELECTION AND CHARACTERIZATION OF BACTERIOPHAGE FOR USE IN
ADSORPTION AND TRANSPORT STUDIES
















By

CHERYL M. BOICE












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003































Copyright 2003

by

Cheryl M. Boice














ACKNOWLEDGMENTS

I would first like to acknowledge my mentor, Dr. Samuel R. Farrah, for his guidance and support throughout my graduate studies. He has encouraged ingenuity in my research. Dr. Farrah has been an inspiration and his vast knowledge on many subjects has been an asset. I would also like to thank the other members of my graduate committee (Dr. Thomas Bobik, Dr. Madeline Rasche, Dr. Ben Koopman, and Dr. Richard Dickinson) for their guidance. I would like to acknowledge Dr. Jerzy Lukasik and Dr. Troy Scott for their assistance. In addition, I would like to thank my husband and son, T.J. and Nathan Boice; and my grandparents, John and Myd Dean for their support and understanding throughout my education. I extend special thanks to the entire Department of Microbiology and the Engineering Research Center (PERC) for their overall support and flexibility.


















iii














TABLE OF CONTENTS



ACKNOW LEDG M ENTS .................. .......... ......... ........................... iii

LIST O F FIG URES . ............................. ...................................................... vii

LIST O F TABLES.................................................... ......................................... ix

ABSTRACT........................................................................................................ xi

1 INTRO DUCTIO N ......................................... .......................................1........

W aterborne Pathogens .............................................................................. 1
Bacteria ....................................................... -- -- ............................................. 2
Viruses ................................... ...... .. .......................................................... 5
Protozoa .......................... . ................... ........... .... 6
Indicators ........................................................................................................ .... 7
M odels .................................- ..... . ............................................................... 9
Tracers ............................ ................. ........ ........................... 12
Interactions between Viruses and Solids ............................................................. 13
Survival ............................................................................................................. 19
Virus Characterization ............................................. .....................................21
Experim ental Rationale .............................................. ...................................23

2 VIRAL INTERACTIONS WITH DEFINED MEDIA.................................26

Introduction ....................... .......................... ...................................................... 26
Adsorption ..................................................................... .........................27
Elution ............................................................. ....................................... 29
Current M ethods ................................... ----... .............................................. 30
M aterials and M ethods .................................................. ................................31
Collection of Raw Sew age Sam ples.............................................................31
M edia.......................................................................................31
Chem icals ..................................................................................................... 31
Solutions.................................................. ...............................................31
Viruses ....................................................................................................... 32
Isolation of Bacteriophage Using Nitrocellulose Filters .................................33
Isolation of Bacteriophage Resistant to High Temperature .......................34
Selection of Bacteriophage for Additional Studies .....................................34


iv









Characterization of Bacteriophage Adsorption to Filters under Varying
C onditions ..................................... . ................................ 35
Bacteriophage Interaction to Particles .... .............................. ..................... 36
R esults ............................................ .................................................37
Isolation of Bacteriophage from Raw Sewage Samples.........................37
Selection of Bacteriophage........................................................38
Characterization of Bacteriophage ................................................ 42
D iscussion ........................................................ ............................................. 4 7

3 VIRAL INTERACTIONS WITH NATURAL MEDIA................................. 52

Introduction ................................. . ... .... ............................ ....................... 52
Adsorption .................................................................53
Tracers .......................................................................54
S u rv iva l ......................................................................................................... 5 5
M aterials and M ethods .........................................................56
M edia..............................................................56
S o lutio ns ....................................................................................................... 56
V iru se s .........................................................................................................56
Adsorption of Viruses to Mixed-Liquor Suspended Solids .........................57
Adsorption of Viruses to Soils............................................... 58
Kinetic Adsorption Studies .......................................................................... 59
Column Adsorption Experiments ..............................................60
Groundwater Tracer Studies.............................. ................ 61
Groundwater Survival Studies ......................... .......62
Results .....................................................................63
Batch Adsorption Studies ......................................... ..........................63
Kinetic A dsorption Studies .................................... .................................. 67
Column Adsorption Experiments ......................................... ......70
Groundwater Studies ............................................................... ......... 73
D iscu ss io n ................................................................................................... 7 4

4 IDENTIFICATION AND PROTEIN COAT CHARACTERIZATION OF
SELECTED BACTERIOPHAGE .....................................................................90

Introd u ctio n ............................... ...........................................................9 0
Materials and Methods ........................................................91
V iruse s .......................................................................... .........................9 1
Cross Reactivity (Host Range) ................................................92
Plaque Morphology.....................................................92
Electron Microscopy .......................................92
Nucleic Acid Determination .................................................. 93
P rotein A nalysis ....................................................................................... ...94
Restriction Enzyme Analysis ..................................................94
Sequencing............................................................95
G e l E xcision ............................................................................................. 95









C lo n in g ....................................................................................................... 9 5
Screening..................... ........ ..... . .......... .............................. 95
Plasmid Isolation and DNA Sequencing ..............................................96
Sequence Analysis of the Capsid and INT Gene ...................... ..............96
Protein Coat Analysis ................................ ............................96
R e su lts ........................................................................ ............................... 97
Cross Reactivity (Host Range) ..................................................................97
Plaque Morphology and Electron Microscopy ........... .................................... 97
Nucleic Acid Determination ................................ ..................................98
Protein Analysis and Restriction Enzyme Analysis...................................... 99
S e q ue ncing .................................................................................................10 0
Protein Coat Analysis .............. ............... . .... 100
D iscussion ..................................... .......................................................... 10 1

5 SUMMARY AND CONCLUSIONS ....................................................................... 119

APPENDIX

A BACTERIOPHAGE ADSORPTION ............................................................ 124

B AMINO ACID SEQUENCES OF VIRAL CAPSIDS..................................127

C audovirales................................................. ........................................ 127
Leviviridae ................................................................................. 127
M icroviridae .......................................................................... 127
Tectiviridae ......................................... - ............................................. 127
M yoviridae ................................................................ ...... 128
P odoviridae ........................................................................................ 128
Siphoviridae ........ ......................................................................... 129
Picom aviridae ................................ ...................................................130
P oliovirus .......................................... - ............................................... 130
Echovirus ............ ........................................................131
Coxsackie virus .................................................................................. 131

LIST O F REFERENCES .................................................................................. 133

BIOGRAPHICAL SKETCH ...........................................................................146











vi















LIST OF FIGURES

Figure page

2-1 Raw sewage bacteriophage survival at 40'C and 500C over time ..........40

2-2 Survival of bacteriophage at 400C in UV dechlorinated water ..............42

2-3 Survival of bacteriophage at 500C in UV dechlorinated water ..............43

2-4 Bacteriophage adsorption to 0.45 pm HA nitrocellulose filters at pH 7
in the presence of M gCI2 ...............................................................44

2-5 Bacteriophage elution from 0.45 pm HA nitrocellulose filters at pH 3.5
in the presence of Tween 80 ................................................................ 45

2-6 Bacteriophage elution from 0.45 pm 1MDS charged modified filters
at pH 7 in the presence of MgCl2 ........................................... 46

2-7 MS-2 adsorption to nanosized filter fibers ...............................................48

2-8 60STP2 adsorption to nanosized filter fibers................................ 49

3-1 Soil colum n setup ................................................................... 61

3-2 Adsorption of model bacteriophage to mixed-liquor suspended solids ....64 3-3 Adsorption at 40C of select bacteriophage to natural media ................70

3-4 Adsorption at 250C of select bacteriophage to natural media ..............71

3-5 Adsorption at 370C of select bacteriophage to natural media ............72

3-6 Arrhenius plot of select bacteriophage adsorption to natural media........74 3-7 Transport in (10 cm) sandy soil column of bacteriophage ....................75

3-8 Transport in (10 cm) forest soil column of bacteriophage ....................76

3-9 Transport in (20 cm) sandy soil column of bacteriophage ....................77



vii









3-10 Transport of viruses in (37 cm) sandy soil column .................................. 78

3-11 Transport in (20 cm) forest soil column of bacteriophage ....................79

3-12 Transport of viruses in (35 cm) forest soil column ...................................80

3-13 Tracer study in well 1 at a 10 ft distance from the system....................82

3-14 Tracer study in well 2 at a 12 ft distance from the system .....................83

3-15 Tracer study in well 3 at a 27 ft distance from the system .....................84

3-16 Tracer study in well 4 at a 49 ft distance from the system .....................85

3-17 Tracer study in well 5 at a 71 ft distance from the system .....................86

3-18 Tracer study in well 6 at a 73 ft distance from the system ....................87

3-19 Bacteriophage inactivation in wells ..................................... .....88

4-1 Electron m icrograph of 60KCK2 ...........................................................99

4-2 Electron m icrograph of 61KCK10 ..........................................................100

4-3 Electron micrograph of 60STP2 ...........................101

4-4 Electron micrograph of 61STP7 ...................................... ................... 102

4-5 Electron micrograph of the bacteriophage 60STP2 with Salmonella
typhim urium ..................................................... ................................ 103

4-6 4-20% SDS-PAGE of bacteriophage .......................................... ...........105

4-7 Restriction enzyme EcoRV analysis of bacteriophage .................... 106

4-8 Restriction enzyme EcoRI and Hincll analysis of bacteriophage .......107 4-9 Restriction enzyme EcoRI and Hincll analysis of 60STP2 .........108 4-10 Screening plasmid restriction enzyme EcoRI and Hincll analysis of
the capsid and integrase genes of 60STP2 .........................................109

4-11 Sequence analysis of the capsid gene of 60STP2 ...........................1...15

4-12 Amino acid residue analysis of the capsid gene of 60STP2 ...............1...16




viii














LIST OF TABLES

Table page

1-1 Sources of pollution in groundwater ..........................................................2

1-2 Solute models frequently used in groundwater studies ...........................14

1-3 Biotracer models frequently used in groundwater studies .......................14

1-4 Factors involved with viral adsorption and transport in soils ................18

1-5 Factors involved with survival of viruses associated with solids..............20

2-1 Adsorption of bacteriophage in filtered raw sewage at pH 7 and
p H 3 .5 ........................................................................ ....................... 39

2-2 Adsorption of selected bacteriophage to microporous filters at pH 7
and pH 3.5....................................................... ............ .............41

2-3 Adsorption of bacteriophage to defined media ........................................47

3-1 Detection of viruses in groundwater ..................................................... 52

3-2 Soil characteristics ......................................................57

3-3 Well characteristics ..................................................... 62

3-4 Weather conditions ..................................................... 63

3-5 Adsorption to mixed-liquor suspended solids by select bacteriophage ...65 3-6 Adsorption to natural area teaching lab soils by select bacteriophage ....66 3-7 Adsorption to soils by select bacteriophage ..................................... 67

3-8 Adsorption to natural media at pH 7 and pH 3.5 by select phage ...........68

3-9 Adsorption of bacteriophage to natural media .................................... 69




ix








3-10 Activation thermodynamics of select bacteriophage adsorption to
natural m edia ...........................................................73

3-11 Bacteriophage in monitoring wells. ......................................................81

3-12 Summary of select bacteriophage ........................................................ 89

4-1 Identification characteristics of select bacteriophage ...........................98

4-2 Similarities of isolated and reference bacteriophage .............................104

4-3 Capsid sequence amino acid hydrophobic residues and adsorption
characterization of various viruses .....................................................117

4-4 Capsid sequence amino acid charged residues and adsorption
characterization of various viruses .....................................................1...18

A-1 Phage adsorption to 0.45 pm nitrocellulose filters at pH 7 and pH 3.5..124






























x














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SELECTION AND CHARACTERIZATION OF BACTERIOPHAGE FOR USE
IN ADSORPTION AND TRANSPORT STUDIES By

Cheryl M. Boice

August 2003

Chairman: Samuel R. Farrah
Major Department: Microbiology and Cell Science

Bacteriophages are frequently used as models of human pathogenic

viruses in studies on virus adsorption. They are also frequently used to examine penetration of barrier materials; and to study viral transport in soil and water. They offer a practical analytical tool in studying numerous applications, such as the effectiveness of water treatment processes. Bacteriophages are used because they are easier, safer, and less expensive to assay than human pathogenic viruses; and they can be produced in large numbers (1014 PFU/mL). However, these models have traditionally been chosen based on similarities shared with human pathogenic viruses including size, shape, nucleic acid type, and chemical composition of the capsid. They are typically not selected based on adsorption or survival characteristics. Previous research indicates that the adsorption mechanisms of some phage models are not comparable to the pathogenic viruses they are supposed to represent.


xi








Our main objectives were to determine if phage models could be chosen based on their adsorption mechanisms; and to clarify these mechanisms. We isolated indigenous bacteriophage from domestic sewage samples based on differences in the strengths of their electrostatic and hydrophobic interactions to charged microporous filters under varying conditions of pH, salt concentration, and concentration of detergents. Bacteriophage adsorption properties were then compared to viruses already studied for their ability to adsorb to defined media and natural solids, such as soils and wastewater sludge. Results indicate that some selected bacteriophages are better models for human pathogenic viruses in adsorption studies than previously used models.

The isolated bacteriophage were identified down to the family level.

Examination of the coat proteins and phage interactions to solids were used to clarify the mechanism of adsorption. It was found that the coat proteins vary in each bacteriophage and may account for the differences in adsorption to the same media.

Finally we used a groundwater tracer study of the bacteriophage selected based on soil column studies. Results indicate that some of the isolated bacteriophage may be better than previously used bacteriophages as models for the movement of viruses in soil.











xii













CHAPTER 1
INTRODUCTION

In the United States, approximately half the population uses groundwater as the major source of drinking water (Craun 1986). Fecal pollution from human or nonhuman origins may affect the quality of water (Keswick and Gerba 1980). Septic tanks are identified as the cause of most of the groundwater contamination (Yates et al. 1985). Studies have also shown that some waterborne pathogens can survive wastewater treatment processes and contaminate the groundwater supply after land application (Keswick et al. 1992, Vaughn et al. 1983). Other sources of possible fecal contamination include polluted surface waters, landfills, and runoff from agriculture, and cesspools (Table 1-1) (Keswick and Gerba 1980). The presence of enteric organisms in groundwater, and well water has been documented (Gerba et al. 1985). Over half of the documented waterborne-disease outbreaks in the United States resulted from consumption of contaminated groundwater, in which an estimated 65% of those cases were caused by enteric viruses (Yates et al. 1985).

Waterborne Pathogens

Several bacterial, viral, and protozoan pathogens may be found in

contaminated waters. Most of these pathogens infect the gastrointestinal tract and are transmitted via person-to-person contact or by contaminated food and water. These organisms may be found in contaminated soil and water.




1






2

Table 1-1. Sources of pollution in groundwater

Sources of pollution Description Land disposal of sewage. Facilities Land disposal of sewage is a
include slow-rate infiltration, rapid form of groundwater recharge
infiltration, overland flow, wetlands, and subsurface
injection.
Sewer line cracks
Land disposal of sewage sludge Sewage sludge is dried and placed on land sites
Septic tanks, and cesspools. Septic systems rank the highest Wastewater (containing pathogenic in the volume of wastewater organisms) is discharged directly discharged directly into
onto soil via onsite disposal groundwater systems. These units. systems are subject to failure These are soil-adsorption treatment leading to groundwater systems. contamination.
Land disposal of solid waste Refuse dumps, landfills.
Microbial waste from diapers and animal feces
Storm water recharge and urban Sewage mixed with rainwater runoff and used for recharge.
Cesspool runoff, or sewage
waste eluted by rainwater or
runoff into streams.
Agricultural Irrigation of crop with sewage effluent

Bacteria

Most of the disease-causing bacteria found in wastewater belong to the

families Enterobacteriaceae and Vibrionaceae; and the genera Campylobacter.

Enterobacteriaceae are gram negative bacilli. Several species are part of the

normal flora of the intestinal tract of warm-blooded animals. Vibrionales members

are gram negative curved bacilli frequently found in aquatic habitats.

Campylobacter genera are gram negative curved bacilli. Some species are part

of the normal flora of the intestinal tract of animals (especially poultry). The

members Escherichia, Salmonella, Shigella, Vibrio, and Campylobacter are






3

frequently the cause of human disease associated with bacteria-contaminated water or food. They are capable of producing a variety of infections including septicemia, gastroenteritis, enteric fever, bacteremia, meningitis, and urinary tract infections (Murray et al. 2001)

Most E coli are considered harmless or opportunistic pathogens. Their presence is most often used as an indicator of the presence of other fecal pathogens. However, some strains of E coli can cause gastroenteritis. These strains are divided into five groups based on specialized fimbriae and the toxins they produce (Murray et al. 2001). The most serious disease is caused by the strain called enterohemorrhagic E. coli (EHEC). Some infections can be mild and uncomplicated; however, one possible presentation of EHEC is hemorrhagic colitis with bloody diarrhea caused by the Shiga-like toxins (Murray et al. 2001). Hemolytic uremic syndrome is another complication of EHEC (Murray et al. 2001). Another strain is enterotoxigenic E. coli (ETEC), which is the primary cause of traveler's diarrhea. This is a noninvasive infection that has an enterotoxin that causes watery diarrhea (Murray et al. 2001). Detection of enterohemorrhagic E. coli (particularly 0157:H7) uses both molecular and biochemical methods.

Salmonella spp. is opportunistic and can cause salmonellosis. Most often the disease is related to contaminated meats and poultry (Murray et al. 2001). Salmonella typhi is only found in humans and can cause typhoid fever. There has been a reduction in typhoid infections because of proper sewage disposal, water treatment, and food sanitation. The methods used in the detection of Salmonella






4

in water are not standardized. Selective enrichment and identification procedures are generally used. PCR and Immunomagnetic separation have been used to detect Salmonella spp. (Hsih and Tsen 2001).

Shigella dysenteriae can cause dysentery by the production of Shiga toxin, an exotoxin (Murray et al. 2001). The detection of Shigella spp. in environmental samples is difficult due to, the low number of organisms present and the presence of a large number of background floras. Although enrichment and direct-detection techniques such as PCR (Frankel et al. 1990) are available, they are difficult to perform. Because the infective dose is so low, however, the presence of any amount of Shigella poses a threat to human health (Stutman 1994).

Vibrio cholerae can cause cholera infections, which are caused by lapses in sanitation practices. Most infections are strongly associated with contaminated water sources (usually from eating contaminated seafood). An enterotoxin is produced that causes secretion of fluids, watery diarrhea, and vomiting resulting in dehydration (Murray et al. 2001). Shock and renal failure can also occur. Vibrio parahaemolyticus and Vibrio vulnificus are usually found in gastroenteritis outbreaks associated with seafood (Murray et al. 2001). These organisms are found in salt water estuaries. Vibrio vulnificus can also cause wound infections after exposure to contaminated seawater; and systemic infections occur after eating contaminated food (Murray et al. 2001). The detection of Vibrio spp.






5

requires water samples to be concentrated and enriched; then plated on selective and differential media for presumptive identification (Murray et al. 2001).

Campylobacterjejuni can cause gastroenteritis associated with food

contamination. The organisms are often found in milk and poultry. The symptoms include fever, cramping, diarrhea, and possibly dysentery (Murray et al. 2001). Guillain-Barre syndrome, with temporary paralysis, is a rare complication of campylobacter infections (Murray et al. 2001). Detection and identification is by plating on selective media under selective conditions. Viruses

It is estimated that over 100 different types of human enteric viruses may be present in human feces (Puig et al. 1994). The enteric viruses associated with humans encompass many viral families such as, Picornaviridae, Adenoviridae, Astroviridae, Caliciviridae, and Reoviridae. The family Picornaviridae includes enteroviruses such as polio, coxsackie A and B, echo, enteroviruses 68-71, and hepatitis A. Adenoviridae includes adenovirus of serotypes 31, 40, and 41. The family Caliciviridae includes Norwalk; small round viruses, and hepatitis E. Reoviridae includes the reoviruses and rotavirus. They are associated with a wide range of diseases such as gastroenteritis, respiratory infections, conjunctivitis, hepatitis, paralysis, meningitis, myocarditis, neonatal, urethritis, rashes, and encephalitis. Generally, the diseases are either asymptomatic; or the symptoms exhibited are gastroenteritis and respiratory infections.






6

Pathogenic viruses of fecal origin are transmitted to water sources via inefficiently treated water. Once in the environment, enteric viruses cannot replicate. However, even a low level of viruses in the environment can pose a risk to human health. Some enteric viruses have a very low (<10) infectious dose (Murray et al. 2001). So detecting viruses in environmental water samples consists of collecting and concentrating viruses from a large sample volume (usually done by filtration) followed by a second concentration step (Clesceri et al. 1989). The viruses can then be detected by cell culturing in conjunction with molecular techniques, primarily reverse transcriptase-polymerase chain reaction (RT-PCR) for RNA viruses (Abbaszadegan et al. 1999, Straub et al. 1995). However, this method is time-consuming, expensive, subject to environmental contamination and background from other viruses (Lewis et al. 2000). Other limitations to detection are the low concentrations in environmental samples; and some are not cultivable (Alvarez et al. 2000, Lewis et al. 2000). Protozoa

The most common Protozoa causing intestinal disease are Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum, and members of the order Microsporidia. The Entamoeba histolytica cyst is infective to humans and has the ability to survive in food and water. Infection may be asymptomatic in some individuals and fatal in others. Acute amebic colitis involves frequent bloody diarrhea with or without fever. Giardia lamblia is prevalent in underdeveloped countries. Waterborne transmission causes giardiasis in travelers in endemic countries. Symptoms include intestinal abdominal cramps, nausea, explosive diarrhea, fever, and chills. Cryptosporidium parvum oocyst is infectious to






7

humans. The oocysts are widely distributed in both sewage and drinking water. Symptoms including diarrhea, fever, abdominal pain, and nausea last for about one week. Environmentally resistant Microsporidia spores have the ability to infect humans. Surface water is the primary environmental residence for Microsporidia. The most common symptoms of Microsporidia infection include diarrhea, dehydration, and weight loss.

Entamoeba histolytica can be detected by microscopic examination or by various molecular assays (Schunk et al. 2001, Zindrou et al. 2001). Characteristic staining patterns identify Giardia and Cryptosporidium. Restriction fragment length polymorphism analysis of PCR amplicons can be used to identify various Microsporidia (Curry and Smith 1998).

Indicators

While pathogen counts in raw sewage can be significant, the

concentration in treated water is usually low. Even at these low concentrations, some enteric organisms are still capable of causing disease. Due to the wide distribution and survival characteristics of waterborne pathogenic organisms, it is necessary to monitor water quality and assess risk factors for waterborne disease. Direct analysis of the pathogens is often difficult due to the low numbers, background from other organisms, and the procedure used. Molecular techniques are quicker and more sensitive. However, they are more expensive; and do not indicate if the organism is viable. Due to these limitations, indicator organisms are often used as an index of water quality; and to test samples for the possible presence of fecal pollution. Indicator organisms are used because






8

they are found in high numbers; and because procedures are easier and faster than assay procedures for pathogens.

Total and fecal coliform organisms are used extensively as indicators for determining the sanitary quality of water (Gerba 1999). The total coliform group includes Escherichia coli, Enterobacter, Klebsiella, and Citrobacter. These organisms ferment lactose and produce gas at 350C within 2 days. However, not all members of this group are of fecal origin. Fecal coliforms include members that can ferment lactose at 44.50C. Members of this group include Escherichia coli and Klebsiella pneumoniae. The presence of fecal coliform indicates the presence of fecal pollution from warm-blooded animals. However, they are not limited to humans. They also differ from pathogenic virus and protozoan organisms in resistance, adsorption, and survival in the environment (Sobsey et al. 1995). E. coli is the most reliable indicator for fecal pollution since it can be differentiated from other coliforms.

Enterococci, and Clostridium perfringens have been considered as alternative indicators. Fecal streptococci are used as an indicator of fecal contamination since they are found in the feces of humans and animals (Gerba 1999). They are more resistant than coliforms, do not reproduce in the environment, and persist longer than coliform organisms. The group Fecal streptococci have a subgroup Enterococci that includes the members S. faecalis and S. faecium. These organisms have been suggested as indicators of the presence of viruses in biosolids (Payment and Franco 1993). Clostridium perfringens produces an anaerobic spore; and has an exclusively fecal origin.






9

The spores are resistant to treatment processes and extreme temperatures (Payment and Franco 1993). The primary use of these spores has been as an indicator to past pollution.

Other organisms such as bacteriophages have also been considered as alternative indicators. Bacteriophages are viruses that infect bacteria. They consist of a nucleic acid molecule (genome) surrounded by a protein coat (capsid). Some bacteriophages may also contain lipids. These organisms may be somatic coliphage, which infect coliforms such as Escherichia coli through cell wall receptors, F+ specific bacteriophage that infect E. coli and other coliforms through the F+-pili, and anaerobic Bacteriodes fragilis bacteriophage (IAWPRC 1991, Leclerc et al. 2000). Bacterial viruses as indicators for fecal pollution have been proposed due to their presence in contaminated waters, and their similarities shared with enteric viruses (Havelaar et al. 1990, Hsu et al. 1995). Bacteriophages are proposed as indicators because their presence indicates the presence of the host organism and possible fecal pollution. The F-specific coliphage are considered the indicator of choice for assessing the potential presence of human enteric viruses since they are a more homogeneous group and generally have a greater resistance to inactivation than somatic coliphages (IAWPRC 1991, Leclerc et al. 2000). Bacteriodes fragilis bacteriophages have been reported to occur in human faeces only (Leclerc et al. 2000).

Models

Pathogens of fecal origin are transmitted through inefficiently treated water. Organisms especially viruses survive current wastewater treatment processes and can be transported back into the groundwater supply. Model





10


organisms for human pathogenic viruses are often used to assess various materials and processes such as filter materials and wastewater treatment processes.

Poliovirus is often used as a representative organism for the pathogenic enteric viruses in numerous applications such as wastewater treatment processes since it is cultivable and more resistant to adverse environmental conditions (Lance and Gerba 1984b, Sobsey et al. 1995, Schijven 2000). However, assessing materials by direct analysis is time consuming and expensive (Dizer et al. 1984, Gerba et al. 1981, Goyal and Gerba 1979, Havelaar et al. 1993). Other model organisms, such as bacteriophage are frequently used to study the adsorption, fate, and transport of human pathogenic viruses due to their availability, low assay cost, and safety (Gerba et al. 1991, Paul et al. 1995, Yates et al. 1985). Bacteriophages are used extensively to evaluate treatment processes, and as tracers (Gantzer et al. 1998, IAWPRC 1991, Leclerc et al. 2000). Frequently studied models for enteric viruses include MS2 (Bales et al. 1989, Farrah 1982, Farrah et al. 1991, Gerba et al. 1991, Shields and Farrah 2002, Sobsey et al. 1995); PRD1 (Bales et al. 1991, Paul et al. 1995, Shields and Farrah 2002); OX174 (Fujito and Lytle 1996, Goyal and Gerba 1979, Shields and Farrah 2002); and other F-specific RNA phages (Havelaar et al. 1993, Havelaar and Sobsey 1995, Sobsey et al. 1986). These models have shown that bacteriophages can be useful under some conditions. However, they are inadequate models for all enteric viruses since their adsorption varies. F-specific bacteriophages, MS2, and PRD 1 are considered worst-case model viruses since








they are poor adsorbers at pH 7 (Schijven 2000). The bacteriophage QX174 is considered a conservative model since it is retained in column studies better than the worst-case models. QX174 has been found to have basically no charge at a neutral pH, low hydrophobicity, and minimal electrostatic interaction; so it has been used in various studies of barrier materials (Dowd et al. 1998, Schijven 2000).

These models have traditionally been chosen based on similarities to

human pathogenic viruses (such as size, shape, and nucleic acid type). Most of the models have not been selected based on their adsorption or survival characteristics. Previous research has shown that the adsorption mechanisms of some models are not comparable to those of the pathogenic viruses they are supposed to represent (Shields and Farrah 1983, Sobsey et al. 1986). For example, the coliphage MS-2 has been used as a model for poliovirus-1 for numerous years. It was primarily selected based on its similarities to polio such as its nucleic acid, size, and shape. MS-2 is an icosahedral ssRNA bacteriophage (for Escherichia coli) of approximately 27 nm size. Polio is an icosahedral ssRNA human virus of approximately 30 nm size. However, it is well documented that MS-2 and poliovirus do not have comparable adsorption characteristics (Gerba et al. 1981, Lukasik et al. 2000, Shields and Farrah 1983). Consequently, the results typically indicate that additional testing of models or possibly further modeling is required; and a greater understanding of viral interactions with surfaces is vital.






12

Tracers

Solutes and biological organisms are used as tracers to delineate the flow path and rate of water in soil and aquifer systems (Bales et al. 1989). Model organisms for pathogenic viruses are also frequently used as tracers to better predict the vulnerability of groundwater sources; and in treatment units (Harvey 1997). Numerous tracers have been used to describe groundwater transport (Tables 1-2 and 1-3). Nonbiological tracers include salts, fluorescent dyes, beads, and radioisotopes. However, there are problems with solutes, such as natural concentrations and adsorption differences (Harvey 1997). Biological tracers include bacteria, yeast, proteins, lipopolysaccarides, viruses, and bacteriophage (Bales et al. 1989). Biological tracers can better predict colloidal transport in a system than solutes can (Bales et al. 1989). The prediction of the adsorption, survival and subsequent elution of colloidal particles is improved by using biological tracers consisting of similar properties. Problems with bacteria, tagged bacteria, protozoa, and yeast include size exclusion, replication in natural environments, background, decay, and inactivation (Harvey 1997). Proteins and lipopolysaccarides can be used for adsorption studies. However, they cannot reveal any details about survival in the various systems. Animal viruses have been used as tracers. However, they can be pathogenic to humans; and detection is time-consuming and expensive. Bacteriophage tracers are frequently used in place of enteric viruses, since they are viruses that are easy to use in experiments. Advantages of using bacteriophage as models include small size, negligible effect on water quality, surface characteristics, high titer, detection of low numbers, and host specificity (Harvey 1997).






13

Interactions between Viruses and Solids

The adsorption characteristics of viruses and bacteriophage have been studied using defined media such as microporous filters (Shields and Farrah 2002, Sobsey and Glass 1984). The adsorption characteristics of viruses have also been studied using natural media, such as soils; and mixed-liquor suspended solids (Bales et al. 1993, Dizer et al. 1984, Dowd et al. 1998, Gerba et al. 1981, Gerba 1984a, Goyal and Gerba 1979, Griffin et al. 1999, Ketratanakul et al. 1991, Powelson and Gerba 1994, Sobsey et al. 1995).

A virus particle is colloidal in nature, so it must be physically adsorbed to a surface in order to be removed. Virus interactions with solids can be described using the DLVO Theory (Derjaguin and Landau 1941, Verwey and Overbeek 1948). Colloidal particles develop a net surface charge, which is dependent on the pH of the environment as well as the presence of ions in the solutions. This layer of surface charges is covered with a layer of counterions (the Stern layer). Another group of counterions extends farther from the particle into solution (the Gouy layer). It is the thickness of this layer that prevents interaction of particles. If the bulk solution of counterions increases by the addition of cationic salts or a decrease in pH, the thickness of this layer decreases because less volume is required to contain enough counterions to neutralize the surface charge. Reducing the thickness results in the articles moving closer to each other, so that van der Waals forces can have an effect. However, frequently hydrophobic and other effects have not been studied.






14




Tablel-2. Solute models frequently used in groundwater studies

Solutes
Models/Tracer Advantages Disadvantages Salts: identify hydrologic Limited by techniques and chloride, bromide, parameters, ease of background, adsorption differences, iodine detection, not no dispersion and porosity hazardous measurements Fluorescent Dyes: identify hydrologic Adsorbed easily on the solid Rhodamine WT, parameters, easy to material, may be toxic, deactivation, Sodium Fluorescein detect adsorption differences Microspheres identify hydrologic Adsorption controlled by coating, parameters, easy to coating stability, adsorption detect, not hazardous differences, micron size Radioisotopes: identify hydrologic Not natural, expensive, decay rate, 180, 3H parameters, easy to water quality hazard, adsorption detect differences

Tablel-3. Biotracer models frequently used in groundwater studies

Biotracers
Models/Tracer Advantages Disadvantages Viruses: Enterovirus, cultivable, Virus types adsorb differently. Poliovirus resistant Direct analysis (time consuming and expensive), low titer, water
quality
Proteins/LPS: Capable of mimicking Degradation by environment Ferritin, myoglobin, adsorption of viruses. cytochrome-c Easy to assay, nonpathogenic, do not
replicate in the
environment
Bacteriophage: Rapid results, resistant Virus types adsorb differently, MS-2, PRD1, x174, to environmental host specificity F+ specific factors, do not replicate in the environment, high
titer, nonpathogenic
Bacteria: Can identify flow rates, Difficult to distinguish from E. coli, Antibiotic rapid results, easy to background, can replicate, resistance, pigmented, assay, potential pathogens, inactivation, unusual bio- types or filterable, transfer of antibiotic spore formers resistance






15

Studies have shown that most viruses may behave differently under

similar conditions (Burge and Enkiri 1978, Gerba et al. 1981, Goyal and Gerba 1979). Numerous factors play a role in the virus-solid interactions (Table 1-4). There are significant factors associated with the virus, solution, and solid. Some of the factors associated with the virus include isoelectric point, size, type, and chemical composition (Dowd et al. 1998, Gerba et al. 1991, Hurst et al. 1980a, Yates et al. 1985). Factors associated with the solution include pH, ionic strength, organic compounds, total dissolved solids, temperature, moisture, flow rate (Bales et al. 1993, Dizer et al. 1984, Dowd et al. 1998, Farrah et al. 1981, Farrah 1982, Fuhs 1987, Gerba et al. 1991, Hurst et al. 1980a, Lance and Gerba 1984a, Lukasik et al. 2000, Lytle and Routson 1995, Lytle et al. 1999, Mix 1974, Powelson et al. 1991, Shields and Farrah 1983, Sobsey and Jones 1979, Sobsey et al. 1980a, Taylor et al. 1981, Wallis et al. 1972, Wallis et al. 1979, Yates et al. 1985). Factors associated with the solid include composition, organics, pore size, type, carbon content, moisture content, surface area, permeability, diffusion, surface charge, ion exchange capacity, and chemical composition (Bitton 1975, Bitton 1980, Dowd et al. 1998, Gerba et al. 1975, Gerba et al. 1981, Gerba 1984b, Gerba et al. 1991, Goyal and Gerba 1979, Hurst et al. 1980a, Kessick and Wagner 1978, Lance and Gerba 1984b, Mix 1974, Shields and Farrah 2002, Sobsey et al. 1980a, Yates et al. 1985).

Virus adsorption to solids has been shown to resemble protein adsorption in that hydrophobic and electrostatic interactions influence adsorption (Bales et al. 1991, Farrah et al. 1981, Farrah 1982, Hatefi and Hanstein 1969, Lukasik et






16

al. 2000, Lytle and Routson 1995, Lytle et al. 1999, Shields and Farrah 1983, Shields and Farrah 2002, Wallis et al. 1979). Shields (2002) reported that there are significant differences in the adsorption properties of numerous bacteriophage and human enteric viruses indicating that viral properties were significant in adsorption. Viruses contain a protein coat, therefore, side chains of amino acids may exhibit charged or hydrophobic groups on the surface, thus solution factors such as pH, ionic strength, and chaotropic agents have been shown to affect adsorption to solids. The surface charge on the virus originates from the ionization of amino acid side chains, and that this charge is a function of pH. When suspended in solutions there may be basic or negatively charged residues that become ionized and give a net surface charge that is pH dependent. A virus will exhibit a negative net surface charge at pH values higher than its isoelectric point, and a net positive surface charge at pH values below its isoelectric point. The results of various studies demonstrate that the adsorptive differences of viruses with similar isoelectric points or characteristics are most likely due to localized groups of negative and positive charges, and possible spans of hydrophobic groups exist on viral surfaces (Mix 1974, Penrod et al. 1996, Schijven 2000, Shields and Farrah 2002). Results have also shown that the isoelectric point is only an indication of the net surface charge at a given pH; it does not convey information about the distribution of charges over the surface of the virus (Penrod et al. 1996). Gerba and colleagues (1981) were able to group viruses adsorbed to nine different soils according to their adsorption characteristics and correlate them with their isoelectric points. Group I viruses






17

adsorbed poorly to soils, and group 2 viruses adsorbed favorably to the soils. Group 2 viruses had lower isoelectric points than group 1 viruses. The results show the surface charges of viruses at a specific pH that determine adsorption (Gerba et al. 1981, Penrod et al. 1996). However, researchers have also shown that size became the predominate factor for particles larger than 60 nm (Dowd et al. 1998). Viral adsorption and survival has been shown to be viral type and site specific (Gerba et al. 1981, Hurst et al.1980a, Yates et al.1985). Solution factors that have a significant affect on adsorption are the pH as previously described and the ionic strength (Dizer et al. 1984, Dowd et al. 1998, Lance and Gerba 1984a, Powelson et al. 1991, Powelson and Gerba 1994). According to the DLVO theory an increase in ionic strength compresses the double layer causing adsorption by van der Waals and hydrophobic forces. Hydrophobic interactions were shown to be involved in adsorption and transport by using detergents that could be present in wastewater that is applied to the soils (Dizer et al. 1984). These detergents eluted viruses such as poliovirus-1 and rotavirus SA11 to an extent. Detergents have been shown to elute phages by making the solution more lipophilic, which allows for the interactions of apolar groups and the solution (Farrah et al. 1981, Farrah 1982).

Viral movement in soils has been found to depend on saturation conditions (Lance and Gerba 1984b, Powelson and Gerba 1994). They found that under saturated conditions viral elution and transport were more likely to occur (Lance and Gerba 1984b). Other solution factors such as suspended solids, and dissolved organic compounds have been found to interfere with adsorption of






18


Table 1-4. Factors involved with viral adsorption and transport in soils

Factors Comments Ref(s)
Increases in the content of Bitton 1980, Gerba et al. 1981, clay, surface area, ion Gerba 1984b, Goyal and Gerba Media type exchange capacity, and 1979, Lance and Gerba 1984b, patches of oxides increase Sobsey et al. 1980a
adsorption of viruses.
Generally, adsorption increase Dizer et al. 1984, Dowd et al. 1998,
with a decrease in pH. Gerba 1984b, Goyal and Gerba
pH 1979, Sobsey et al. 1980a, Taylor et al. 1981
Generally there is an increase Dizer et al. 1984, Gerba 1984b,
Ionic in adsorption with an increase Lance and Gerba 1984a, Sobsey et
strength in the concentration or valence al. 1980a, Taylor et al. 1981 (conductivity) of cations
Organics such as humus tend Dizer et al. 1984, Gerba et al. 1981,
Soluble to interfere with adsorption of Goyal and Gerba 1979, Powelson organic viruses by competing with et al. 1991, Powelson and Gerba
material viruses for adsorption sites on 1994, Sobsey et al. 1980a
a solid.
Adsorption of viruses vary Dowd et al. 1998, Gerba et al.
Virus type according to viral type and 1981, Gerba 1984b, Goyal and
strain, pl differences Gerba 1979
In unsaturated conditions, Powelson et al. 1991, Powelson
where water flows through the and Gerba 1994, Schaub and
Flow small pores and forms a film Sorber 1977
around the particles, viruses
tend to adsorb better.
The slower the infiltration rate Gerba et al. 1975, Lance and
of water through soils allows Gerba 1984b
Flow rate for a greater retention time for
adsorption to occur.


viruses to solids as well (Sobsey and Glass 1984). However, it has been found

that the degree of viral interference was dependent on virus type and adsorption

conditions (Sobsey and Glass 1984). Suspended solids can interfere

with adsorption and elution by adsorbing to the viruses resulting in enhanced

adsorption or adsorbing to the filters resulting in clogging (Sobsey and Cromeans

1985a). A high content of dissolved organic compounds can retard adsorption to






19

solids by competing for adsorption sites, associating with the viruses, or by accumulating on the filter material (Sobsey and Hickey 1985b). Organic matter found in solutions, such as humic materials, are negatively charged particles or soluble compounds that compete with the viruses for adsorption sites on the solid (Gerba 1984b, Sobsey et al. 1980a, Wallis and Melnick 1967). Various soils have been studied for their ability to adsorb and retain viruses. It was concluded clay soils enhance adsorption better than sandy, organic, and mucky soils (Goyal and Gerba 1979, Lance and Gerba 1984b, Powelson et al. 1991, Sobsey et al 1980a). It was shown that viruses could be eluted from sandy soils with rainwater (Sobsey et al 1980a). Other studies have shown that oxides enhance adsorption (Goyal and Gerba 1979, Lance and Gerba 1984b).

Survival

Considering viral contamination is possible, understanding the survival mechanisms of viruses in groundwater systems is of importance. Many studies have examined viral survival in various systems (Alvarez et al. 2000, Blanc and Nasser 1996, Gerba et al. 1975, Hurst et al. 1980b, Nasser et al. 1993, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995, Ward and Ashley 1978, Ward and Ashley 1979, Yahya et al. 1993). Viral inactivation occurs by protein coat disruption and nucleic acid degradation. There are numerous factors involved with the inactivation of viruses in the environment (Table 1-5) (Gerba et al. 1991, Hurst et al. 1980a, Hurst et al. 1980b, Powelson and Gerba 1994, Schaub and Sorber 1977, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985, Yeager and O'Brien 1979). Temperature has been found to be a significant factor in virus






20


Table 1-5. Factors involved with survival of viruses associated with solids

Factors Comments Ref(s)
At lower temperatures Alvarez et al. 2000, Blanc and Nasser
viral survival time 1996, Gerba et al. 1975, Hurst et al.
Temperature increases 1980a, Nasser et al. 1993, Yahya et al.
1993, Yates et al. 1985
May stabilize viruses Hurst et al. 1980a, Sobsey et al. 1980a,
Cations Yates et al. 1985
Viruses survive longer Gerba et al. 1975, Hurst et al. 1980a, Soil moisture with some soil Yeager and O'Brien 1979 moisture content.

Minor inactivation at Gerba et al. 1975
Sunlight a soil surface or in water
systems.
Increased adsorption Blanc and Nasser 1996, Gerba et al.
will increase survival 1975, Gerba 1984b, Hurst et al. 1980a,
Soil time possibly due to Sobsey et al. 1980a, Ward and Ashley characteristics viral stabilization. (pH, 1978, Ward and Ashley 1979, Yates et soil type, cations) al. 1985
Viruses have been Gerba et al. 1975, Hurst et al. 1980a,
Biological shown to survive Sobsey et al. 1980a, Sobsey et al. 1986
longer in sterile soils
a Inactivation by biological predators and sunlight seem to be minimal.

survival (Alvarez et al. 2000, Blanc and Nasser 1996, Hurst et al. 1980a, Nasser

et al. 1993, Sobsey et al. 1995, Yahya et al. 1993, Yates et al. 1985, Yeager and

O'Brien 1979). Studies have found that viruses survive better at temperatures of

100C or below, and that there is significant inactivation at temperatures of 230C

and higher (Alvarez et al. 2000, Blanc and Nasser 1996, Nasser et al. 1993).

Yates et al. (1985) reported that the only factor tested that was significant to

survival of the viruses MS-2, poliovirus 1, and echovirus 1 was temperature.

They also reported that there is no significant difference in the decay rates of

tested viruses MS-2, poliovirus 1, and echovirus 1 in groundwater samples

(Yates et al. 1985). The ionic strength of the system seems to affect the system.






21

An increase in ionic strength seems to stabilize the viruses by enhancing adsorption, which enhances survival (Hurst et al. 1980a, Sobsey 1980a, Yates et al. 1985). High moisture content has been found to increase survival as well (Hurst et al. 1980a, Hurst et al. 1980b, Yeager and O'Brien 1979). Minor inactivation at the soil surface has been found to occur by sunlight (Gerba et al. 1975). Increased adsorption by pH, soil type, and ionic strength will increase survival because of viral stabilization (Blanc and Nasser 1996, Gerba 1984b, Gerba et al. 1991, Hurst et al. 1980a, Sobsey et al. 1980a, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985). Biological predators in natural soils may inactivate viruses (Gerba et al. 1975, Hurst et al. 1980a, Sobsey et al. 1980a, Sobsey et al. 1986). Blanc and Nasser (1996) found PRD-1 was more persistent in natural soil systems with applied treated wastewater than MS-2. Detergents have been reported to alter the thermal stability of viruses therefore affecting survival (Ward and Ashley 1978).

Virus Characterization

Historically, the classification of viruses has been based on physical parameters approved by the International Committee on the Taxonomy of Viruses (ICTV) (Rohwer and Edwards 2002, Van Regenmortel et al. 1999). Some of the techniques used to identify viruses include electron microscopy for structural information, host range, plaque formation, mode of replication, and genomic type (Ackermann 2001, Murphy et al. 1995, Yanko et al. 1999). However, it has been found that some bacteriophage classifications based on morphology and mode of replication are completely different than classifications based on the genome level (Haggard-Ljungquist et al. 1992, Monod et al. 1997,






22

Tetart et al. 1996). Frequently viral genomes are sequenced and the phages are not characterized based on the physical parameters or vice versa. Due to these problems, bacteriophage can be difficult to classify as indicated by the numerous unclassified phage in the ICTV database http://www.ncbi.nlm.nih.qov/ICTVdb/ (Van Regenmortel et al.1999). Current research has identified other methods of classification that are based on genome organization using protein, and molecular techniques (Clark et al. 2001, Ford et al. 1998, Hendrix et al. 1999; Juhala et al. 2000, Rohwer and Edwards 2002). Research has shown that there is no one gene, such as the 16s RNA of prokaryotes that can be used for classification. However, the dinucleotide relative abundance measures of distance > 20Kb provide a unique signature for each genome and can be used to measure similarities in the viruses (Blaisdell et al. 1996). In this study, they have found that each viral genome has a distinctive signature of short oligonucleotide abundances in the entire genome. This indicates that viral genomes can be grouped based on the abundance of short nucleic acid base sequences. Current research has shown the possibility of using "signature genes" of the phage families as a method of classification (Rohwer and Edwards 2002). Hypothetically, structural proteins such as the capsid could be used as a "signature" gene (Bamford et al. 2002, Le Marrec et al. 1997). However, even though structures and functions of the capsid may be conserved viruses may contain no similarity in sequences for easy identification (Rohwer and Edwards 2002). There is evidence that there is structural and functional conservation limited to core viral functions but no sequence conservation (Baker et al. 1999,






23

Bamford et al. 2002). Even though there is significant diversity in the capsid sequences within a family; there are some surprising correlations among viruses, for instance, the similarity in genome organization, replication, and capsid structure of adenoviruses and PRD-1 (Bamford et al. 2002).

Experimental Rationale

Previous studies have used known bacteriophages as models for enteric viruses to study the adsorption, and transport phenomena. The studies have shown that the adsorption properties of enteric viruses vary under similar conditions (Dowd et al 1998, Goyal and Gerba 1979, Gerba et al 1981, Sobsey et al 1995). Researchers have also shown that different strains of the same type of enteric virus have varying adsorption under similar conditions (Goyal and Gerba 1979, Gerba et al 1981). Based on the research it is apparent that no one virus is a good model for determining the interactions of the entire enteric virus group to solids.

There are a few studies that have examined the adsorption properties of viruses as one of the predominate factors for modeling enteric viruses (Gerba et al. 1981, Penrod et al. 1996, Shields and Farrah 2002). Gerba and colleagues studied viral adsorption to nine different soil types and reported that viruses could be grouped according to their adsorption properties (1981). Shields and Farrah examined the adsorption of viruses to DEAE-cellulose and octyl sepharose materials and classified the viruses based on the strength of their hydrophobic and electrostatic adsorptive properties (2002). Other research has focused on the adsorption of viruses to media and correlated it to the surfaces charges over






24


a viral surface (Penrod et al. 1996). The study indicated that the surface charges are significant in the adsorption mechanism (Penrod et al. 1996).

In the first part of our study, bacteriophages were selected from filtered raw sewage isolates based on specific adsorption properties using a variety of hosts commonly found in contaminated water. The bacteriophages were then characterized based on properties such as their hydrophobic or electrostatic nature, and the strength of the interactions to solids to identify a range of phages based on their adsorption characteristics.

We also isolated bacteriophages from raw sewage based on their survival at elevated temperatures of 400C and 50'C after three weeks. These temperatures are more selective for phages of fecal coliforms (44.50C) found in warm-blooded animals. Bacteriophages were then selected based on their survival in UV dechlorinated water at these elevated temperatures over an extended period of time.

The second part of our study investigated the factors affecting adsorption of selected bacteriophage to natural media such as mixed-liquor suspended solids and various soils. Variations in pH, salt concentrations, and detergent concentrations were also used in conjunction with the natural media to identify the mechanism of adsorption to natural media. The bacteriophages were then compared with enteric viruses of previous studies (Dizer et al. 1984, Gerba et al. 1981, Goyal and Gerba 1979).

The final part of our study involved identifying the families that the

selected bacteriophages belong to by various methodologies. The techniques






25

used were electron microscopy, host range, plaque morphology, nucleic acid determination, protein, and molecular (Ackermann and Nguyen 1983, Clark et al. 2001, Liu et al. 2000, Loessner et al. 1993, Yanko et al. 1999, Zink and Loessner 1992). The phages were compared to known members of the family. The diversity of the capsid sequences of select bacteriophages and viruses from our study and previous studies (Gerba et al. 1981, Penrod et al. 1996, Shields and Farrah 2002) were examined to assist in explaining the differences in adsorption. The phage 60STP2 was studied further considering it represents a worst-case model. Another worst-case model MS-2 has been previously studied (Penrod et al. 1996). The results from our study will be compared against previous work to identify the mechanism or mechanisms involved in adsorption (Penrod et al. 1996, Shields and Farrah 2002).













CHAPTER 2
VIRAL INTERACTIONS WITH DEFINED MEDIA Introduction

There are many methods that have been used to concentrate viruses from water samples. Adsorption to and elution from solids, adsorption to organic or inorganic precipitates, and ultrafiltration are some methods used for concentrating viruses. However, membrane adsorption-elution methodology is most frequently used (Farrah et al. 1976, Rao and Labzoffsky 1969, Wallis and Melnick 1967). The filtration process involves running a sample of water through a filter that adsorbs viruses. The viruses are subsequently eluted off the filter by using a smaller volume of high pH eluant. The first reported use of membrane filters to concentrate viruses from water samples was in 1967 (Wallis and Melnick 1967). Since then there have been numerous studies utilizing many types of filters including, pleated epoxy-fiberglass Filterite filters (Farrah et al. 1976, Goyal et al. 1980, Sobsey and Jones 1979), nitrocellulose HA millipore filters (Farrah et al. 1976, Shields and Farrah 1983), zeta-plus AMF Cuno microporous filters (Farrah et al. 1981, Goyal et al. 1980, Sobsey and Jones 1979), asbestoscellulose Seitz S filters (Sobsey and Jones 1979), and 1MDS surface modified fiberglass-cellulose resin filters (Shields et al. 1986b, Sobsey and Glass 1980). Chemical modification of filters has been used to increase viral recoveries from water samples (Farrah and Preston 1985, Preston et al. 1988b). Diatomaceous




26






27

earth and sand filters coated with metallic peroxides, and metallic hydroxides were used to increase adsorption for recovery of viruses from water (Farrah et al. 1991, Lukasik et al. 1996, Lukasik et al. 1999). Adsorption

Electrophoretic studies have shown that the various filters used in

membrane adsorption-elution methodology have varying net charges. Filterite, millipore HA have net negative charge throughout a pH range of 2-7, and Zetaplus, Seitz S, and 1MDS have a net positive charge over the same pH range (Kessick and Wagner 1978, Sobsey and Jones 1979). Electrophoretic studies have also shown that at neutral pH many viruses have a net negative charge because they have isoelectric points below pH 7 (Brinton and Lauffer 1959). Previous studies have reported that the positively charged filters adsorb viruses at neutral pH and negatively charged filters require the addition of salts at pH 7 or a decrease in pH for significant adsorption of viruses (Farrah et al. 1976, Gerba et al. 1978, Goyal et al. 1980, Shields et al. 1986b, Sobsey and Jones 1979, Sobsey and Glass 1980, Wallis and Melnick 1967, Wallis et al. 1972). Nitrocellulose filters, such as Millipore HA, have been shown to have a net negative charge and hydrophobic characteristics (Farrah et al. 1981, Farrah 1982, Kessick and Wagner 1978, Lukasik et al. 2000, Mix 1974, Shields and Farrah 1983). At pH 3.5, nitrocellulose filters have a net negative charge. This pH is below the isoelectric point of many viruses so they have a net positive charge (Hatefi and Hanstein 1969). Therefore, adsorption may be by electrostatic and hydrophobic interactions (Lukasik et al. 2000, Shields and Farrah 1983, Wallis et al. 1979). The addition of increasing concentrations of chaotropic ions or neutral






28

detergents has been found to decrease the structure making the solution more lipophilic. These solutions can solubilize viruses, there by influencing hydrophobic associations of the viruses to the filters (Farrah et al. 1981, Fujito and Lytle 1996, Hatefi and Hanstein 1969, Lukasik et al. 2000, Shields and Farrah 1983). At neutral pH values, often above the viral isoelectric points, viruses exhibit a net negative charge. The nitrocellulose filters are also negatively charged so minimal adsorption occurs mainly by hydrophobic interactions. Increasing the ionic strength of certain antichaotropic salts, such as MgCl2, can screen out electrostatic charges and promote hydrophobic and van der Waals interactions (Farrah et al. 1981, Lukasik et al. 2000, Shields and Farrah 1983, Wallis et al. 1979).

Other defined media have been used for studies pertaining to the

theoretical viral adsorption phenomena. Organically coated silicates have been used to study the roles hydrophobicity, pH, and ionic strength in adsorption of viruses to solids (Bales et al. 1993, Zerda et al. 1985). The results show that both electrostatic and hydrophobic interactions are involved in adsorption. The studies also indicated that the pH of the solution determines the surface charges of the viruses and adsorbent media. DEAE-sepharose and octyl sepharose materials (Shields and Farrah 2002) were used to investigate the adsorption characteristics of various bacteriophage and viruses. The results show that the viruses can be grouped according to the strength of their adsorption properties. The viruses were shown to adsorb to the materials by predominately electrostatic forces, predominately hydrophobic forces, or a combination of forces.





29

Inorganic and organic flocculation procedures have been used for concentrating or reconcentrating viruses from water samples. Inorganic flocculation procedures such as, the addition of aluminum hydroxide at a low pH and then the subsequent increase in pH by the addition of 1M sodium carbonate resulted in a floc that adsorbs viruses (Farrah et al. 1976). However, the drawbacks included filter clogging and inefficient recoveries. Organic flocculation procedures such as decreasing the pH of a protein solution to 3.5 produce flocs of proteins. The floc is then isolated by centrifugation and solubilized by sodium phosphate solution at pH 9 (Katzenelson et al. 1976). Elution

Elution of viruses from solids has been studied to maximize viral

recoveries and explain viral adsorption mechanisms. Elution studies used different solutions that altered the ionic strength, or increased the pH. Some of the most efficient methods include, the introduction of beef extract supplemented with glycine and NaOH to obtain a high pH (Preston et al. 1988b, Scott et al. 2002), urea with the addition of lysine at a high pH (Farrah et al. 1981, Hatefi and Hanstein 1969), and glycine at a high pH by addition of NaOH (Farrah et al. 1976). These solutions tend to interfere with the hydrophobic and electrostatic interactions involved in the adsorption phenomena. Chaotropic agents, large singly charged ions (such as trichloroacetate) or neutral detergents disrupt hydrophobic interactions. Antichaotropic agents are small singly charged ions such as fluoride and chloride, or multivalent ions such as phosphate or sulfate. Solution of metal chelators such as EDTA, an antichaotropic agent have failed to elute viruses off negatively charged filters at a high pH indicating that






30

electrostatic interactions play a minimal role in adsorption at this pH and hydrophobic interactions are the predominate forces (Farrah et al. 1981, Kessick and Wagner 1978, Lukasik et al. 2000, Mix 1974, Shields and Farrah 1983). These solutions also suggest that cationic bridging, or reversal of charge on the filter media are not factors in adsorption at neutral pH. Current Methods

However, studies have shown that the extremes in pH for the

electronegative filter adsorption method and other techniques inactivate some bacteriophage (Goyal et al. 1980, Primrose et al. 1981, Scott et al. 2002). This is a problem since bacteriophage are often used in viral tracer studies, as proposed indicators of their bacterial host (possible presence of fecal pollution), and as models for water treatment processes (Griffin et al. 1999, Harvey 1997, Havelaar et al. 1993, Paul et al. 1995, Paul et al. 1997). Current methods include concentrating by filtration with the addition of salts, enrichment, and direct plating (US EPA 2001). To overcome this limitation investigators have proposed to use electropositive filters (Goyal et al. 1980, Shields et al. 1986b, Sobsey and Jones 1979), chemical modification of filters (Farrah and Preston 1985, Preston et al. 1988b), or stabilizing the bacteriophage at a low pH with the addition of manganese chloride to the sample (Scott et al. 2002). Another flocculation procedure for the recovery of bacteriophage from small volumes or as a reconcentration step at pH 7 involves the addition of saturated ammonium sulfate to beef extract for the formation of flocs that adsorb viruses (Shields and Farrah 1986a).






31

Materials and Methods

Collection of Raw Sewage Samples

Raw sewage was collected at the University of Florida Wastewater Reclamation Facility in Gainesville, Florida. The samples were filtered through 0.2 pm porosity filter (Millipore GS; Millipore Corp., Bedford, Mass.) to remove debris. Media

The defined media used in adsorption studies were the microporous filters, 0.45 pm nitrocellulose membrane filters (Millipore type HA, Millipore Corp., Bedford, Mass.) and 0.45 pm 1-MDS (charge-modified resin filters, AMF Cuno, Meridan, Conn.), diatomaceous earth (grade 1; Sigma Chemical Co. St. Louis, Mo.), and aluminum hydroxide coated nanosized filter fibers (Argonide Sanford, FL). Chemicals

The chemicals used in our study include imidazole, glycine, magnesium chloride, sodium chloride, ferric chloride, aluminum chloride, ammonium hydroxide, and Tween 80 from Sigma Co.; the chemicals obtained from Fischer Scientific Co. were sodium chloride and sodium citrate. The beef extract was obtained from Difco.

Solutions

The buffer solution used in our study was 0.02M imidazole plus 0.02M glycine made in deionized water. All solutions in our study were made using 0.02M imidazole-glycine buffer. The buffer and solutions were adjusted to the required pH by the addition of NaOH or HCI. Elution of the viruses from the filter material were performed using Beef Extract solutions (Difco) or 0.1% Tween 80 and 1M






32


NaCI. Diatomaceous earth (Sigma Chemical Co., St. Louis, MO.) was coated with solutions of ferric chloride, aluminum chloride, and ammonium hydroxide as described by Lukasik et al. (1996).

Viruses

Sources

The following phages with their hosts were used in these studies: MS-2 (ATCC 15597-B1) was assayed using Escherichia coli 3000 (ATCC15597), PRD1 was assayed using Salmonella typhimurium (ATCC 19585), 0X1 74 (13706-B1) was assayed using Escherichia coli C (ATCC 13706). Indigenous bacterial viruses were isolated from raw sewage, grown, and assayed using the bacterial hosts Eschenchia coli 3000 (ATCC15597), Escherichia coli C (ATCC 13706), Salmonella typhimurium (ATCC 19585), Proteus vulgans, Citrobacter freundii, Enterobacter aerogenes, and Klebsiella pneumoniae were obtained from the Microbiology and Cell Science culture collection, UF. Preparation of phage stocks

An isolated plaque was excised and added to 3% Tryptic Soy Broth (Difco) with the appropriate host and incubated overnight at 370C. Phages solutions were plated using the standard agar overlay procedure (Snustad and Dean 1971). The next day the lysates plates were scraped and centrifuged at 12,100xg (Beckman J2-HS centrifuge) for 10 minutes (Sambrook et al. 1989). Deionized water was used to scrape lysates for the electron microscopy studies of adsorption. The supernatant was filtered with 0.45pm pore sized filters (Millipore Corp, Bedford Mass.) to create a stock solution. Phage stocks were concentrated by a 10% PEG 8000 (polyethylene glycol) procedure (Sambrook et






33

al. 1989). Phage were plated and assayed as plaque-forming units (PFU/mL) using the standard agar overlay procedure (Snustad and Dean 1971). Poliovirus-1 LSc strain (ATCC VR-59) was assayed as PFU on BGM cells using an agar overlay method (Smith and Gerba 1982). Isolation of Bacteriophage Using Nitrocellulose Filters Procedure 1

A 10-mL filtered raw sewage sample with the addition of imidazole glycine

buffer to a final concentration of 0.02 M or the addition of buffer and NaCI, MgCI2, or Na3C6HsO7-2H20 to a final concentration of 0.01M was passed through a series of three 0.45 pm HA filters (Millipore) at pH 7. Viruses that adsorbed to the first in the series of three filters were eluted with 3% Beef Extract at pH 9 and isolated. Viruses that were not adsorbed after passage through three filters were also isolated. The samples were then plaque assayed using bacterial hosts previously mentioned according to standard procedure. Procedure 2

A 1 O-mL filtered raw sewage sample in imidazole glycine buffer to a final concentration of 0.02 M, or buffer with the addition of 0.01 % Tween 80 was passed through a series of three 0.45 pm HA filter (Millipore) at pH 3.5. Viruses that were not adsorbed after passage through three filters were isolated, and viruses that adsorbed to the last of the series of three filters were eluted with 3% Beef Extract at pH 9 and isolated. The samples were then plaque assayed using bacterial hosts previously mentioned according to standard procedure.






34


Isolation of Bacteriophage Resistant to High Temperature Initially, raw sewage was placed in a 400C or 500C water bath for 4 weeks, and assayed weekly. Bacteriophages were isolated after 4 weeks and plaque assayed using bacterial hosts previously mentioned according to standard procedure.

Selection of Bacteriophage for Additional Studies Procedure 1

The isolated bacteriophage at a 105 PFU/ mL concentration in 0.02 M

imidazole glycine buffer, or buffer with the addition of 0.1M MgCI2 was passed through a 0.45 micron HA filter (Millipore) at pH 7 to select phage based on hydrophobic adsorption properties. Bacteriophage were selected that adsorbed to the filter in buffer or buffer and salt solutions as predominately hydrophobic. Bacteriophages were also selected that did not adsorb to the filter in the presence of buffer and salt solutions as weakly hydrophobic. The samples were then plaque assayed using standard procedure. Procedure 2

Isolated bacteriophage at a 105PFU/I mL concentration in 0.02 M imidazole glycine buffer, or buffer with the addition of 0.1% Tween 80 was passed through a 0.45 gm HA filter (Millipore) at pH 3.5 to select bacteriophage based on electrostatic adsorption properties. Bacteriophages were selected that did adsorb to the filter in the presence of buffer and detergents as predominately electrostatic. Bacteriophages were also selected that did not adsorb to the filter in buffer solutions as weakly electrostatic. The samples were then plaque assayed using standard procedure.






35

Procedure 3

The isolated bacteriophage at a concentration of 10 PFU/ mL were placed in UV dechlorinated water and, incubated in a 40oC or 50'C water bath for extended time periods and assayed weekly. The absence of residual chlorine was ascertained by the addition of orthotoulidine (Clesceri et al. 1998). Characterization of Bacteriophage Adsorption to Filters under Varying
Conditions

Procedure 1

The selected isolates at a 105 PFU/mL concentration in 0.02M imidazole

glycine buffer at pH 7, with the addition of 0.001M MgCI2, 0.01M MgCI2, or 0.1M MgCI2 was passed through 0.45 p.m HA filter (Millipore). Elution of the filters was done with 3% Beef Extract at pH 9. The samples were then plaque assayed using standard procedure.

Procedure 2

The selected isolates at a 105 PFU/mL concentration in 0.02M imidazole

glycine buffer at pH 7 were passed through a 0.45 ptm 1 MDS filter. The filter was serial eluted with 10 mL 0.02M imidazole glycine buffer, and then varying concentrations of 0.001 M MgCI2, 0.01 M MgCI2, or 0.1M MMgCI2. The samples were then plaque assayed using standard procedure. Procedure 3

The selected isolates at a 105 PFU/ mL concentration in 0.02M imidazole glycine were also passed through a 0.45 plm HA filter (Millipore) at pH 3.5 and the filter was serial eluted with 10 mL 0.02M imidazole glycine buffer, and then varying concentrations of a neutral detergent, 0.001% Tween80, 0.01%






36


Tween80, and 0.1% Tween80. Final elution was with 3% Beef Extract or 0.1% Tween 80 and 1M MgCI2. The resultant samples were then plaque assayed as previously mentioned.

Bacteriophage Interactions to Particles Diatomaceous earth coating

Diatomaceous earth was coated according to a two-step procedure (Lukasik et al. 1996). Diatomaceous earth was mixed with 0.25M FeC13 for 30 minutes, drained and air-dried. Then the solid was soaked in 3M ammonium hydroxide for 10 minutes, rinsed and dried. The second part of coating consisted of soaking the solid in 0.5M AICI3 for 30 minutes, drained and dried, soaked in 3M ammonium hydroxide for 10 minutes, rinsed and dried. Adsorption procedure

A 0.1g sample of Argonide (Sanford, FL) aluminum hydroxide coated

nanoparticles (filter fibers) or coated diatomaceous earth (Lukasik et al. 1996) were added to a concentration of 106 PFU/ mL bacteriophage in deionized water, agitated on shaker (Reliable Scientific) for 15minutes, 30 minutes, and 1 hour. The samples were centrifuged at 1200xg (Dynac II centrifuge) for 15 minutes. A sample of the supernatant was plaque assayed using standard procedure. Electron microscopy procedure

A stock solution of 1011 PFU/ mL bacteriophage and 0.1g of coated filter

fibers were agitated on shaker (Reliable Scientific) for 1 hour. The samples were centrifuged at 1200xg (Dynac II centrifuge) for 15 minutes. A supernatant sample was removed and studied using electron microscopy (Ackermann and Nguyen 1983, Yanko et al. 1999). One drop of sample was placed on a 300 mesh






37

formvar coated grid for 1 minute. The excess was removed using filter paper. The grid was rinsed with two drops distilled water and the excess was removed using filter paper. Two drops of 1% Uranyl acetate was applied to the grid for 1 minute and the excess was removed using filter paper. The grid was air dried and examined on a Zeiss electron microscope operating at 80kV or 100 kV (Bozzola and Russell 1999).

Results

Isolation of Bacteriophage from Raw Sewage Samples

We used the standard method of adsorption-elution using negatively

charged microporous filters under the two conditions described in the Materials and Methods section to isolate indigenous bacteriophage from raw sewage. This procedure resulted in the isolation of 86 bacteriophages (Table A-I). We also isolated other phages based on their survival in raw sewage sample at elevated temperatures over a 4-week period.

1st set of conditions

At pH 7, viruses suspended in buffer or buffer and salts solutions that adsorbed to the filters were eluted with 3% beef extract at pH 9 and exhibit strong hydrophobic characteristics. Viruses from the resulting salt solutions that did not adsorb were isolated and considered to exhibit weak hydrophobic characteristics (Table 2-1). The data indicates that MgCl2 and Na3C6H507-2H20 enhance the adsorption of indigenous bacteriophage to the filters at a pH 7 by approximately 34%. Based on the T-test there is no significant difference between adsorption in the presence of MgCI2 and Na3C6H5O7-2H20 (sodium citrate) (Table 2-1).






38

2nd set of conditions

At pH 3.5, viruses in buffer solutions that did not adsorb to the filters were isolated and considered to exhibit weak electrostatic characteristics. Viruses in the Tween 80 solution that adsorbed to the third filter were eluted with 3% Beef Extract at pH 9 and considered to exhibit strong electrostatic characteristics (Table 2-1). The data indicates that adsorption of indigenous bacteriophage to the filters at pH 3.5 is influenced by both hydrophobic and electrostatic forces with phage adsorption at approximately 94% with a standard deviation of 2%. Although there is a decrease in adsorption with the addition of neutral detergent to approximately 54% with a standard deviation of 13%, a significant portion of indigenous phage remain adsorbed in the presence of a neutral detergent. Survival at elevated temperatures

The survival of indigenous phages in raw sewage was studied over a

period of four weeks. There was a steady significant decrease in the population of indigenous bacteriophage (using the hosts E. coli C3000, E. coli KC, S. typhimurium, and K. pneumoniae) at the elevated temperatures of 400C and 500C. The survival of phages at 40C indicates that a significant decrease in the phage population is due to the increase in temperature and not the composition of the raw sewage solution. At the end of the four-week period, phages were isolated as being more resistant to environmental factors (Figure 2-1). Selection of Bacteriophage

The isolated bacteriophages were then selected based on the adsorption characteristics, either strong or weak hydrophobic and either strong or weak electrostatic interactions. Adsorption of the phages 60KCK2 (80%) and 61KCK10





39


Table 2-1. Adsorption of bacteriophage in filtered raw sewage at pH 7 and
pH 3.5 a

SOLUTIONS % ADSORPTION b Buffer pH 7 61 +/- 5 Cc 0.01M NaCI pH 7 82 +/- 7 B 0.01M MgCI2 pH 7 97 +/- 3 A
0.01M Na3C6H507-2H20 pH 7 94 +/- 4 A Buffer pH 3.5 94 +/- 2 A 0.01% Tween 80 pH 3.5 56 +/- 13 B
a Prefiltered raw sewage with an initial phage concentration of approximately 5x105 PFU/mL was passed through a series of three 0.45pm microporous filters in various solutions. The hosts used include Escherichia coli 3000, Escherichia coli C, Salmonella typhimurium, Proteus vulgaris, Citrobacter freundii, Enterobacter aerogenes, and Klebsiella pneumoniae.
bAverage of adsorption to the three-0.45pm Millipore HA filters. oValues at the same pH followed by the same letter are not significantly different at P=0.05.

(82%) to 0.45pm HA millipore filters at pH 7 in 0.02M imidazole glycine buffer suggests strong hydrophobic interactions (Table 2-2). However, the minimal adsorption of the phage 60STP2 (46%) to the filters at pH 7 with the addition of

0.1M MgCI2 salts indicates weak hydrophobic interactions (Table 2-2). At pH 3.5, we found that hydrophobic and electrostatic interactions play a role in adsorption. At pH 3.5, the poor adsorption of the bacteriophage 60STP2 (3%) in the presence of 0.02M imidazole glycine buffer suggests weak electrostatic interactions (Table 2-2). The adsorption of the bacteriophage 61 KCK10 (54%) in the presence of 0.1% Tween 80, which disrupts hydrophobic interactions at pH

3.5 indicates predominate electrostatic interactions (Table 2-2). We selected bacteriophage that exhibited weak or strong hydrophobic characteristics at pH 7,





40


6
T
T T T



E4 U.

o I
3
0


2




0 1 2 3 4 TIME (Week)

""* APhage 40C -* *PHAGE 400C * PHAGE 50C


Figure 2-1. Raw sewage bacteriophage survival at 400C and 500C over time aBacteriophage in raw sewage at various temperatures were assayed, and isolated at 4 weeks using the hosts Escherichia coli 3000, Escherichia coli C, Salmonella typhimurium, Proteus vulgaris, Citrobacter freundii, Enterobacter aerogenes, and Klebsiella pneumoniae. and phage that exhibited weak or strong electrostatic characteristics at pH 3.5. Some bacteriophage such as 61STP7 and 61KCK10 were also selected based on their resemblance to poliovirusl adsorption properties (Table 2-2).

The isolated bacteriophage at a concentration of 106 PFU/mL were placed in UV dechlorinated water and, incubated in a 400C or 500C water bath for extended time periods. At 400C, all the isolated phage but 60STP2 had a 2





41

Table 2-2. Adsorption of selected bacteriophage to microporous filters at pH 7
and pH 3.5a


%ADSORPTION % ADSORPTION SOURCE OF OF VIRUSES AT OF VIRUSES AT PHAGE HOST PHAGE pH 7: pH 3.5:
Bufferb 0.1M Buff b 0.1% MgCI2 b u Tween 80b
E. ColiC3000 60C3F2 36+ 10 91 +6 93+ 10 33+8

E. Coli C 60KCK2 80 + 4 100 + 2 92 + 6 37 + 8
ISOLATED _FROM 61KCK9 31 + 10 91 +7 90+ 10 9+ 12 SEWAGE(Our 61KCK10 82+6 99+3 59+6 54+9
study) S.
Typhimurium 60STP2 0+3 46+6 3+2 3+6 61STP7 0+2 69+4 29+6 14+4 E. Coli C3000 MS2 18 + 8 75 + 8 100 + 8 27 + 5

CULTURE E. ColiC OX174 0+5 87+5 82+6 45+5 COLLECTION S.
Typhimurium PRD1 22+6 62+6 99+3 33+9
phimurium I I
BGM Cell line POLIO 1 0 + 3 72 + 3 98 + 4 60 + 12 aThe solutions containing 105 PFU/mL viruses were passed through 0.45 pm filter.
bAll solutions are in 0.02M imidazole-glycine at pH 7 or pH 3.5. log or less reduction over a 12-week time span indicating an insignificant reduction in the phage population (Figure 2-2). The laboratory cultures and 60STP2 had a more significant reduction in population over the six week time period (Figure 2-2). At 50�C, the results show the only phage not to show a significant reduction in population is 61KCK10 (Figure 2-3). The laboratory cultures and other selected phages showed a substantial decrease in numbers over the six week time period (Figure 2-3).






42


7 6-5









2
1U




0 4 8 12 TIME(Week)
- 60STP2 *M*61STP7 - 60KCK2 -*61KCKI0
SMS2 - PRD1 --K PHIX

Figure 2-2. Survival of bacteriophage at 400C in UV dechlorinated water aBacteriophage at a concentration of 106 PFU/mL were placed in tubes of 9 mL UV dechlorinated water and placed in 400C water bath. Characterization of Bacteriophage

The characterization of selected bacteriophages at a 105 PFU/mL

concentration were carried out using 0.45 pm HA millipore filters and 0.02M imidazole-glycine buffer and various concentrations of MgCI2 at pH 7. At neutral pH, the filter and bacteriophage exhibit negative surface charges. Due to the charges, bacteriophages tend to adsorb poorly in the presence of buffer. The results show that the previous results obtained in our study are accurate, the bacteriophages 60KCK2 and 61KCK10 adsorbed in buffer alone indicating strong hydrophobic interactions. The electrostatic repulsion decreases





43


7 T



25 u 4 03 02



0
0 2 4 6 TIME(Week)
U60STP2 in*****61STP7 - 60KCK2 *** 61KCK10
-C MS2 ** "PRD1 *** PHIX

Figure 2-3. Survival of bacteriophage at 500C in UV dechlorinated water aBacteriophage at a concentration of 106 PFU/mL were placed in tubes of 9 mL UV dechlorinated water and placed in 50�C water bath. as the ionic strength increases due to screening of the surfaces charges, which generally promotes adsorption. The bacteriophage 60STP2 adsorbed poorly in the presence of salts indicating that it exhibits weak hydrophobic interactions (Figure 2-4). Characterization of selected phages by adsorption to microporous filters were carried out using 0.45 pm HA millipore filters and subsequent rinses with 0.02M imidazole- glycine buffer and increasing concentrations of Tween 80 (a neutral detergent) at pH 3.5. At a low pH, the filter exhibits a negative charge and generally bacteriophages exhibit positive surface charges. Due to the charges, bacteriophages tend to adsorb to the filter in the presence of buffer at





44


100
90 80

z 70


o 50o
040

20
30 - Ol 61STP7
10
0
0.02 0.0225 0.045 0.27 Ionic Strength (MgCI2)
-1*+60KCK2 - 61KCK9 ~-- 61KCK10 -4-60STP2
-Z*61STP7 --) *MS2 wI 'POLIO


Figure 2-4. Bacteriophage adsorption to 0.45 pm HA nitrocellulose filters at pH 7
in the presence of MgCI2a
aAll solutions contain 105PFU/mL viruses in 0.02M imidazole glycine buffer and increasing concentrations of MgCI2 were passed through a 0.45 pm filter. All solutions were adjusted to pH 7. low pH due to electrostatic and hydrophobic charges. The bacteriophage 60STP2 adsorbed initially at 3 % and did not elute off with increasing detergent concentration indicating weak electrostatic interactions (Figure 2-5). As the concentration of Tween 80 increases; hydrophobic interactions are disrupted. The phage 61KCK10 adsorbed initially at 59 % and was not eluted from the filter in the presence of high Tween 80 concentrations. This indicates that electrostatic interactions predominate (Figure 2-5).





45


100
90 GOGTP2
80 1K9 61S 7


-0 o60KCK2 S5061KK0
= 40 30MS-2
UJ0 - - pVl


10


0% T 0.001%T 0.005%T 0.01%T 0.05% T 0.1%T %[TWEEN]
*4*'60KCK2 - 61KCK9 ""*61KCK10 -4--60STP2
***61STP7 - W* 'MS2 - POLIO Figure 2-5. Bacteriophage elution from 0.45 pm HA nitrocellulose filters at pH
3.5 in the presence of Tween 80a
aA concentration of 10 PFU/ mL bacteriophage in 0.02M imidazole glycine buffer was passed through a 0.45 pm filter and subsequently rinsed with 0.02M buffer and increasing concentrations of Tween 80. All solutions are in 0.02M imidazole glycine buffer and were adjusted to pH 3.5.

Further characterization of the selected bacteriophages was done using 1MDS electropositive filters at a neutral pH. At pH 7, bacteriophages exhibit negative surface charges and readily adsorb to 1MDS filters. Subsequent rinses with 0.02M imidazole glycine buffer and increasing concentrations of MgCI2 indicated that the antichaotropic agent, MgCI2 is capable of eluting adsorbed viruses. Based on the results, the bacteriophage 61KCK10 and poliovirus-1 exhibit predominate electrostatic interactions and the bacteriophage 60STP2 and 61STP7 exhibit weak electrostatic interactions (Figure 2-6).





46


100 1
60STP2-61 STP7 -"
90
80 61KCK9
60K 2
70
40 - ! F


30
20 - PV1
20 FMS-2
10


0.020 0.0225 0.033 0.045 0.270 Ionic Strength (MgCI2)

'M60STP2 - 61STP7 "---60KCK2 - 61KCK9
*61KCK10 - * 'MS2 - "POLIO 1

Figure 2-6. Bacteriophage elution from 0.45 pm 1MDS charged modified filters
at pH 7 in the presence of MgCI2a
aAll solutions are in 0.02M imidazole glycine buffer and were adjusted to pH 7. A concentration of 105PFU/ mL bacteriophage in 0.02M imidazole glycine buffer was passed through a 0.45 pm filter and subsequently rinsed with 0.02M buffer and increasing concentrations of MgCI2.

The diatomaceous earth was modified with a mixture of 0.25MFeCI3 and 0.5M AICI3 and rinsed with 3N ammonium hydroxide to give a metallic hydroxide coating (Lukasik et al. 1996). The modified nanosized filter fibers from Argonide (Sanford, FL) have an aluminum hydroxide coating. Batch adsorption results show that most adsorption occurs within the first 15 minutes (Table 2-3). The bacteriophage MS2 adsorbs poorly to unmodified diatomaceous earth, and over 80% to the other modified materials (Table2-3). The phage 60STP2 adsorbed poorly to all the materials (Table 2-3). The electron micrograph of MS2 to the






47


Table 2-3. Adsorption of bacteriophage to defined media ADSORPTION TO DEFINED MEDIA Percent Adsorption
MEDIA PHAGE 15min. 30min. 60min.
MS2 0 10 27 Diatomaceous Earth
60STP2 0 0 2 Modified Diatomaceous Earth MS2 87 93 100 60STP2 0 13 32 Modified Filter Fibers MS2 81 92 99 60STP2 27 38 44
aA 0.lg sample of diatomaceous earth, modified diatomaceous earth, or coated filter fibers (Argonide) was suspended in deionized water with an initial concentration of 106 PFU/mL bacteriophage and mixed for 15, 30, or 60minutes. Argonide coated filter fibers show that the MS2 phage adsorbs to the material (Figure 2-7). The electron micrograph of 60STP2 to the Argonide coated filter fibers indicates minimal adsorption to the coated filter fibers by the head (capsid) portion of the phage at 50K (125,000x) (Figure 2-8).

Discussion

Bacteriophages were isolated using membrane adsorption-elution filtration methodology (Farrah et al. 1976, Rao and Labzoffsky 1969, Wallis and Melnick 1967). The results have shown that bacteriophages can be isolated, selected, and characterized based on their adsorption properties. It is thought that the different types of viruses adsorb differently due to, the differences in the surface proteins of the capsid (Hatefi and Hanstein 1969, Shields and Farrah 2002). Viruses contain a protein coat therefore they may exhibit charged or hydrophobic groups on the surface, and thus factors such as pH, ionic strength, and chaotropic agents have been shown to affect adsorption to solids. By using






48








MS












100nm


Figure 2-7. MS-2 adsorption to nanosized filter fibersa aA stock solution of 1011 PFU/mL bacteriophage and 0.lg of coated filter fibers were agitated for 1 hour in deionized water, centrifuged, and a sample of supernatant was removed and studied using electron microscopy. Electron micrographs were taken at 50K (125,000x). defined media we were able to alter the properties of the solution to change viral characteristics. At pH 7, nitrocellulose filters exhibit a negative charge and viruses exhibit a negative charge so adsorption by electrostatic interactions should be minimal. We were able to show the extent of viral hydrophobic interactions by increasing the concentration of an antichaotropic agent such as MgCI2. Antichaotropic agents have been shown to disrupt electrostatic interactions by screening the charges, which would promote hydrophobic and van der Waals interactions (Farrah et al. 1981). Our results show that there was






49





* V










100nm











Figure 2-8. 60STP2 adsorption to nanosized filter fibers aA stock solution of 1011 PFU/mL bacteriophage and 0.1g of coated filter fibers were agitated for 1 hour in deionized water, centrifuged, and a sample of supernatant was removed and studied using electron microscopy. Electron micrographs were taken at 50K (125,000x). a significant difference in adsorption of bacteriophage by filters in the presence of monovalent and multivalent salts are in agreement with previous studies (Lukasik et al. 2000). However, the results also show that there was no significant difference in adsorption of bacteriophage by filters in the presence of divalent or trivalent salts, which is in opposition to previous reports (Gerba 1984b, Mix 1974, Wallis et al. 1972, Wallis et al. 1979). At pH 3.5, nitrocellulose filters exhibit a negative charge and viruses exhibit a positive charge therefore electrostatic and hydrophobic interactions are possible. We were able to show the extent of viral





50

electrostatic interactions by increasing the concentration of a neutral detergent such as Tween80. The addition of a chaotropic agent or neutral detergent promote elution of phages by making the solution more lipophilic which allows for the interactions of apolar groups and the solution (Farrah et al. 1981, Farrah 1982). Our results show that Tween 80 (a neutral detergent) does promote elution of viruses to an extent by disrupting hydrophobic interactions. However, the amount of elution is dependent on the viral properties. Further elution can be accomplished by using a combination of a chaotropic and antichaotropic agents, or a protein solution such as beef extract, which disrupts both electrostatic and hydrophobic interactions.

Adsorption of select viruses such as MS2, a predominantly hydrophobic bacteriophage to modified defined media has been previously studied (Bales et al. 1993, Farrah et al. 1991, Lukasik et al. 1996, Lukasik et al. 1999, Penrod et al. 1996, Shields and Farrah 2002, Zerda et al. 1985). In the current study we used hydroxide coated diatomaceous earth and filter fibers to examine the difference in adsorption between MS2 a frequently used model, and a phage 60STP2. Our studies were in agreement with previous studies that MS2 adsorbed well to the modified material (Farrah et al. 1991, Lukasik et al. 1996, Lukasik et al. 1999). However, our studies showed that the phage 60STP2 adsorbed poorly to these modified materials. When the phage 60STP2 did adsorb it was by the head (protein capsid).

Environmental influences have also been shown to influence adsorption and survival. These factors include; dissolved organic compounds (Sobsey and






51

Hickey 1985b), suspended solids (Sobsey and Cromeans 1985a), temperature, and biological predators. The raw sewage used for isolation based on survival at elevated temperatures was not prefiltered to remove debris. However, our results of bacteriophage survival at 4�C, 40"C, and 50�C show that the decrease in the phage population was primarily due to an increase in temperature and not the composition of the raw sewage samples.

The results show that bacteriophage can be selected as models for

human pathogenic viruses based on their adsorption characteristics. We have isolated a range of bacteriophage that can be used as models for other enteric organisms for various applications based on their adsorption and survival properties. The results show that understanding the properties of the virus, solid, and environment are important parameters for identifying the mechanism of adsorption.














CHAPTER 3
VIRAL INTERACTIONS WITH NATURAL MEDIA Introduction

The presence of enteric viruses in groundwater has been documented (Table 3-1) (Griffin et al. 1999, Keswick and Gerba 1980, Vaughn et al. 1978, Yates et al. 1985). As previously stated contamination of groundwater occurs through a variety of sources including septic tanks, landfills, artificial recharge, inefficient water treatment processes, polluted surface waters, runoff, and drainfields (Yates et al. 1985). Viruses have been known to travel several meters in a variety of systems (Bales et al. 1989, Schaub and Sorber 1977, Skilton and Wheeler 1988, Vaughn et al. 1978, Vaughn et al. 1983, Yanko et al. 1999). Since water treatment processes do not completely remove enteric viruses; understanding the adsorption properties of the soil system is important. Table 3-1. Detection of viruses in groundwater

ENTERIC
TYPES References VIRUSES
Polio 1,2,3 Griffin et al. 1999, Vaughn et al. 1978
Coxsackie A16, B3, B4 Griffin et al. 1999, Vaughn et al. 1978
Echo 6, 7,11,12,21,23,24, Griffin et al. 1999, Vaughn et al. 1978 25
Hepatitis A Griffin et al. 1999
Norwalk Griffin et al. 1999 Unknown Vaughn et al. 1978





52






53

Adsorption

Viral adsorption in natural media has been well studied (Bales et al. 1989, Bitton 1975, Bitton 1980, Dizer et al. 1984, Dowd et al. 1998, Fuhs 1987, Funderburg et al.1981, Gerba et al. 1975, Gerba et al. 1981, Gerba 1984b, Goyal and Gerba 1979, Ketratanakul et al. 1991, Lance and Gerba 1984b, Powelson et al. 1991, Powelson and Gerba 1994, Schaub and Sorber 1977, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995). The behavior of bacteriophage adsorption to natural media such as mixed-liquor suspended solids and soils are complex. The results show that adsorption to natural media seems to be due to various forces such as van der Waals forces, hydrophobic, and electrostatic interactions (Bitton 1975, Gerba 1984b, Dizer et al. 1984, Dowd et al. 1998, Lance and Gerba 1984a, Powelson et al. 1991, and Taylor et al. 1981).

At a neutral pH, soils generally have a net negative charge and many

viruses have a net negative charge. Numerous studies have shown that there is less adsorption at higher pH values (Burge and Enkiri 1978, Sobsey et al. 1980a, Taylor et al. 1981). This can be explained according to the DLVO theory that at a higher pH there is an increased electrostatic repulsion between the solid and virus particles. Research has shown that the addition of antichaotropic agents such as MgSO4 promotes adsorption primarily by strengthening hydrophobic interactions (Farrah 1982, Hatefi and Hanstein 1969). According to the DLVO theory an increase in ionic strength compresses the double layer causing adsorption by van der Waals and hydrophobic forces. Hydrophobic interactions were studied using octyl sepharose columns, which contain hydrophobic groups with varying concentrations of NaCl (Shields and Farrah 2002). As the






54

concentration of salts decrease the double layer is less compressed and allows for electrostatic repulsion. This allows viruses with the least hydrophobicity to be eluted first (Shields and Farrah 2002). It has also been found in various studies that chaotropic agents, urea, ethanol, and neutral detergents tend to solubilize hydrophobic molecules and disrupt hydrophobic interactions (Farrah et al. 1981, Hatefi and Hanstein 1969). However, it is also important to note that natural media are heterogeneous surfaces composed of patches of positive charges on negatively charged particles. These sites are often composed of oxides that carry a positive charge and can adsorb negatively charged viruses at near neutral pH.

The activation thermodynamics of viral adsorption to various filter media have also been studied (Preston and Farrah 1988a). They studied kinetics as a function of temperature, and the results indicated that adsorption was a physical process and describes the initial stages of adsorption to microporous filters (Preston and Farrah 1988a). They also have shown that correlation between the percent of viral adsorption and the energy of activation were dependent on the solids, and that these differences may influence the mechanism of adsorption (Preston and Farrah 1988a).

Tracers

Theoretically under certain conditions groundwater contamination is possible by viral transport through the soils. Many investigators have studied transport by using tracers (Tables 1-2 and 1-3) (Bales et al. 1989, Gerba 1984a, Griffin et al. 1999, Harvey 1997, Paul et al. 1995, Paul et al. 1997, Schaub and Sorber 1977, Skilton and Wheeler 1988, Vaughn et al. 1978, Vaughn et al. 1983, Yanko et al. 1999). Bacteriophage tracers are frequently used in place of enteric






55

viruses since they are easier and safer to work with. Models that have been used frequently include MS-2, PRD-1, other F+ specific bacteriophage, and QX174 (Schijven 2000). F+ specific phage such as MS-2, and PRD-1 are hydrophobic bacteriophage that do not adsorb well to solids and are relatively stable are used as worst-case models for possible contamination (Goyal and Gerba 1979, Havelaar et al. 1993, Schijven 2000). Biological tracers can better predict colloidal transport in a system than solutes (Bales et al. 1989). The prediction of the adsorption, survival and subsequent elution of colloidal virus particles is improved by using biological tracers consisting of similar properties. Survival

Considering viral contamination is possible, understanding the survival mechanisms of viruses in groundwater systems is of importance. There are numerous factors involved with the inactivation of viruses in the environment; such as temperature, ionic strength, moisture content, pH, soil type, sunlight, and biological predators as previously stated (Table 1-5) (Gerba et al. 1991, Hurst et al. 1980a, Hurst et al. 1980b, Powelson and Gerba 1994, Schaub and Sorber 1977, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985, Yeager and O'Brien 1979). At low temperatures, viruses survive for a longer period of time than at higher temperatures. Other components such as detergents, and pH changes can affect the stability of the viruses (Ward and Ashley 1979). It is important to note that inactivation is generally negligible within the time scale of the






56


experiments. However for field experiments viral inactivation may be significant, especially under some conditions such as unsaturated, low flow rate, high adsorption.

Materials and Methods

Media

Samples of mixed-liquor suspended solids were obtained from the University of Florida Wastewater Reclamation Facility in Gainesville, Florida. Soils were obtained from the University of Florida Natural Area Teaching Lab (NATL), Gainesville, Florida. Soil cores were obtained from Joan Rose at the University of South Florida Tampa, Florida. The soils have been previously characterized and marked (Table 3-2).

Solutions

The buffer solution used in our study was 0.02M imidazole and 0.02M glycine from Sigma Co. made in deionized water. The solutions used in our study were as previously described.

Viruses

The following phages with their hosts were used in these studies: MS2 (ATCC 15597-B1) assayed using Escherichia col 3000 (ATCC15597), PRD1 assayed using Salmonella typhimurium (ATCC 19585), QX174 (13706-B1) assayed using Escherichia col C (ATCC 13706). Bacterial viruses isolated in our study and their hosts are: 60C3F2 assayed using Escherichia coli 3000 (ATCC15597), 60KCK2, 61KCK9, and 61KCK10 assayed using Escherichia coli C (ATCC 13706), 60STP2 and 61 STP7 assayed using Salmonella typhimurium (ATCC 19585).






57

Table 3-2. Soil characteristics

TYPE Composition pH Depth Comments

1 Silty sandy 6.23 53cm upland pine sandy UF NATL
2 Silty sand 7.0 54cm hammock, UF NATL
organics
3 Clayey silty sand 6.1 60cm Sandy Hammock, UF
organics NATL
4 Clayey silty sand 6.32 57cm hammock forest, UF NATL
organics
17-19ft
5" Silty pale orange 6.8 Top
to yellow brown 17-19ft
6a sand 5.41
Bottom
28-30ft
7a 6.10 2830ft
Fine grain clayey Top Upper clay- plastic, stiff to
sand 28-30ft medium stiff
8a 5.72
Bottom
S 4.16 42-44ft
9" 4.16
Soft to medium Top Lower clay- Primary
10a stiff sandy clay 4.77 42-44ft confining unit Bottom
a Consists of 3 undisturbed (Shelby tube) soil samples from boring SB-27 at depths of 17 ft. bls, 28 ft. bls, 42ft. bls (bls-below land surface) These core were
2 ft. in length that were divided in half (a top half and a bottom half).

Phage stocks were prepared and assayed as previously described.

Preparation of phage stocks for groundwater and survival studies was done

using deionized water to avoid nutrient contamination.


Adsorption of Viruses to Mixed-Liquor Suspended Solids

Procedure 1

Concentrations of 106 PFU/ mL of selected bacteriophages, or poliovirusl

were placed in mixed-liquor suspended solids to a final volume of 50mL. The

suspension was agitated on a shaker (Reliable Scientific) at low speed for 1, 5,






58

15, 30, or 60 minutes at room temperature, centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed. Samples were then plaque assayed according to standard procedure. Procedure 2

Approximately 106 PFU/mL phage were added to biosolids to a final

volume of 50mL. The pH was adjusted with HCI or NaOH solutions to pH 3.5 or pH 7. The suspension was agitated on a shaker (Reliable Scientific) at low speed for sixty minutes, centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed. The supernatant was removed and the solids left were then serial treated at low speed for 10 minutes with 0.1% Tween 80, 0.1% Tween 80 and 1M MgCI2, and 10% Beef extract pH 7. The samples were centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed for each elution. The samples were then plaque assayed according to standard procedure. Adsorption of Viruses to Soils

Procedure I

A two gram sample of material was sieved to 25 mesh, shaken (Reliable Scientific) for 30 minutes with 2 mL deionized water to breakup aggregates and stabilize the temperature. The sample was centrifuged at 1200xg for 10 minutes (Dynac II centrifuge) and the solution removed. Then a 2 mL solution of deionized water; which contains 106 PFU/ mL bacteriophage or virus, was added. The pH was taken, then the suspension was agitated (Reliable Scientific) at low speed for thirty minutes, centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed. The samples were then plaque assayed.





59

Procedure 2

A two gram sample of material sieved to 25 mesh, was added to deionized water and shaken (Reliable Scientific) for 30 minutes to breakup aggregates and stabilize the temperature. The sample was centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes and the solution removed. Then a 2 mL solution of deionized water; which contains 106 PFU/mL bacteriophage, or polio 1 virus, was added. The pH was adjusted with HCI or NaOH solutions to pH 3.5 or 7. Then the suspension was agitated at low speed for sixty minutes, centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes, and a sample of supernatant was removed. The samples were then serial treated at low speed on a shaker (Reliable Scientific) for 10 minutes with 0.1% Tween 80, 0.1% Tween 80 and 1M MgCI2, and 10% Beef extract pH 7. The samples were centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes, and a sample of supernatant was removed for each elution. The samples were then plaque assayed. Kinetic Adsorption Studies

The adsorption of selected bacteriophage to natural media was examined to identify adsorption rates at varying temperatures. Approximately 106 PFU/ mL phage were added to mixed-liquor suspended solids at a final volume of 50mL, a

2 mL solution of deionized water and 2 grams soil, or a 10 mL solution of deionized water at various temperatures of 4�C, 25�C, and 370C. The pH was taken, then the suspension was agitated at low speed on a shaker (Reliable Scientific) for specified times, centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes, and a sample of supernatant was removed and plaque assayed.






60

Column Adsorption Experiments

Miniature columns

One set of column soil adsorption experiments were carried out using columns of 2.5 cm diameter X 13 cm length in a 60 mL syringe with the tip packed with glass beads and poly propylene fibers at room temperature (Figure 3-1). Dry soil was sieved with a 25 mesh sieve (710 pm), made into a slurry and poured into columns at a depth of 10 cm and stirred to prevent layering and stabilized. The packed columns were rinsed with 5 equal bed volumes of deionized water at pH 7 to reach equilibrium. An initial sample was taken and a solution of deionized water containing 106 PFU/mL bacteriophage or polio 1 virus was passed through the column by gravity flow. The effluent samples were collected until 4 pore volumes had passed and plaque assayed. Large columns

Another set of column soil adsorption experiments were carried out using larger columns of 5 cm diameter X 30 cm length in PVC pipe with screen and poly propylene fibers at room temperature (Figure 3-1). Dry soil was sieved with a 25 mesh sieve (710 pm), made into a slurry and poured into columns at a depth of 20 cm and stirred to prevent layering and stabilized. The packed columns were rinsed with 10 equal bed volumes of deionized water at pH 7 until at equilibrium. An initial sample was taken and then a solution of deionized water containing 106 PFU/mL bacteriophage or virus was passed through the column by gravity flow. The effluent samples were collected until 8 pore volumes had passed and plaque assayed.






61

A. B.


























Figure 3-1. Soil Column Setup
A. The miniature column 2.5 cm diameter X 13 cm length on the left is packed at the tip with polypropylene fibers and glass beads. The column has a soil depth of 10 cm. B. The large column 5 cm diameter X 30 cm length PVC pipe with screen and polypropylene on the right. The column has a soil depth of 20 cm. Groundwater Tracer Studies

Six observation wells were augured to reach groundwater and into the saturated zone for sampling (Table 3-3). The wells consist of 5ft. PVC pipe (2") coupled with a 2" coupler to a 3ft. screen (2") with tip for flow through. Well water samples were taken with a bailer, which has a one way flow valve was used in sampling. Background samples were taken and plaque assayed at 0.1 mL, and 1mL with






62


Table 3-3. Well Characteristics

Wells Distance From Septic Systema Depth pH
1 10ft 6ft. 9in. 5.51 2 12 ft 7ft. lin. 6.39 3 27 ft 6ft. 8 in. 5.74 4 49 ft 7ft. 5.64 5 71 ft 6ft. 11in. 5.6 6 73 ft 6ft. 9in. 5.82 aDistance from septic tank around the soil adsorption field. the appropriate hosts. Then 100mL 109 PFU/mL 60STP2, and 100mL 1011 PFU/mL MS2 were flushed down (poured into) the septic system. Well water samples were taken every 4-8 hours. The samples were pooled as a composite of each day for initial readings and plaque assayed. Plaque assays of well water samples were corrected for background and individual samples were assayed further if counts in the composite were high. Wells 1 and 3 were used for a repeat (second) study using the same techniques Groundwater Survival Studies

The groundwater characteristics were measured and shown in Table 3-4. Then duplicate samples of 10 mL deionized water with 106 PFU/mL 60STP2, and 10 mL deionized water with 106 PFU/mL MS2 were placed in polypropylene tubes in monitoring wells 1 and 3. Initial samples were taken and plaque assayed. Then samples were taken every 2 days and plaque assayed.






63


Table 3-4. Weather conditions

Groundwater Water Temp. Rain inches Max. Temp.a Min. Temp.a
Characteristics (oC) b (oC) (oC) DAYS OF EXPERIMENTS
3/9/2003 21 1.55 19.4 14.4 3/10/2003 22 0 26.1 12.8 3/11/2003 22 0 26.7 11.7 3/12/2003 22 0 26.1 12.8 3/13/2003 22 1.27 26.7 15.6 3/14/2003 22 T 23.9 15 3/15/2003 22 0.05 17.8 13.9 3/16/2003 22 0 18.9 13.9 3/23/2003 22 T 22.8 14.4 3/24/2003 22 0 25 11.1 3/25/2003 22 0 24.4 9.4 3/26/2003 22 0 27.2 13.3 3/27/2003 22 0 22.8 17.2 3/28/2003 22 0.33 26.1 15.6 3/29/2003 22 0 28.3 13.3 3/30/2003 22 0.20 19.4 8.3 3/31/2003 21 0 14.4 2.8 4/1/2003 21 0 20.6 1.7 4/2/2003 21 0 23.9 5.6 4/3/2003 21 0 25.6 8.3 4/4/2003 22 0 27.8 13.3 4/5/2003 22 0 28.9 13.9 4/6/2003 22 0 29.4 16.1 aObtained from the NOAA NWS Jacksonville, FL (www.srh.noaa.gov/iax) bMeasured experimentally and is an average of the 6 wells

Results

Batch Adsorption Studies

The results of the batch adsorption studies using model viruses shows

that significant adsorption with various bacteriophage occur over an extended

period of time (Figure 3-2). The results of thirty minute batch adsorption of

selected bacteriophage to mixed-liquor suspended solids at pH 7 show that the

phage 60KCK2 (91%) and 61KCK10 (85%) show significant adsorption while the






64


100
90

0
6o
0

0 40
30
20 -10
0
1 5 15 30 45 60 TIME (MIN.)

-4-MS2 UPRD" -A, PHIX PV Figure 3-2. Adsorption of model bacteriophage to mixed-liquor suspended
solidsa
aThe experiments were carried out mixing 106 PFU/mL bacteriophage in 50mL mixed-liquor suspended solids at pH 7 for specified times on shaker (Reliable Scientific).

phage 60STP2 (0%) has poor adsorption (Table 3-5). The frequently used model phages MS-2, PRD-1, poliovirus-1 and the select phage 61STP7 adsorbed to the mixed-liquor suspended solids (Table 3-5). The thirty minute batch adsorption results of selected bacteriophages to the ten soils, four from NATL Natural Area Teaching Lab at UF Gainesville, FL and six obtained from J. Rose at USF Tampa, FL at pH 7 show that the bacteriophage 60KCK2, 61KCK10, X174 adsorbed well and the phage 60STP2 had the poorest adsorption to all the soils (Tables 3-6 and 3-7). There was a significant increase in adsorption as the depth of the soil samples increased, which was due to the






65

Table 3-5. Adsorption to mixed-liquor suspended solids by select bacteriophagea

PERCENT ADSORPTION TO MIXED LIQUOR SUSPENDED SOLIDS
BACTERIOPHAGE % ADSORPTION
60STP2 0+0
61STP7 49 + 2 Db 60KCK2 91 +3A 61KCK9 89 + 1 A 61KCK10 85 + 2B
MS2 63 + 6 C PRD1 56 + 5 C 4X174 92 + 7 A POLIO 60 + 6 C
aThe experiments were carried out mixing 105 PFU/mL bacteriophage in 50mL mixed-liquor suspended solids at pH 7 for thirty minutes. bValues followed by the same letter are not significantly different at P=0.05. increase in the content of clay. The batch adsorption experiments for 60 minutes of MS2 and 60STP2 at pH 3.5 and pH 7 using mixed-liquor suspended solids or soils show that at pH 3.5 the bacteriophage MS-2 adsorbs well to all media, and the phage 60STP2 adsorbs poorly except to the mixed-liquor suspended solids (Table 3-8). At pH 7, the phage 60STP2 adsorbs poorly and MS-2 adsorption varies depending on the media (Table 3-8). The detergent disrupts hydrophobic interactions and elutes 60STP2 from most media except sludge (Table 3-8). The detergent solutions elute MS-2 from the forest soil samples at pH 3.5 and 7 (Table 3-8). There is some elution of MS-2 from other media with the neutral detergent. Solutions of 1% Tween 80 detergent and 1M MgCl2 eluted MS-2 from most media types (Table 3-8). However MS-2 remains adsorbed to sandy soil at pH 3.5, clay soils at pH 3.5 and 7, and mixed-liquor suspended solids at pH 3.5 and 7 (Table 3-8). The phage 60STP2 is significantly eluted from most materials






66

Table 3-6. Adsorption to natural area teaching lab soils by select bacteriophage


Phage PERCENT ADSORPTION TO NATLb SOILS

SANDY FOREST SANDY-CLAY FOREST-CLAY

60KCK2 51 + 3 Bc 70+3 B 55+4 B 48+7 B 61KCK10 33+7C 73+8B 35+4D 52+5B
60STP2 0+0 0+0 0+0 0+0
61STP7 30 + 4 C 27 + 5 D 23 + 7 E 27 + 2 D

MS-2 5+9D 36+3D 22+3E 0+0

PRD1 70 + 3A 57+4 C 42+2 C 61 + 5A OX174 70 + 6 A 84 + 6 A 70 + 7 A 64 + 8 A Polio 0 + 6 E 24 + 4 D 49 + 3 B 35 + 2 C
aThe experiments were carried out mixing 106 PFU/mL bacteriophage in 2mL deionized water and 2 gram soil solution at pH 7 for thirty minutes. bNATL- Natural Area Teaching Lab at the University of Florida Gainesville, FL cValues in the same column followed by the same letter are not significantly different at P=0.05.

using a detergent. However, the phage was eluted from all other media using a solution containing 1% Tween 80 detergent and 1M MgCI2 (Table 3-8). The results indicate that most of the interactions are due to hydrophobic and electrostatic interactions.

The results of the batch adsorption experiments show that an expected trend of adsorption occurs based on the viral adsorption properties the phages were selected for. Variation in the adsorption pattern are most likely due to the effects of interfering particles such as organics and particulates in the media such as clays and oxides. The factors found to be significant in the adsorption






67

Table 3-7. Adsorption to soils by select bacteriophagea


PERCENT ADSORPTION TO SOILSb PHAGE

5_ 6c 7c 8c 9C 10C 60KCK2 97+3 92+3 100+2 99+3 100+0 100+0 61KCK10 87+6 99+1 99+1 99+0 100+0 100+0 60STP2 32 + 2 17 + 4 93 + 9 77 + 1 95 + 1 95 + 2 61STP7 0+0 69+5 93+7 48+2 100+0 100+0
MS-2 60+9 82+3 74+2 76+1 99+1 99+1 PRD-1 45 + 4 34 + 2 44 + 5 69 + 6 100 + 0 100 + 0 4X174 99+1 100+0 100+0 98+1 100+0 100+0 Polio 49 + 4 66 + 6 56 + 5 54 + 4 94 + 0 75 + 3 aThe experiments were carried out mixing 10" PFU/mL bacteriophage in 2mL deionized water and 2 gram soil solution at pH 7 for thirty minutes. bJoan Rose, University of South Florida, Tampa, FL c See Table 3-2 for specific soil characteristics of the samples. There is an increase in clay content as the sample number increases. phenomena of the batch adsorption experiments are time (Figure 3-2), viral adsorption properties (Figure 3-2 and Tables 3-5, 3-6, 3-7, 3-8), composition of the solutions (Table 3-8), pH (Table 3-8), and composition of the solids (Tables 3-5, 3-6, 3-7, 3-8) are significant factors in the adsorption phenomena. Kinetic Adsorption Studies

The kinetics of adsorption of the selected bacteriophage to natural media was determined as a function of temperature to study the thermodynamic parameters. The percent of adsorption reported here is at 2 hours, the maximum amount of time we allowed for adsorption to occur (Table 3-9). At 4�C, MS-2





68

Table 3-8. Adsorption to natural media at pH 7 and pH 3.5 by select phagea

PERCENT ADSORPTION TO NATURAL MEDIA

BACTERIOPHAGE SANDY FOREST SOIL CLAY SOIL MLSSb SOIL
pH 3.5 7 3.5 7 3.5 7 3.5 7

MS2-Buffer 100 73 5 21 100 100 95 21 60STP2-Buffer 1 62 32 35 27 35 86 27 MS-2-Tween 79 1 0 0 79 74 89 21 60STP2-Tween 0 20 0 0 0 3 79 27 MS-2-Tween&MgCI 59 0 0 0 76 47 24 13 60STP2-Tween&
TP2T n 0 0 0 0 0 0 0 0 aThe experiments were carried out mixing 10" PFU/mL concentration of bacteriophage in 50 mL mixed-liquor suspended solids or 2mL deionized water and 2 gram soil solutions at pH 3.5 or 7 for sixty minutes. bMLSS is an abbreviation for mixed liquor suspended solids. (87%) adsorbed well and the phage 60STP2 (58%) adsorbed to the mixed-liquor suspended solids media (Figure 3-3). However, there was a significant difference in adsorption to the forest soil. The bacteriophage MS-2 (72%) adsorbed well but the phage 60STP2 adsorbed minimally (2%)(Figure 3-3). At 25�C, MS-2 (91%) and 60STP2 (64%) phages adsorbed to the sludge, MS-2 (75%) adsorbed to the forest soil, but the phage 60STP2 (7%) adsorbed poorly to forest soils (Figure 34). At 37�C, MS-2 adsorbed to the mixed-liquor suspended solids and soils at 99% and 97% respectively (Figure 3-5). There was increased adsorption of MS-2 to the forest soil. The bacteriophage 60STP2 adsorbed well to sludge (99%) and there was increased adsorption to the forest soils (40%) (Figure 3-5). The Arrhenius plot uses the adsorption rate constant (-k) versus the inverse of





69

Table 3-9. Adsorption of bacteriophage to natural mediaa

PERCENT ADSORPTION TO NATURAL MEDIA AT VARIOUS TEMPERATURE

TEMPERATURE
C NATURAL MEDIA MS-2 60STP2

4 MLSSb 87 + 2 Ac 58 + 7 B 4 Forest soil 63 + 6 A 2 + 2 B 25 MLSS 91 + 4 A 64 + 9 B 25 Forest soil 64 + 8 A 5 + 5 B 37 MLSSa 99 + 2 A 99 + 0 A 37 Forest soil 89 + 3 A 36 + 5 B aA 10 PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL, or a 2 mL solution of deionized water and 2grams soil at various temperatures and agitated for 2 hours. bMLSS is an abbreviation for mixed liquor suspended solids. c/alues in the same row followed by the same letter are not significantly different at P=0.05.

temperature in Kelvin. The adsorption rate constant is the slope of the rate constant plot, which uses the natural log of viruses in the supernatant versus time in seconds (Figures 3-3, 3-4, 3-5). Activation thermodynamic comparisons show that phage adsorption to natural medias at various temperatures is a physical process with a low energy of activation of <40 Kcal/mol (Table 3-10). The results also suggest that adsorption of the phage 60STP2 is minimal to the forest soil. The Arrhenius plot of phage to natural media shows that 60STP2 adsorbed poorer to natural media than the model MS-2 (Figure 3-6).






70


100
90 80
Z 70
- 60 s
0 50
0 40
S30
20
10
0
0 5 10 15 30 60 120 TIME (min.)

--- Sludge MS-2 ----Sludge 60STP2
- I Soil MS-2 C Soil 60STP2

Figure 3-3. Adsorption at 40C of select bacteriophage to natural media a aA 106 PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL or 2mL solution of deionized water and forest soil at 4�C and agitated for 2 hours. Column Adsorption Experiments

The experiments were carried out at room temperature using 10 cm soil depth x 2.5 cm diameter in a 60 cm syringe or a 20 cm soil depth x 5 cm diameter in a 50 cm PVC column with gravitational flow. Breakthrough plot is concentration supernatant/ initial concentration versus time. Effluent samples were collected, and assayed. The results show that a significant difference in soil column breakthrough requires an optimal distance for adsorption and soil retardation factors to affect the curves. At 10 cm soil depth, there is a minimal






71


100
90

80
z 70

0
O 50
S40


2 0 -,
0


0 5 10 15 30 60 120 TIME (min.)
*-Sludge MS-2 --Sludge 60STP2
- 'Soil MS-2 - Soil 60STP2Figure 3-4. Adsorption at 250C of select bacteriophage to natural media a aA 106 PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL or 2mL solution of deionized water and forest soil at 25oC and agitated for 2 hours. difference in the passage of column effluent between MS2 and 60STP2 (Figures 3-7 and 3-8). The bacteriophage 60KCK2 adsorbed well to both soils but was found in the column effluent of sandy soil after time (Figures 3-7 and 3-8). However, if the distance is increased to a 20 cm soil depth there is a significant difference in breakthrough, possibly due to an increase in length that increases contact time and binding sites. The phage 60STP2 had a significant breakthrough as compared to MS-2 (Figure 3-9). In sandy soil columns (5 cm diameter X 37 cm depth) with a flow rate of 1.5mL/min., both phages were found






72


100
90 80
z 70
0
o s
60


o 40 ,
30 -
20
10 4
20


0 5 10 15 30 60 120 TIME (min.)

-* Sludge MS-2 * *Sludge 60STP2 3- W'Soil MS-2 ' Soil 60STP2



Figure 3-5. Adsorption at 37oC of select bacteriophage to natural media aA 106 PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL or 2mL solution of deionized water and forest soil at 370C and agitated for 2 hours. in column effluent in 15 minutes (Figure 3-10). In forest soil columns with a flow rate of 0.98 mL/min the results show that approximately a 60% of phage 60STP2 were found in the column effluent in 1 and 1/2 hours, whereas 2 and 1/2 hours were required for significant numbers of MS-2 to be found in the column effluent (Figure 3-11). In a column of forest soil (5 cm diameter X 35 cm depth) with a flow rate of 1.2 mL/min. approximately 50% of the phage 60STP2 was found in the column effluent in an hour, whereas only 10% of MS-2 was found in the effluent in 2 hours (Figure 3-12). MS-2 required a longer amount of time for






73

Table 3-10. Activation thermodynamics of select bacteriophage adsorption to
natural media

ACTIVATION THERMODYNAMICS OF SELECT BACTERIOPHAGE
EA AG AH AS 25C (298kcal/mol) (kcal/mol) (kcal/mol) (caVmol per K)
Sludge MS2 3.34 22.24 2.75 -65.4

Sludge 60STP2 9.13 22.74 8.54 -47.65

Soil MS2 4.03 22.32 3.44 -63.36

Soil 60STP2 18.31 24.04 17.72 -21.21
aComparative activation thermodynamics at 250C for phage adsorption to solids. A 106 PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL, or a 2 mL solution of deionized water and 2grams soil at various temperatures and agitated for 2 hours. passage through the column, and poliovirus-1 adsorbed well to the columns (Figure 3-12).

Groundwater Studies

Six observation wells were sampled to monitor the transport of tracer bacteriophage through soils. The results were assayed and corrected for background counts (Table 3-11). Our results show that the first observation of 60STP2 and MS-2 in well water occurred within the first 8 hours in well 1 (Figure 3-13). It is also obvious from the results that 60STP2 shows up first followed closely by MS-2, and that a greater amount of 60STP2 was found (Figures 3-13, 3-14, 3-15, 3-16, 3-17, 3-18). MS-2 and 60STP2 were found in the same wells at approximately the same time (Figures 3-13, 3-14, 3-15, 3-16, 3-17, 3-18). The results observed show that the phage traveled in similar paths through the field, and to a horizontal distance of at least 73 feet from the tank through the field in a






74


-5
-6
-7


-9 r .
--10
-11 -12
-13 -14 -15
3.23E-03 3.36E-03 3.61 E-03
TEMP (1/K)

* Sludge MS2 **W =Sludge 60STP2
-***Soil MS2 -Soil 6OSTP2

Figure 3-6. Arrhenius plot of select bacteriophage adsorption to natural mediaa aThe Arrhenius plot uses the adsorption rate constant (-k) versus the inverse of temperature in Kelvin. A 106 PFU/mL phage concentration was added to mixedliquor suspended solids at a final volume of 50mL, or a 2 mL solution of deionized water and 2grams soil at various temperatures and agitated. minimal amount of time (Figure 3-18). However, our study was carried out under rainy conditions where the soil was saturated. The results of the second study under dryer conditions using wells 1, 3, and 5 have shown similar results. The survival study has shown that the phage 60STP2, and MS2 were inactivated between 8 and 10 days (Figure 3-19).

Discussion

The viruses we selected based on specific adsorption characteristics

primarily adsorbed as we expected. The bacteriophages 60KCK2 was selected based on strong hydrophobic interactions; 60STP2 was selected for its weak






75




0.9 0.8
0.7
0.6
S0.5
0.4
0.3
0.2 0.1


0 15 30 45 60 75 90 120 TIME (minutes)
- 60STP2 --MS2 60KCK2

Figure 3-7. Transport in (10 cm) sandy soil column of bacteriophage aThe experiment was carried out at room temperature using a 10 cm sandy soil depth column in a 60 cm syringe with gravitational flow and 106 PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. hydrophobic and electrostatic characteristics, and 61STP7 was selected based on moderate hydrophobic and electrostatic characteristics, and 61KCK10 was selected based on strong hydrophobic and electrostatic properties. However, the entire system including the virus, solution, and solid defines the adsorption pattern. Due to the numerous factors involved in these complex systems, it is necessary to define the entire system each time to determine the mechanism of adsorption. The parameters that have been found to affect adsorption have been previously stated in the introduction (Bitton 1980, Blanc and Nasser 1996, Gerba et al. 1981, Gerba 1984b, Goyal and Gerba 1979, Lance and Gerba 1984a,






76



1
0.9 0.8 0.7
0.6
S0.5
S0.4
0.3
0.2 0.1
0
0 15 30 45 60 75 90 120 Time (minutes)
*4- 60STP2 - MS-2 * 60KCK2

Figure 3-8. Transport in (10 cm) forest soil column of bacteriophagea aThe experiment was carried out at room temperature using a 10 cm forest soil depth column in a 60 cm syringe with gravitational flow and 106 PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. Lance and Gerba 1984b, Nasser et al. 1993, Sobsey et al. 1995, Yahya et al. 1993, Yates et al. 1985). As seen in Figures 3-2 to 3-8 and Tables 3-5 to 3-9, variation in temperature, pH, solids, and phages affect adsorption of phages to the solids. Nevertheless, knowledge of the significant parameters can give a predictive adsorption model. At a neutral pH, soils generally have a net negative charge and many viruses have a net negative charge. Most studies have found enhanced adsorption with the addition of a high concentration of salts or a decrease in pH, large soil surface area, or a high cation exchange capacity (Dowd et al. 1998, Lance and Gerba 1984a, Taylor et al. 1981).






77




0.9
0.8
0.7


0.6
0.4
0.3
0.2
0.1 Flow rate= 0.98mLimin.


0 30 60 105 120 180 240 TIME (min)

460STP2 - MS2


Figure 3-9. Transport in (20 cm) sandy soil column of bacteriophagea aThe experiment was carried out at room temperature using a 20 cm soil depth in a 50 cm PVC column with gravitational flow and 106 PFU/mL phage concentration. The abbreviations Cs =concentration supernatant and Co =initial concentration.

Previous studies have shown that an increase in the clay content of soils, increases adsorption of the viruses, and that organic materials interfere with viral adsorption to soils (Gerba et al. 1981, Goyal and Gerba 1979, Sobsey et al. 1980a). Our results were in agreement that an increase in adsorption is mainly due to the increase in clay content as the depth of the sample increases. Other studies have shown that the length of the column, and that the flow rate can affect viral adsorption (Lance and Gerba 1984b). We also found high






78




0.9 0.8

0.7

0.6
o
o0.5

0.4 0.3

0.2 0.1
Flow rate= 1.5mLimin.

0 15 30 45 60 90 120 150 180 TIME (min.)

*****60STP2 -MS2 -PV' Figure 3-10. Transport of viruses in (37 cm) sandy soil column aThe experiment was carried out at room temperature using a 37cm sandy soil depth in a 50 cm PVC column with gravitational flow and 106 PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. variability in adsorption to mixed-liquor suspended solid (MLSS) samples, which we believe occurs due to interfering particles such as organics in the media. The batch adsorption studies with the natural media have shown that as the time allowed for viral adsorption increased there was an increase in adsorption at pH 7, and that an increase in column size from a 10 cm soil depth to 20 cm soil depth allowed for a difference in transport time between viruses. Another factor minimally affecting time dependent viral adsorption was flow rate.






79


1
Flow rate=0.88mLmin.
0.9

0.8

0.7 0.6

0 0.5

0.4

0.3

0.2 0.1

0 I
0 30 60 105 120 180 240 TIME (min.)

--O60STP2 - MS2


Figure 3-11. Transport in (20 cm) forest soil column of bacteriophage aThe experiment was carried out at room temperature using a 20 cm soil depth in a 50 cm PVC column with gravitational flow and 106 PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration.

Several studies have shown that an increase in ionic strength of a solution was found to enhance adsorption and detergents interfered with adsorption of several enteroviruses and rotavirus SA11 (Dizer et al. 1984, Lance and Gerba 1984a, Lytle and Routson 1995, Sobsey et al. 1980a, and Taylor et al. 1981). These observations are in agreement with what we observed in various natural media systems (Tables 3-8 and 3-9).






80



Flow rate=1.2mUmin.
0.9 0.8 0.7 0.6
0
U 0.5

0.4

0.3

0.2

0.1


0 15 30 45 60 90 120 150 180 TIME (min.)

60STP2 - MS2 PV


Figure 3-12. Transport of viruses in (35 cm) forest soil columna aThe experiment was carried out at room temperature using a 35 cm forest soil depth in a 50 cm PVC column with gravitational flow and 106 PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration.

Previous studies and our results also show that viral adsorption to natural media at a neutral pH is dependent on the viral surface composition (Dowd et al 1998, Penrod et al. 1996, Schijven 2000). Studies have shown that most viruses may behave differently under similar conditions (Burge and Enkiri 1978, Goyal and Gerba 1979, Shields and Farrah 2002). It is believed that their differences in






81

Table 3-11. Bacteriophage in monitoring wells

Well # PFUlmLa
E. coli C-3000 S. Typhimurium
1 5 1 2 7 3 3 6 2 4 4 6 5 3 12 6 7 2
aBefore the addition of bacteriophage, a sample of well water was removed and a 1mL sample was assayed as PFU/mL using the hosts Escherichia coli C-3000 and Salmonella typhimurium.

adsorption are due to differences in their hydrophobicity and electrical charges on the capsid surfaces (Shields and Farrah 2002). The results for the most part follow the DLVO theory so an increase in ionic strength screens electrostatic charges and promotes hydrophobic and van der Waals interactions. Our results show that the pH had a significant effect on adsorption of viruses to media. Previous studies show that adsorption varies based on pH dependent adsorption properties related to the isoelectric point of the viruses and solids (Dowd et al. 1998, Gerba et al. 1981, Gerba 1984b, Mix 1974, Mix 1987, Sobsey et al. 1980a, Taylor et al. 1981). A virus particle is colloidal in nature so it must be physically adsorbed to a surface in order to be removed. The kinetics of adsorption of the selected bacteriophage to natural media was determined as a function of temperature to study the thermodynamic parameters. Results of previous studies and our study show that phage adsorption to natural medias at various temperatures is a physical process with a low energy of activation of <40 Kcal/mol (Table 3-10) (Adamson 1982, Preston and Farrah 1988a). The






82


75 70
65 1 60 55 50
45 1 -E 40 u. 35


20
15
10


8 16 24 32 40 48 56 64 72 80 88 96
TIME (Hour)

* MS-2 N60STP2


Figure 3-13. Tracer study in well 1 at a 10 ft distance from the system a aBacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). Arrhenius plot uses the adsorption rate constant (-k) versus the inverse of temperature in Kelvin. The adsorption rate constant is the slope of the rate constant plot, which uses the natural log of viruses in the supernatant versus time in seconds (Figures 3-3 to 3-5). The Arrhenius plot of the phage to natural media indicates that the phage 60STP2 adsorbed poorer to natural media than the model MS-2 (Figure 3-6). The results show that adsorption was dependent on temperature, and solid composition. Adsorption of MS-2 and 60STP2 increased significantly at 370C to all media. This phenomenon was confirmed by






83


75
70
65
60
55 50
J 45 E 40

a- 30
25
20
15
10 *
5 0
8 16 24 32 40 48 56 64 72 80 88 96 TIME (Hour)

" MS-2 60TP2


Figure 3-14. Tracer study in well 2 at a 12 ft distance from the systema aBacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF).

controls of bacteriophage in solution without the biosolids that inactivation was

not a significant factor over the time frame of the experiments.

Due to the presence of enteric viruses in groundwater many investigators

have studied the transport phenomena by using tracers (Bales et al. 1989, Gerba

1984a, Griffin et al. 1999, Harvey 1997, Paul et al. 1995, Paul et al. 1997,

Schaub and Sorber 1977, Skilton and Wheeler 1988, Vaughn et al. 1978,

Vaughn et al. 1983, Yanko et al. 1999). The bacteriophage MS-2, and PRD-1 are

hydrophobic bacteriophage that have been found to adsorb poorly to various






84


75 70 65 60 55 50
45
E 40
U.35
30
25
20
15 10
5
0
8 16 24 32 40 48 56 64 72 80 88 96
TIME (Hour)

'-4 -MS-2 --- 60STP21


Figure 3-15. Tracer study in well 3 at a 27 ft distance from the system a aBacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). solids so they are frequently used as worst-case models for possible

contamination (Goyal and Gerba 1979, Schijven 2000). However, we found that

MS-2 and PRD-1 do not adsorb that poorly to all media, and that adsorption was

dependent on the various factors in the systems. Our results also show that by

selecting bacteriophage based on their adsorption properties, and the system

parameters we can predict better models for transport systems. The

bacteriophage 60STP2 selected based on its weak hydrophobic and electrostatic

properties was shown to adsorb poorly to virtually all media and in transport






85



75 70 65 60 55 50 45
E40 U- 35
a.30

25 20 15 10



8 16 24 32 40 48 56 64 72 80 88 96
TIME (Hour)

- *MS-2 *-60STP2


Figure 3-16. Tracer study in well 4 at a 49 ft distance from the systema aBacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). studies had increased flow with less reduction than the previously used MS-2 phage. It is well known that viruses can flow through the soils from waste disposal systems into the water supplies.

Studies have found that viruses survive better at temperatures of 100C or below and that there is significant inactivation at temperatures of 230C and higher (Alvarez et al. 2000, Blanc and Nasser 1996, Nasser et al. 1993). Our results indicated that it took between eight and ten days for complete inactivation of the bacteriophage at an average temperature of 220C. These results are in






86


75 70 65 60 55 50
45
E 40 ,, 35
30
25 20
15 .
10
5
0
8 16 24 32 40 48 56 64 72 80 88 96
TIME (Hour)

i-** *MS-2 --60STP2


Figure 3-17. Tracer study in well 5 at a 71 ft distance from the systema aBacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). agreement with previous studies (Alvarez et al. 2000). It is important to note that

inactivation is generally negligible within the time scale of the experiments.

However for field experiments viral inactivation may be significant, especially

under some conditions such as unsaturated, low flow rate, high adsorption. The

results also show that there was approximately a 5 logo reduction in the phage

tracers. The reduction occurred mainly due to adsorption since the results of the

survival study show a low rate of inactivation.





87


75 70 65 60 55 50 45
E40
u. 35 S30
25 20 15
10 "T T T" T T
10
0 . .. .
8 16 24 32 40 48 56 64 72 80 88 96
TIME (Hour)

*i** *MS-2 I*-60STP2


Figure 3-18. Tracer study in well 6 at a 73 ft distance from the system aBacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF).

In summary, the results show that viral adsorption to media is based on

specific adsorption properties that for the most part follow the DLVO theory. The

results also show that adsorption or transport is dependent on specific factors

such as, retention time, composition of the solutions, ionic strength, pH, viral coat

composition, viral type, the composition of the solids, and temperature. It is

fundamental to understand the interactions involved in the adsorption, survival

and transport of viruses. Using our results and results from other studies we have





88



10

9 8 7

26 M5 o 4
-j

3





0
0 1 2 4 6 8 10
TIME (days)
- "-MS-2 -0-60STP2


Figure 3-19. Bacteriophage inactivation in wellsa aBacteriophage 60STP2 and MS-2 at 106PFU/mL in deionized water were placed in polypropylene tubes in the monitoring wells and assayed daily for inactivation. compared the selected indigenous bacteriophage to enteric viruses (Table 3-12) (Gerba et al. 1981, Goyal and Gerba 1979, Shields and Farrah 2002).




Full Text

PAGE 1

SELECTION AND CHARACTERIZATION OF BACTERIOPHAGE FOR USE ADSORPTION AND TRANSPORT STUDIES By CHERYL M. BOICE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Cheryl M. Boice

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ACKNOWLEDGMENTS I would first like to acknowledge my mentor, Dr. Samuel R. Farrah, for his guidance and support throughout my graduate studies. He has encouraged ingenuity in my research. Dr. Farrah has been an inspiration and his vast knowledge on many subjects has been an asset. I would also like to thank the other members of my graduate committee (Dr. Thomas Bobik, Dr. Madeline Rasche, Dr. Ben Koopman, and Dr. Richard Dickinson) for their guidance. I would like to acknowledge Dr. Jerzy Lukasik and Dr. Troy Scott for their assistance. In addition, I would like to thank my husband and son, T.J. and Nathan Boice; and my grandparents, John and MyrI Dean for their support and understanding throughout my education. I extend special thanks to the entire Department of Microbiology and the Engineering Research Center (PERC) for their overall support and flexibility. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS LIST OF FIGURES vii LIST OF TABLES ABSTRACT ^' 1 INTRODUCTION ^ Waterborne Pathogens Bacteria ^ Viruses ^ Protozoa ^ Indicators ^ Models Tracers l~ interactions between Viruses and Solids 13 Survival Virus Characterization 21 Experimental Rationale 23 2 VIRAL INTERACTIONS WITH DEFINED MEDIA 26 Introduction • 26 Adsorption 27 Elution 29 Current Methods 30 Materials and Methods 31 Collection of Raw Sewage Samples 31 Media 31 Chemicals 31 Solutions 31 Viruses 32 Isolation of Bacteriophage Using Nitrocellulose Filters 33 Isolation of Bacteriophage Resistant to High Temperature 34 Selection of Bacteriophage for Additional Studies 34 IV

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Characterization of Bacteriophage Adsorption to Filters under Varying Conditions Bacteriophage interaction to Particles j-Jg Isolation of Bacteriophage from Raw Sewage Samples 37 Selection of Bacteriophage ^° Characterization of Bacteriophage Discussion 3 VIRAL INTERACTIONS WITH NATURAL MEDIA 52 52 Introduction Adsorption Tracers Survival Materials and Methods Media Solutions Viruses ^ Adsorption of Viruses to Mixed-Liquor Suspended Solids o/ Adsorption of Viruses to Soils 58 Kinetic Adsorption Studies 59 Column Adsorption Experiments ^ Groundwater Tracer Studies Groundwater Survival Studies °2 Results II Batch Adsorption Studies Kinetic Adsorption Studies ^' Column Adsorption Experiments ]P Groundwater Studies Discussion 4 IDENTIFICATION AND PROTEIN COAT CHARACTERIZATION OF SELECTED BACTERIOPHAGE 90 Introduction 90 Materials and Methods 91 Q1 Viruses ^ ' Cross Reactivity (Host Range) 92 Plaque Morphology 92 Electron Microscopy 92 Nucleic Acid Determination 93 Protein Analysis 94 Restriction Enzyme Analysis 94 Sequencing 95 Gel Excision 95

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Cloning 95 Screening Plasnaid Isolation and DNA Sequencing 96 Sequence Analysis of the Capsid and INT Gene 96 Protein Coat Analysis 96 Results ^ Cross Reactivity (Host Range) 97 Plaque Morphology and Electron Microscopy 97 Nucleic Acid Deternnination 98 Protein Analysis and Restriction Enzyme Analysis 99 Sequencing Protein Coat Analysis ''00 Discussion ' ^ 5 SUMMARY AND CONCLUSIONS 119 APPENDIX A BACTERIOPHAGE ADSORPTION 1 24 B AMINO ACID SEQUENCES OF VIRAL CAPSIDS 127 Caudovirales 127 Leviviridae 12^ Microviridae 127 Tectiviridae 12^ Myoviridae 128 Podoviridae 12^ Siphoviridae 129 Picomaviridae 1^^ Poliovirus 1^^ Echovirus 1*^1 Coxsackie virus 1^1 LIST OF REFERENCES 133 BIOGRAPHICAL SKETCH 146 vi

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LIST OF FIGURES Figure Eige 2-1 Raw sewage bacteriophage survival at 40°C and 50°C over time 40 2-2 Survival of bacteriophage at 40°C in UV dechlorlnated water 42 2-3 Survival of bacteriophage at SOX in UV dechlorinated water 43 2-4 Bacteriophage adsorption to 0.45 Mm HA nitrocellulose filters at pH 7 in the presence of MgCIa 2-5 Bacteriophage elution from 0.45 pm HA nitrocellulose filters at pH 3.5 in the presence of Tween 80 45 2-6 Bacteriophage elution from 0.45 pm 1 MDS charged modified filters at pH 7 in the presence of MgCl2 2-7 MS-2 adsorption to nanosized filter fibers 48 28 60STP2 adsorption to nanosized filter fibers 49 31 Soil column setup 3-2 Adsorption of model bacteriophage to mixed-liquor suspended solids. ...64 3-3 Adsorption at 4°C of select bacteriophage to natural media 70 3-4 Adsorption at 25°C of select bacteriophage to natural media 71 3-5 Adsorption at 37°C of select bacteriophage to natural media 72 3-6 Arrhenius plot of select bacteriophage adsorption to natural media 74 3-7 Transport in (1 0 cm) sandy soil column of bacteriophage 75 3-8 Transport in (1 0 cm) forest soil column of bacteriophage 76 3-9 Transport in (20 cm) sandy soil column of bacteriophage 77 vii

PAGE 8

3-1 0 Transport of viruses in (37 cm) sandy soil column 78 3-1 1 Transport in (20 cm) forest soil column of bacteriophage 79 3-12 Transport of viruses in (35 cm) forest soil column 80 3-1 3 Tracer study in well 1 at a 1 0 ft distance from the system 82 3-14 Tracer study in well 2 at a 12 ft distance from the system 83 3-1 5 Tracer study in well 3 at a 27 ft distance from the system 84 3-16 Tracer study in well 4 at a 49 ft distance from the system 85 3-17 Tracer study in well 5 at a 71 ft distance from the system 86 3-1 8 Tracer study in well 6 at a 73 ft distance from the system 87 319 Bacteriophage inactivation in wells 88 41 Electron micrograph of 60KCK2 99 4-2 Electron micrograph of 61KCK10 100 4-3 Electron micrograph of 60STP2 101 4-4 Electron micrograph of 61 STP7 1 02 4-5 Electron micrograph of the bacteriophage 60STP2 with Salmonella typhimurium 1 03 4-6 4-20% SDS-PAGE of bacteriophage 105 4-7 Restriction enzyme EcoRV analysis of bacteriophage 1 06 4-8 Restriction enzyme EcoRI and Hindi analysis of bacteriophage 107 4-9 Restriction enzyme EcoRI and Hindi analysis of 60STP2 108 4-10 Screening plasmid restriction enzyme EcoRI and Hindi analysis of the capsid and integrase genes of 60STP2 109 4-1 1 Sequence analysis of the capsid gene of 60STP2 115 4-12 Amino acid residue analysis of the capsid gene of 60STP2 116 viii

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LIST OF TABLES Table eaae 1-1 Sources of pollution in groundwater 2 1-2 Solute models frequently used in groundwater studies 14 1-3 Biotracer models frequently used in groundwater studies 14 14 Factors involved with viral adsorption and transport in soils 18 1 -5 Factors involved with survival of viruses associated with solids 20 21 Adsorption of bacteriophage in filtered raw sewage at pH 7 and pH 3.5 39 2-2 Adsorption of selected bacteriophage to microporous filters at pH 7 and pH 3.5 23 Adsorption of bacteriophage to defined media 47 31 Detection of viruses in groundwater 52 3-2 Soil characteristics 57 3-3 Well characteristics ^2 3-4 Weather conditions 63 3-5 Adsorption to mixed-liquor suspended solids by select bacteriophage ...65 3-6 Adsorption to natural area teaching lab soils by select bacteriophage.... 66 3-7 Adsorption to soils by select bacteriophage 67 3-8 Adsorption to natural media at pH 7 and pH 3.5 by select phage 68 3-9 Adsorption of bacteriophage to natural media 69 ix

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3-1 0 Activation thermodynamics of select bacteriophage adsorption to natural media 3-1 1 Bacteriophage in monitoring wells ^'I 312 Summary of select bacteriophage 89 41 Identification characteristics of select bacteriophage 98 4-2 Similarities of isolated and reference bacteriophage 1 04 4-3 Capsid sequence amino acid hydrophobic residues and adsorption characterization of various viruses 'I ^ 4-4 Capsid sequence amino acid charged residues and adsorption characterization of various viruses 1 18 A-1 Phage adsorption to 0.45 pm nitrocellulose filters at pH 7 and pH 3.5.. 124 X

PAGE 11

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SELECTION AND CHARACTERIZATION OF BACTERIOPHAGE FOR USE IN ADSORPTION AND TRANSPORT STUDIES By Cheryl M. Boice August 2003 Chairman: Samuel R. Farrah Major Department: Microbiology and Cell Science Bacteriophages are frequently used as models of human pathogenic viruses in studies on virus adsorption. They are also frequently used to examine penetration of barrier materials; and to study viral transport in soil and water. They offer a practical analytical tool in studying numerous applications, such as the effectiveness of water treatment processes. Bacteriophages are used because they are easier, safer, and less expensive to assay than human pathogenic viruses; and they can be produced in large numbers (lO^'' PFU/mL). However, these models have traditionally been chosen based on similarities shared with human pathogenic viruses including size, shape, nucleic acid type, and chemical composition of the capsid. They are typically not selected based on adsorption or survival characteristics. Previous research indicates that the adsorption mechanisms of some phage models are not comparable to the pathogenic viruses they are supposed to represent. xi

PAGE 12

Our main objectives were to determine if phage models could be chosen based on their adsorption mechanisms; and to clarify these mechanisms. We isolated indigenous bacteriophage from domestic sewage samples based on differences in the strengths of their electrostatic and hydrophobic interactions to charged microporous filters under varying conditions of pH, salt concentration, and concentration of detergents. Bacteriophage adsorption properties were then compared to viruses already studied for their ability to adsorb to defined media and natural solids, such as soils and wastewater sludge. Results indicate that some selected bacteriophages are better models for human pathogenic viruses in adsorption studies than previously used models. The isolated bacteriophage were identified down to the family level. Examination of the coat proteins and phage interactions to solids were used to clarify the mechanism of adsorption. It was found that the coat proteins vary in each bacteriophage and may account for the differences in adsorption to the same media. Finally we used a groundwater tracer study of the bacteriophage selected based on soil column studies. Results indicate that some of the isolated bacteriophage may be better than previously used bacteriophages as models for the movement of viruses in soil. xii

PAGE 13

CHAPTER 1 INTRODUCTION In the United States, approximately half the population uses groundwater as the major source of drinking water (Craun 1986). Fecal pollution from human or nonhuman origins may affect the quality of water (Keswick and Gerba 1980). Septic tanks are identified as the cause of most of the groundwater contamination (Yates et al. 1985). Studies have also shown that some waterborne pathogens can survive wastewater treatment processes and contaminate the groundwater supply after land application (Keswick et al. 1992, Vaughn et al. 1983). Other sources of possible fecal contamination include polluted surface waters, landfills, and runoff from agriculture, and cesspools (Table 1-1) (Keswick and Gerba 1980). The presence of enteric organisms in groundwater, and well water has been documented (Gerba et al. 1985). Over half of the documented waterborne-disease outbreaks in the United States resulted from consumption of contaminated groundwater, in which an estimated 65% of those cases were caused by enteric viruses (Yates et al. 1985). Waterborne Pathogens Several bacterial, viral, and protozoan pathogens may be found in contaminated waters. Most of these pathogens infect the gastrointestinal tract and are transmitted via person-to-person contact or by contaminated food and water. These organisms may be found in contaminated soil and water. 1

PAGE 14

2 Table 1-1. Sources of pollution in groundwater Sources of pollution Description Land disposal of sewage. Facilities in^iiirto cln\A/-ratp infiltration rdDld inCIUU" olUW Idle II II iili aiiwi 1 , lapi^^ infiltration, overland flow, wetlands, and subsurface injection. Sewer line cracks Land disposal of sewage is a form of groundwater recharge Land disposal of sewage sludge Sewage sludge is dried and placed on land sites Septic tanks, and cesspools. Wastewater (containing pathogenic organisms) is discharged directly onto soil via onsite disposal units. These are soil-adsorption treatment systems. Septic systems rank the highest in the volume of wastewater discharged directly into groundwater systems. These systems are subject to failure leading to groundwater contamination. Land disposal of solid waste Refuse dumps, landfills. Microbial waste from diapers ana animal feces Storm water recharge and urban runoff QavA/ono miY
PAGE 15

3 frequently the cause of human disease associated with bacteria-contaminated water or food. They are capable of producing a variety of infections including septicemia, gastroenteritis, enteric fever, bacteremia, meningitis, and urinary tract infections (Murray et al. 2001) Most E. coli are considered harmless or opportunistic pathogens. Their presence is most often used as an indicator of the presence of other fecal pathogens. However, some strains of £. coli can cause gastroenteritis. These strains are divided into five groups based on specialized fimbriae and the toxins they produce (Murray et al. 2001). The most serious disease is caused by the strain called enterohemorrhagic E. coli (EHEC). Some infections can be mild and uncomplicated; however, one possible presentation of EHEC is hemorrhagic colitis with bloody diarrhea caused by the Shiga-like toxins (Murray et al. 2001). Hemolytic uremic syndrome is another complication of EHEC (Murray et al. 2001). Another strain is enterotoxigenic E. coli (ETEC), which is the primary cause of traveler's diarrhea. This is a noninvasive infection that has an enterotoxin that causes watery diarrhea (Murray et al. 2001). Detection of enterohemorrhagic E. coli (particularly 0157:H7) uses both molecular and biochemical methods. Salmonella spp. is opportunistic and can cause salmonellosis. Most often the disease is related to contaminated meats and poultry (Murray et al. 2001). Salmonella typhi is only found in humans and can cause typhoid fever. There has been a reduction in typhoid infections because of proper sewage disposal, water treatment, and food sanitation. The methods used in the detection of Salmonella

PAGE 16

4 in water are not standardized. Selective enrichment and identification procedures are generally used. PGR and Innmunomagnetic separation have been used to detect Salmonella spp. (Hsih and Tsen 2001). Shigella dysenteriae can cause dysentery by the production of Shiga toxin, an exotoxin (Murray et al. 2001). The detection of Shigella spp. in environmental samples is difficult due to, the low number of organisms present and the presence of a large number of background floras. Although enrichment and direct-detection techniques such as PGR (Frankel et al. 1990) are available, they are difficult to perform. Because the infective dose is so low, however, the presence of any amount of Shigella poses a threat to human health (Stutman 1994). Vibho cholerae can cause cholera infections, which are caused by lapses in sanitation practices. Most infections are strongly associated with contaminated water sources (usually from eating contaminated seafood). An enterotoxin is produced that causes secretion of fluids, watery diarrhea, and vomiting resulting in dehydration (Murray et al. 2001). Shock and renal failure can also occur. Vibno parahaemolyticus and Vibho vulnificus are usually found in gastroenteritis outbreaks associated with seafood (Murray et al. 2001). These organisms are found in salt water estuaries. Vibho vulnificus can also cause wound infections after exposure to contaminated seawater; and systemic infections occur after eating contaminated food (Murray et al. 2001). The detection of Vibrio spp.

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5 requires water samples to be concentrated and enriched; then plated on selective and differential media for presumptive identification (Murray et al. 2001). Campylobacter jejuni can cause gastroenteritis associated with food contamination. The organisms are often found in milk and poultry. The symptoms include fever, cramping, diarrhea, and possibly dysentery (Murray et al. 2001). Guillain-Barre syndrome, with temporary paralysis, is a rare complication of Campylobacter infections (Murray et al. 2001). Detection and identification is by plating on selective media under selective conditions. Viruses It is estimated that over 100 different types of human enteric viruses may be present in human feces (Puig et al. 1994). The enteric viruses associated with humans encompass many viral families such as, Picornaviridae, Adenoviridae, Astroviridae, Caliciviridae, and Reoviridae. The family Picornaviridae includes enteroviruses such as polio, coxsackie A and B, echo, enteroviruses 68-71, and hepatitis A. Adenoviridae includes adenovirus of serotypes 31, 40, and 41. The family Caliciviridae includes Nonwalk; small round viruses, and hepatitis E. Reoviridae includes the reoviruses and rotavirus. They are associated with a wide range of diseases such as gastroenteritis, respiratory infections, conjunctivitis, hepatitis, paralysis, meningitis, myocarditis, neonatal, urethritis, rashes, and encephalitis. Generally, the diseases are either asymptomatic; or the symptoms exhibited are gastroenteritis and respiratory infections.

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6 Pathogenic viruses of fecal origin are transmitted to water sources via inefficiently treated water. Once in the environment, enteric viruses cannot replicate. However, even a low level of viruses in the environment can pose a risk to human health. Some enteric viruses have a very low (<10) infectious dose (Murray et al. 2001). So detecting viruses in environmental water samples consists of collecting and concentrating viruses from a large sample volume (usually done by filtration) followed by a second concentration step (Clesceri et al. 1989). The viruses can then be detected by cell culturing in conjunction with molecular techniques, primarily reverse transcriptase-polymerase chain reaction (RT-PCR) for RNA viruses (Abbaszadegan et al. 1999, Straub et al. 1995). However, this method is time-consuming, expensive, subject to environmental contamination and background from other viruses (Lewis et al. 2000). Other limitations to detection are the low concentrations in environmental samples; and some are not cultivable (Alvarez et al. 2000, Lewis et al. 2000). Protozoa The most common Protozoa causing intestinal disease are Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum, and members of the order Microsporidia. The Entamoeba histolytica cyst is infective to humans and has the ability to survive in food and water. Infection may be asymptomatic in some individuals and fatal in others. Acute amebic colitis involves frequent bloody diarrhea with or without fever. Giardia lamblia is prevalent in underdeveloped countries. Waterborne transmission causes giardiasis in travelers in endemic countries. Symptoms include intestinal abdominal cramps, nausea, explosive diarrhea, fever, and chills. Cryptosporidium parvum oocyst is infectious to

PAGE 19

7 humans. The oocysts are widely distributed in both sewage and drinking water. Symptoms including diarrhea, fever, abdominal pain, and nausea last for about one week. Environmentally resistant Microsporidia spores have the ability to infect humans. Surface water is the primary environmental residence for Microsporidia. The most common symptoms of Microsporidia infection include diarrhea, dehydration, and weight loss. Entamoeba histolytica can be detected by microscopic examination or by various molecular assays (Schunk et al. 2001 , Zindrou et al. 2001). Characteristic staining patterns identify Giardia and Cryptosporidium. Restriction fragment length polymorphism analysis of PGR amplicons can be used to identify various Microsporidia (Curry and Smith 1998). Indicators While pathogen counts in raw sewage can be significant, the concentration in treated water is usually low. Even at these low concentrations, some enteric organisms are still capable of causing disease. Due to the wide distribution and survival characteristics of waterborne pathogenic organisms, it is necessary to monitor water quality and assess risk factors for waterborne disease. Direct analysis of the pathogens is often difficult due to the low numbers, background from other organisms, and the procedure used. Molecular techniques are quicker and more sensitive. However, they are more expensive; and do not indicate if the organism is viable. Due to these limitations, indicator organisms are often used as an index of water quality; and to test samples for the possible presence of fecal pollution. Indicator organisms are used because

PAGE 20

8 they are found in high numbers; and because procedures are easier and faster than assay procedures for pathogens. Total and fecal coliform organisms are used extensively as indicators for determining the sanitary quality of water (Gerba 1999). The total coliform group includes Escherichia coli, Enterobacter, Klebsiella, and Citrobacter. These organisms ferment lactose and produce gas at 35°C within 2 days. However, not all members of this group are of fecal origin. Fecal colifomis include members that can ferment lactose at 44.5°C. Members of this group include Escherichia coli and Klebsiella pneumoniae. The presence of fecal coliform indicates the presence of fecal pollution from warm-blooded animals. However, they are not limited to humans. They also differ from pathogenic virus and protozoan organisms in resistance, adsorption, and survival in the environment (Sobsey et al. 1995). E. coli is the most reliable indicator for fecal pollution since it can be differentiated from other coliforms. Enterococci, and Clostridium perfringens have been considered as alternative indicators. Fecal streptococci are used as an indicator of fecal contamination since they are found in the feces of humans and animals (Gerba 1999). They are more resistant than coliforms, do not reproduce in the environment, and persist longer than coliform organisms. The group Fecal streptococci have a subgroup Enterococci that includes the members S. faecalis and S. faecium. These organisms have been suggested as indicators of the presence of viruses in biosolids (Payment and Franco 1993). Clostridium perfringens produces an anaerobic spore; and has an exclusively fecal origin.

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9 The spores are resistant to treatment processes and extreme temperatures (Payment and Franco 1993). The primary use of these spores has been as an indicator to past pollution. Other organisms such as bacteriophages have also been considered as alternative indicators. Bacteriophages are viruses that infect bacteria. They consist of a nucleic acid molecule (genome) surrounded by a protein coat (capsid). Some bacteriophages may also contain lipids. These organisms may be somatic coliphage, which infect conforms such as Escherichia co// through cell wall receptors, F+ specific bacteriophage that infect £. coli and other coliforms through the F+-pili, and anaerobic Bactehodes fragilis bacteriophage (lAWPRC 1991, Leclerc et al. 2000). Bacterial viruses as indicators for fecal pollution have been proposed due to their presence in contaminated waters, and their similarities shared with enteric viruses (Havelaar et al. 1 990, Hsu et al. 1 995). Bacteriophages are proposed as indicators because their presence indicates the presence of the host organism and possible fecal pollution. The F-specific coliphage are considered the indicator of choice for assessing the potential presence of human enteric viruses since they are a more homogeneous group and generally have a greater resistance to inactivation than somatic coliphages (lAWPRC 1991, Leclerc et al. 2000). Bactehodes fragilis bacteriophages have been reported to occur in human faeces only (Leclerc et al. 2000). Models Pathogens of fecal origin are transmitted through inefficiently treated water. Organisms especially viruses survive current wastewater treatment processes and can be transported back into the groundwater supply. Model

PAGE 22

10 organisms for human pathogenic viruses are often used to assess various materials and processes such as filter materials and wastewater treatment processes. Poliovirus is often used as a representative organism for the pathogenic enteric viruses in numerous applications such as wastewater treatment processes since it is cultivable and more resistant to adverse environmental conditions (Lance and Gerba 1984b, Sobsey et al. 1995, Schijven 2000). However, assessing materials by direct analysis is time consuming and expensive (Dizeret al. 1984, Gerba et al. 1981, Goyal and Gerba 1979, Havelaar et al. 1993). Other model organisms, such as bacteriophage are frequently used to study the adsorption, fate, and transport of human pathogenic viruses due to their availability, low assay cost, and safety (Gerba et al. 1991 , Paul et al. 1995, Yates et al. 1985). Bacteriophages are used extensively to evaluate treatment processes, and as tracers (Gantzer et al. 1998, lAWPRC 1991, Leclerc et al. 2000). Frequently studied models for enteric viruses include MS2 (Bales et al. 1989, Farrah 1982, Farrah et al. 1991, Gerba et al. 1991, Shields and Farrah 2002, Sobsey et al. 1995); PRD1 (Bales et al. 1991, Paul et al. 1995, Shields and Farrah 2002); OX174 (Fujito and Lytle 1996, Goyal and Gerba 1979, Shields and Farrah 2002); and other F-specific RNA phages (Havelaar et al. 1993, Havelaar and Sobsey 1995, Sobsey et al. 1986). These models have shown that bacteriophages can be useful under some conditions. However, they are inadequate models for all enteric viruses since their adsorption varies. F-specific bacteriophages, MS2, and PRD 1 are considered worst-case model viruses since

PAGE 23

11 they are poor adsorbers at pH 7 (Schijven 2000). The bacteriophage 0X174 is considered a conservative model since it is retained in column studies better than the worst-case models. 0X174 has been found to have basically no charge at a neutral pH, low hydrophobicity, and minimal electrostatic interaction; so it has been used in various studies of barrier materials (Dowd et al. 1998, Schijven 2000). These models have traditionally been chosen based on similarities to human pathogenic viruses (such as size, shape, and nucleic acid type). Most of the models have not been selected based on their adsorption or survival characteristics. Previous research has shown that the adsorption mechanisms of some models are not comparable to those of the pathogenic viruses they are supposed to represent (Shields and Farrah 1983, Sobsey et al. 1986). For example, the coliphage MS-2 has been used as a model for poliovirus-1 for numerous years. It was primarily selected based on its similarities to polio such as its nucleic acid, size, and shape. MS-2 is an icosahedral ssRNA bacteriophage (for Escherichia coli) of approximately 27 nm size. Polio is an icosahedral ssRNA human virus of approximately 30 nm size. However, it is well documented that MS-2 and poliovirus do not have comparable adsorption characteristics (Gerba et al. 1981, Lukasik et al. 2000, Shields and Farrah 1983). Consequently, the results typically indicate that additional testing of models or possibly further modeling is required; and a greater understanding of viral interactions with surfaces is vital.

PAGE 24

12 Tracers Solutes and biological organisms are used as tracers to delineate the flow path and rate of water in soil and aquifer systems (Bales et al. 1989). Model organisms for pathogenic viruses are also frequently used as tracers to better predict the vulnerability of groundwater sources; and in treatment units (Harvey 1997). Numerous tracers have been used to describe groundwater transport (Tables 1-2 and 1-3). Nonbiological tracers include salts, fluorescent dyes, beads, and radioisotopes. However, there are problems with solutes, such as natural concentrations and adsorption differences (Harvey 1997). Biological tracers include bacteria, yeast, proteins, lipopolysaccarides, viruses, and bacteriophage (Bales et al. 1989). Biological tracers can better predict colloidal transport in a system than solutes can (Bales et al. 1989). The prediction of the adsorption, survival and subsequent elution of colloidal particles is improved by using biological tracers consisting of similar properties. Problems with bacteria, tagged bacteria, protozoa, and yeast include size exclusion, replication in natural environments, background, decay, and inactivatlon (Harvey 1997). Proteins and lipopolysaccarides can be used for adsorption studies. However, they cannot reveal any details about survival in the various systems. Animal viruses have been used as tracers. However, they can be pathogenic to humans; and detection is time-consuming and expensive. Bacteriophage tracers are frequently used in place of enteric viruses, since they are viruses that are easy to use in experiments. Advantages of using bacteriophage as models include small size, negligible effect on water quality, surface characteristics, high titer, detection of low numbers, and host specificity (Harvey 1997).

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13 Interactions between Viruses and Solids The adsorption characteristics of viruses and bacteriophage have been studied using defined media such as microporous filters (Shields and Farrah 2002, Sobsey and Glass 1984). The adsorption characteristics of viruses have also been studied using natural media, such as soils; and mixed-liquor suspended solids (Bales et al. 1993, Dizer et al. 1984, Dov\/d et al. 1998, Gerba et al. 1981, Gerba 1984a, Goyal and Gerba 1979, Griffin et al. 1999, Ketratanakul et al. 1991, Powelson and Gerba 1994, Sobsey et al. 1995). A virus particle is colloidal in nature, so it must be physically adsorbed to a surface in order to be removed. Virus interactions with solids can be described using the DLVO Theory (Derjaguin and Landau 1941, Venwey and Overbeek 1948). Colloidal particles develop a net surface charge, which is dependent on the pH of the environment as well as the presence of ions in the solutions. This layer of surface charges is covered with a layer of counterions (the Stern layer). Another group of counterions extends farther from the particle into solution (the Gouy layer). It is the thickness of this layer that prevents interaction of particles. If the bulk solution of counterions increases by the addition of cationic salts or a decrease in pH, the thickness of this layer decreases because less volume is required to contain enough counterions to neutralize the surface charge. Reducing the thickness results in the articles moving closer to each other, so that van der Waals forces can have an effect. However, frequently hydrophobic and other effects have not been studied.

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14 Table1-2. Solute models frequently used In groundwater studies Solutes Models/ 1 racer Muvanidyco Disadvantaaes Salts: chloride, bromide, inHinp IWUI 1 Iw identify hydrologic parameters, ease of detection, not hazardous Limited by techniques and background, adsorption differences, no dispersion and porosity measurements Fluorescent Dyes: Rhodamine WT, Sodium Fluorescein identify hydrologic parameters, easy to detect Adsorbed easily on the solid material, may be toxic, deactivation, adsorption differences Microspheres identify hydrologic parameters, easy to detect, not hazardous Adsorption controlled by coating, coating stability, adsorption differences, micron size Radioisotopes: identify hydrologic parameters, easy to detect Not natural, expensive, decay rate, water quality hazard, adsorption differences Table1-3. Biotracer models frequently used in groundwater studies Biotracers Models/Tracer Advantages Disadvantages Viruses: Poliovirus Enterovirus, cultivable, resistant Virus types adsorb differently. Direct analysis (time consuming and expensive), low titer, water quality Proteins/LPS: Ferritin, myoglobin, cytochrome-c Capable of mimicking adsorption of viruses. Easy to assay, nonpathogenic, do not replicate in the environment Degradation by environment Bacteriophage: MS-2, PRD1, x174, F+ specific Rapid results, resistant to environmental factors, do not replicate in the environment, high titer, nonpathogenic Virus types adsorb differently, host specificity Bacteria: E. coll. Antibiotic resistance, pigmented, unusual biotypes or spore formers Can identify flow rates, rapid results, easy to assay, Difficult to distinguish from background, can replicate, potential pathogens, inactivation, filterable, transfer of antibiotic resistance

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15 Studies have shown that most viruses may behave differently under similar conditions (Burge and Enkiri 1978, Gerba et al. 1981, Goyal and Gerba 1979). Numerous factors play a role in the virus-solid interactions (Table 1-4). There are significant factors associated with the virus, solution, and solid. Some of the factors associated with the virus include isoelectric point, size, type, and chemical composition (Dowd et al. 1998, Gerba et al. 1991, Hurst et al. 1980a, Yates et al. 1985). Factors associated with the solution include pH, ionic strength, organic compounds, total dissolved solids, temperature, moisture, flow rate (Bales et al. 1993, Dizer et al. 1984, Dowd et al. 1998, Farrah et al. 1981, Farrah 1982, Fuhs 1987, Gerba et al. 1991, Hurst et al. 1980a, Lance and Gerba 1984a, Lukasik et al. 2000, Lytle and Routson 1995, Lytle et al. 1999, Mix 1974, Powelson et al. 1991, Shields and Farrah 1983, Sobsey and Jones 1979, Sobsey et al. 1980a, Taylor et al. 1981, Wallis et al. 1972, Wallis et al. 1979, Yates et al. 1985). Factors associated with the solid include composition, organics, pore size, type, carbon content, moisture content, surface area, pemneability, diffusion, surface charge, ion exchange capacity, and chemical composition (Bitton 1975, Bitton 1980, Dowd et al. 1998, Gerba et al. 1975, Gerba et al. 1981, Gerba 1984b, Gerba et al. 1991, Goyal and Gerba 1979, Hurst et al. 1980a, Kessick and Wagner 1978, Lance and Gerba 1984b, Mix 1974, Shields and Farrah 2002, Sobsey et al. 1980a, Yates et al. 1985). Virus adsorption to solids has been shown to resemble protein adsorption in that hydrophobic and electrostatic interactions influence adsorption (Bales et al. 1991, Farrah et al. 1981, Farrah 1982, Hatefi and Hanstein 1969, Lukasik et

PAGE 28

16 al. 2000, Lytle and Routson 1995, Lytle et al. 1999, Shields and Farrah 1983, Shields and Farrah 2002, Wallis et al. 1979). Shields (2002) reported that there are significant differences in the adsorption properties of numerous bacteriophage and human enteric viruses indicating that viral properties were significant in adsorption. Viruses contain a protein coat, therefore, side chains of amino acids may exhibit charged or hydrophobic groups on the surface, thus solution factors such as pH, ionic strength, and chaotropic agents have been shown to affect adsorption to solids. The surface charge on the virus originates from the ionization of amino acid side chains, and that this charge is a function of pH. When suspended in solutions there may be basic or negatively charged residues that become ionized and give a net surface charge that is pH dependent. A virus will exhibit a negative net surface charge at pH values higher than its isoelectric point, and a net positive surface charge at pH values below its isoelectric point. The results of various studies demonstrate that the adsorptive differences of viruses with similar isoelectric points or characteristics are most likely due to localized groups of negative and positive charges, and possible spans of hydrophobic groups exist on viral surfaces (Mix 1974, Penrod et al. 1996, Schijven 2000, Shields and Farrah 2002). Results have also shown that the isoelectric point is only an indication of the net surface charge at a given pH; it does not convey information about the distribution of charges over the surface of the virus (Penrod et al. 1996). Gerba and colleagues (1981) were able to group viruses adsorbed to nine different soils according to their adsorption characteristics and con-elate them with their isoelectric points. Group I viruses

PAGE 29

17 adsorbed poorly to soils, and group 2 viruses adsorbed favorably to the soils. Group 2 viruses had lower isoelectric points than group 1 viruses. The results show the surface charges of viruses at a specific pH that determine adsorption (Gerba et ai. 1981, Penrod et al. 1996). However, researchers have also shown that size became the predominate factor for particles larger than 60 nm (Dowd et al. 1998). Viral adsorption and survival has been shown to be viral type and site specific (Gerba et al. 1981, Hurst et al. 1980a, Yates et al.1985). Solution factors that have a significant affect on adsorption are the pH as previously described and the ionic strength (Dizer et al. 1984, Dowd et al. 1998, Lance and Gerba 1984a, Powelson et al. 1991, Powelson and Gerba 1994). According to the DLVO theory an increase in ionic strength compresses the double layer causing adsorption by van der Waals and hydrophobic forces. Hydrophobic interactions were shown to be involved in adsorption and transport by using detergents that could be present in wastewater that is applied to the soils (Dizer et al. 1984). These detergents eluted viruses such as poliovirus-1 and rotavirus SA1 1 to an extent. Detergents have been shown to elute phages by making the solution more lipophilic, which allows for the interactions of apolar groups and the solution (Farrah etal. 1981, Farrah 1982). Viral movement in soils has been found to depend on saturation conditions (Lance and Gerba 1984b, Powelson and Gerba 1994). They found that under saturated conditions viral elution and transport were more likely to occur (Lance and Gerba 1984b). Other solution factors such as suspended solids, and dissolved organic compounds have been found to interfere with adsorption of

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18 Table 1^. Factors involved with viral adsorption and transport in soils Factors Comments Ref(s) Media type Increases in the content of clay, surface area, ion exchange capacity, and patches of oxides increase adsorption of viruses. Bitton 1980, Gerba et al. 1981, Gerba 1984b, Goyal and Gerba 1979, Lance and Gerba 1984b, Sobsey et al. 1980a pH Generally, adsorption increase with a decrease in pH. Dizer et al. 1984, Dowd et al. 1998, Gerba 1984b, Goyal and Gerba 1979, Sobsey et al. 1980a, Taylor etal. 1981 Ionic strength (conductivity) Generally there is an increase in adsorption with an Increase in the concentration or valence of cations Dizer et al. 1984, Gerba 1984b, Lance and Gerba 1984a, Sobsey et al. 1980a, Taylor et al. 19oi Soluble organic material Organics such as humus tend to interfere with adsorption of viruses by competing with viruses for adsorption sites on a solid. Dizer et al. 1984, Gerba et al. 1981, Goyal and Gerba 1979, Powelson et al. 1991, Powelson and Gerba 1994, Sobsey et al. 1980a Virus type Adsorption of viruses vary according to viral type and strain, pi differences Dowd et al. 1998, Gerba et al. lyol, ueroa nyon-D, ooydi dnu Gerba 1979 Flow In unsaturated conditions, where water flows through the small pores and forms a film around the particles, viruses tend to adsorb better. Powelson et al. 1991, Powelson and Gerba 1994, Schaub and Qnrhor 1Q77 Flow rate The slower the infiltration rate of water through soils allows for a greater retention time for adsorption to occur. Gerba et al. 1975, Lance and Gerba 1984b viruses to solids as well (Sobsey and Glass 1984). However, it has been found that the degree of viral Interference was dependent on virus type and adsorption conditions (Sobsey and Glass 1984). Suspended solids can interfere with adsorption and elution by adsorbing to the viruses resulting in enhanced adsorption or adsorbing to the filters resulting in clogging (Sobsey and Cromeans 1985a). A high content of dissolved organic compounds can retard adsorption to

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19 solids by competing for adsorption sites, associating with the viruses, or by accumulating on the filter material (Sobsey and Hickey 1985b). Organic matter found in solutions, such as humic materials, are negatively charged particles or soluble compounds that compete with the viruses for adsorption sites on the solid (Gerba 1984b, Sobsey et al. 1980a, Wallis and Melnick 1967). Various soils have been studied for their ability to adsorb and retain viruses. It was concluded clay soils enhance adsorption better than sandy, organic, and mucky soils (Goyal and Gerba 1979, Lance and Gerba 1984b, Powelson et al. 1991. Sobsey et al 1980a). It was shown that viruses could be eluted from sandy soils with rainwater (Sobsey et al 1980a). Other studies have shown that oxides enhance adsorption (Goyal and Gerba 1979, Lance and Gerba 1984b). Survival Considering viral contamination is possible, understanding the survival mechanisms of viruses in groundwater systems is of importance. Many studies have examined viral survival in various systems (Alvarez et al. 2000, Blanc and Nasser 1996, Gerba et al. 1975, Hurst et al. 1980b, Nasser et al. 1993, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995, Ward and Ashley 1978, Ward and Ashley 1979, Yahya et al. 1993). Viral inactivation occurs by protein coat disruption and nucleic acid degradation. There are numerous factors involved with the inactivation of viruses in the environment (Table 1-5) (Gerba et al. 1991, Hurst et al. 1980a, Hurst et al. 1980b, Powelson and Gerba 1994, Schaub and Sorber 1977, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985, Yeager and O'Brien 1979). Temperature has been found to be a significant factor in virus

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20 Table 1-5. Factors involved with survival of viruses associated with solids Factors Comments Ref(s) Temperature At lower temperatures viral survival time increases Alvarez et al. 2000, Blanc and Nasser 1996, Gerba et al. 1975, Hurst et al. 1980a, Nasser et al. 1993, Yahya et al. 1993, Yates etal. 1985 Cations May stabilize viruses Hurst et al. 1980a, Sobsey et al. 1980a, Yates etal. 1985 Soil moisture Viruses survive longer with some soil moisture content. Gerba et al. 1975, Hurst et al. 1980a, Yeager and O'Brien 1979 Sunlight ^ Minor inactivation at ^nil surface or in water systems. Gerba etal. 1975 Soil characteristics Increased adsorption will increase survival time possibly due to viral stabilization. (pH, soil type, cations) Blanc and Nasser 1996, Gerba et al. 1975, Gerba 1984b, Hurst et al. 1980a, Sobsey et al. 1980a, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985 Biological Viruses have been shown to survive longer in sterile soils Gerba et al. 1975, Hurst et al. 1980a, Sobsey et al. 1980a, Sobsey et al. 1986 ^ Inactivation by biological predators and sunlight seem to be minimal. survival (Alvarez et al. 2000, Blanc and Nasser 1996, Hurst et al. 1980a, Nasser et al. 1993, Sobsey et al. 1995, Yahya et al. 1993, Yates et al. 1985, Yeager and O'Brien 1979). Studies have found that viruses survive better at temperatures of 10°C or below, and that there is significant inactivation at temperatures of 23°C and higher (Alvarez et al. 2000, Blanc and Nasser 1996, Nasser et al. 1993). Yates et al. (1985) reported that the only factor tested that was significant to survival of the viruses MS-2, poliovirus 1, and echovirus 1 was temperature. They also reported that there is no significant difference in the decay rates of tested viruses MS-2, poliovirus 1 , and echovirus 1 in groundwater samples (Yates et al. 1985). The ionic strength of the system seems to affect the system.

PAGE 33

21 An increase in ionic strength seems to stabilize the viruses by enhancing adsorption, which enhances survival (Hurst et al. 1980a, Sobsey 1980a, Yates et al. 1985). High moisture content has been found to increase survival as well (Hurst et al. 1980a, Hurst et al. 1980b, Yeager and O'Brien 1979). Minor inactivation at the soil surface has been found to occur by sunlight (Gerba et al. 1975). Increased adsorption by pH, soil type, and ionic strength will increase survival because of viral stabilization (Blanc and Nasser 1996, Gerba 1984b, Gerba et al. 1 991 , Hurst et al. 1 980a, Sobsey et al. 1 980a, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985). Biological predators in natural soils may inactivate viruses (Gerba et al. 1975, Hurst et al. 1980a, Sobsey et al. 1980a, Sobsey et al. 1986). Blanc and Nasser (1996) found PRD-1 was more persistent in natural soil systems with applied treated wastewater than MS-2. Detergents have been reported to alter the thermal stability of viruses therefore affecting survival (Ward and Ashley 1978). Virus Characterization Historically, the classification of viruses has been based on physical parameters approved by the International Committee on the Taxonomy of Viruses (ICW) (Rohwer and Edwards 2002, Van Regenmortel et al. 1999). Some of the techniques used to identify viruses include electron microscopy for structural information, host range, plaque formation, mode of replication, and genomic type (Ackermann 2001, Murphy et al. 1995, Yanko et al. 1999). However, it has been found that some bacteriophage classifications based on morphology and mode of replication are completely different than classifications based on the genome level (Haggard-Ljungquist et al. 1992, Monod et al. 1997,

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22 Tetart et al. 1996). Frequently viral genomes are sequenced and the phages are not characterized based on the physical parameters or vice versa. Due to these problems, bacteriophage can be difficult to classify as indicated by the numerous unclassified phage in the ICW database httD://www.nc bi.nlm.nih.qov/ICTVdb/ (Van Regenmortel et al.1999). Current research has identified other methods of classification that are based on genome organization using protein, and molecular techniques (Clark et al. 2001 , Ford et al. 1998, Hendrix et al. 1999; Juhala et al. 2000, Rohwer and Edwards 2002). Research has shown that there Is no one gene, such as the 16s RNA of prokaryotes that can be used for classification. However, the dinucleotide relative abundance measures of distance > 20Kb provide a unique signature for each genome and can be used to measure similarities in the viruses (Blaisdell et al. 1996). In this study, they have found that each viral genome has a distinctive signature of short oligonucleotide abundances in the entire genome. This indicates that viral genomes can be grouped based on the abundance of short nucleic acid base sequences. Current research has shown the possibility of using "signature genes" of the phage families as a method of classification (Rohwer and Edwards 2002). Hypothetically, structural proteins such as the capsid could be used as a "signature" gene (Bamford et al. 2002, Le Marrec et al. 1997). However, even though structures and functions of the capsid may be conserved viruses may contain no similarity in sequences for easy identification (Rohwer and Edwards 2002). There is evidence that there is structural and functional conservation limited to core viral functions but no sequence conservation (Baker et al. 1999,

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23 Bamford et al. 2002). Even though there is significant diversity in the capsid sequences within a family; there are some surphsing correlations among viruses, for instance, the similarity in genome organization, replication, and capsid structure of adenoviruses and PRD-1 (Bamford et al. 2002). Experimental Rationale Previous studies have used known bacteriophages as models for enteric viruses to study the adsorption, and transport phenomena. The studies have shown that the adsorption properties of enteric viruses vary under similar conditions (Dowd et al 1998, Goyal and Gerba 1979, Gerba et al 1981, Sobsey et al 1995). Researchers have also shown that different strains of the same type of enteric virus have varying adsorption under similar conditions (Goyal and Gerba 1979, Gerba et al 1981). Based on the research it is apparent that no one virus is a good model for determining the interactions of the entire enteric virus group to solids. There are a few studies that have examined the adsorption properties of viruses as one of the predominate factors for modeling enteric viruses (Gerba et al. 1981, Penrod etal. 1996, Shields and Farrah 2002). Gerba and colleagues studied viral adsorption to nine different soil types and reported that viruses could be grouped according to their adsorption properties (1981). Shields and Farrah examined the adsorption of viruses to DEAE-cellulose and octyl sepharose materials and classified the viruses based on the strength of their hydrophobic and electrostatic adsorptive properties (2002). Other research has focused on the adsorption of viruses to media and correlated it to the surfaces charges over

PAGE 36

24 a viral surface (Penrod et al. 1996). The study indicated that the surface charges are significant in the adsorption mechanism (Penrod et al. 1996). In the first part of our study, bacteriophages were selected from filtered raw sewage Isolates based on specific adsorption properties using a variety of hosts commonly found in contaminated water. The bacteriophages were then characterized based on properties such as their hydrophobic or electrostatic nature, and the strength of the interactions to solids to identify a range of phages based on their adsorption characteristics. We also isolated bacteriophages from raw sewage based on their survival at elevated temperatures of 40°C and SOX after three weeks. These temperatures are more selective for phages of fecal coliforms (44.5°C) found in warm-blooded animals. Bacteriophages were then selected based on their survival in UV dechlorinated water at these elevated temperatures over an extended period of time. The second part of our study investigated the factors affecting adsorption of selected bacteriophage to natural media such as mixed-liquor suspended solids and various soils. Variations in pH, salt concentrations, and detergent concentrations were also used in conjunction with the natural media to identify the mechanism of adsorption to natural media. The bacteriophages were then compared with enteric viruses of previous studies (Dizer et al. 1984, Gerba et al. 1981, Goyal and Gerba 1979). The final part of our study involved identifying the families that the selected bacteriophages belong to by various methodologies. The techniques

PAGE 37

25 used were electron microscopy, host range, plaque morphology, nucleic acid determination, protein, and molecular (Ackermann and Nguyen 1983, Clark et al. 2001, Liu et al. 2000, Loessner et al. 1993, Yanko et al. 1999, Zink and Loessner 1992). The phages were compared to known members of the family. The diversity of the capsid sequences of select bacteriophages and viruses from our study and previous studies (Gerba et al. 1981, Penrod et al. 1996, Shields and Farrah 2002) were examined to assist in explaining the differences in adsorption. The phage 60STP2 was studied further considering it represents a worst-case model. Another worst-case model MS-2 has been previously studied (Penrod et al. 1996). The results from our study will be compared against previous work to identify the mechanism or mechanisms involved in adsorption (Penrod et al. 1996, Shields and Farrah 2002).

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CHAPTER 2 VIRAL INTERACTIONS WITH DEFINED MEDIA Introduction There are many methods that have been used to concentrate viruses from water samples. Adsorption to and elution from solids, adsorption to organic or inorganic precipitates, and ultrafiltration are some methods used for concentrating viruses. However, membrane adsorption-elution methodology is most frequently used (Farrah et al. 1976, Rao and Labzoffsky 1969, Wallis and Melnick 1967). The filtration process involves running a sample of water through a filter that adsorbs viruses. The viruses are subsequently eluted off the filter by using a smaller volume of high pH eluant. The first reported use of membrane filters to concentrate viruses from water samples was in 1967 (Wallis and Melnick 1967). Since then there have been numerous studies ufilizing many types of filters including, pleated epoxy-fiberglass Filterite filters (Farrah et al. 1976, Goyal et al. 1980, Sobsey and Jones 1979), nitrocellulose HA millipore filters (Farrah et al. 1976, Shields and Farrah 1983), zeta-plus AMF Cuno microporous filters (Farrah et al. 1981, Goyal et al. 1980, Sobsey and Jones 1979), asbestoscellulose Seitz S filters (Sobsey and Jones 1979), and 1MDS surface modified fiberglass-cellulose resin filters (Shields et al. 1986b, Sobsey and Glass 1980). Chemical modification of filters has been used to increase viral recoveries from water samples (Farrah and Preston 1985, Preston et al. 1988b). Diatomaceous 26

PAGE 39

27 earth and sand filters coated with metallic peroxides, and metallic hydroxides were used to increase adsorption for recovery of viruses from water (Farrah et al. 1991, Lukasiketal. 1996, Lukasiketal. 1999). Adsorption Electrophoretic studies have shown that the various filters used in membrane adsorption-elution methodology have varying net charges. Filterite, millipore HA have net negative charge throughout a pH range of 2-7, and Zetaplus, Seitz S, and 1MDS have a net positive charge over the same pH range (Kessick and Wagner 1978, Sobsey and Jones 1979). Electrophoretic studies have also shown that at neutral pH many viruses have a net negative charge because they have isoelectric points below pH 7 (Brinton and Lauffer 1959). Previous studies have reported that the positively charged filters adsorb viruses at neutral pH and negatively charged filters require the addition of salts at pH 7 or a decrease in pH for significant adsorption of viruses (Farrah et al. 1976, Gerba et al. 1978, Goyal et al. 1980, Shields et al. 1986b, Sobsey and Jones 1979, Sobsey and Glass 1980, Wallis and Melnick 1967, Wallis et al. 1972). Nitrocellulose filters, such as Millipore HA, have been shown to have a net negative charge and hydrophobic characteristics (Farrah et al. 1981, Farrah 1982, Kessick and Wagner 1978, Lukasik et al. 2000, Mix 1974, Shields and Farrah 1983). At pH 3.5, nitrocellulose filters have a net negative charge. This pH is below the isoelectric point of many viruses so they have a net positive charge (Hatefi and Hanstein 1969). Therefore, adsorption may be by electrostatic and hydrophobic interactions (Lukasik et al. 2000, Shields and Farrah 1983, Wallis et al. 1979). The addition of increasing concentrations of chaotropic ions or neutral

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28 detergents has been found to decrease the stmcture making the solution more lipophilic. These solutions can solubilize viruses, there by influencing hydrophobic associations of the viruses to the filters (Farrah et al. 1981, Fujito and Lytle 1996, Hatefi and Hanstein 1969, Lukasiketal. 2000, Shields and Farrah 1983). At neutral pH values, often above the viral isoelectric points, vimses exhibit a net negative charge. The nitrocellulose filters are also negatively charged so minimal adsorption occurs mainly by hydrophobic interactions. Increasing the ionic strength of certain antichaotropic salts, such as MgCb, can screen out electrostatic charges and promote hydrophobic and van der Waals interactions (Farrah et al. 1981, Lukasik et al. 2000, Shields and Farrah 1983, Wallis etal. 1979). Other defined media have been used for studies pertaining to the theoretical viral adsorption phenomena. Organically coated silicates have been used to study the roles hydrophobicity, pH, and ionic strength in adsorption of viruses to solids (Bales et al. 1993, Zerda et al. 1985). The results show that both electrostatic and hydrophobic interactions are involved in adsorption. The studies also indicated that the pH of the solution determines the surface charges of the viruses and adsorbent media. DEAE-sepharose and octyl sepharose materials (Shields and Farrah 2002) were used to investigate the adsorption characteristics of various bacteriophage and viruses. The results show that the viruses can be grouped according to the strength of their adsorption properties. The viruses were shown to adsorb to the materials by predominately electrostatic forces, predominately hydrophobic forces, or a combination offerees.

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29 Inorganic and organic flocculation procedures have been used for concentrating or reconcentrating viruses from water samples. Inorganic flocculation procedures such as, the addition of aluminum hydroxide at a low pH and then the subsequent increase in pH by the addition of 1M sodium carbonate resulted in a floe that adsorbs viruses (Farrah et al. 1976). However, the drawbacks included filter clogging and inefficient recoveries. Organic flocculation procedures such as decreasing the pH of a protein solution to 3.5 produce floes of proteins. The floe is then isolated by centrifugation and solubilized by sodium phosphate solution at pH 9 (Katzenelson et al. 1976). Elution Elution of viruses from solids has been studied to maximize viral recoveries and explain viral adsorption mechanisms. Elution studies used different solutions that altered the ionic strength, or increased the pH. Some of the most efficient methods include, the introduction of beef extract supplemented with glycine and NaOH to obtain a high pH (Preston et al. 1988b, Scott et al. 2002), urea with the addition of lysine at a high pH (Farrah et al. 1981 , Hatefi and Hanstein 1969), and glycine at a high pH by addition of NaOH (Farrah et al. 1976). These solutions tend to interfere with the hydrophobic and electrostatic interactions involved in the adsorption phenomena. Chaotropic agents, large singly charged ions (such as trichloroacetate) or neutral detergents disrupt hydrophobic interactions. Antichaotropic agents are small singly charged ions such as fluoride and chloride, or multivalent ions such as phosphate or sulfate. Solution of metal chelators such as EDTA, an antichaotropic agent have failed to elute viruses off negatively charged filters at a high pH indicating that

PAGE 42

30 electrostatic interactions play a minimal role in adsorption at this pH and hydrophobic interactions are the predominate forces (Farrah et al. 1981, Kessick and Wagner 1978, Lukasik et al. 2000, Mix 1974, Shields and Farrah 1983). These solutions also suggest that cationic bridging, or reversal of charge on the filter media are not factors in adsorption at neutral pH. Current Methods However, studies have shown that the extremes in pH for the electronegative filter adsorption method and other techniques inactivate some bacteriophage (Goyal et al. 1980, Primrose et al. 1981 , Scott et al. 2002). This is a problem since bacteriophage are often used in viral tracer studies, as proposed indicators of their bacterial host (possible presence of fecal pollution), and as models for water treatment processes (Griffin et al. 1999, Harvey 1997, Havelaar et al. 1993, Paul et al. 1995, Paul et al. 1997). Current methods include concentrating by filtration with the addition of salts, enrichment, and direct plating (US EPA 2001). To overcome this limitation investigators have proposed to use electropositive filters (Goyal et al. 1980, Shields et al. 1986b, Sobsey and Jones 1979), chemical modification of filters (Farrah and Preston 1985, Preston et al. 1988b), or stabilizing the bacteriophage at a low pH with the addition of manganese chloride to the sample (Scott et al. 2002). Another flocculation procedure for the recovery of bacteriophage from small volumes or as a reconcentration step at pH 7 involves the addition of saturated ammonium sulfate to beef extract for the formation of floes that adsorb viruses (Shields and Farrah 1986a).

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31 Materials and Methods Collection of Raw Sewage Samples Raw sewage was collected at the University of Florida Wastewater Reclamation Facility in Gainesville, Florida. The samples were filtered through 0.2 [im porosity filter (Millipore GS; Millipore Corp., Bedford, Mass.) to remove debris. Media The defined media used in adsorption studies were the microporous filters, 0.45 pm nitrocellulose membrane filters (Millipore type HA, Millipore Corp., Bedford, Mass.) and 0.45 pm 1-MDS (charge-modified resin filters, AMF Cuno, Meridan, Conn.), diatomaceous earth (grade 1; Sigma Chemical Co. St. Louis, Mo.), and aluminum hydroxide coated nanosized filter fibers (Argonide Sanford, FL). Chemicals The chemicals used in our study include imidazole, glycine, magnesium chloride, sodium chloride, ferric chloride, aluminum chloride, ammonium hydroxide, and Tween 80 from Sigma Co.; the chemicals obtained from Fischer Scientific Co. were sodium chloride and sodium citrate. The beef extract was obtained from Difco. Solutions The buffer solution used in our study was 0.02M imidazole plus 0.02M glycine made in deionized water. All solutions in our study were made using 0.02M imidazole-glycine buffer. The buffer and solutions were adjusted to the required pH by the addition of NaOH or HCI. Elution of the viruses from the filter material were performed using Beef Extract solutions (Difco) or 0.1% Tween 80 and 1M

PAGE 44

32 NaCI. Diatomaceous earth (Sigma Chemical Co., St. Louis, MO.) was coated with solutions of ferric chloride, aluminum chloride, and ammonium hydroxide as described by Lukasik et al. (1996). Viruses Sources The following phages with their hosts were used in these studies: MS-2 (ATCC 15597-B1) was assayed using Escherichia coli 3000 (ATCC15597), PRD1 was assayed using Salmonella typhimurium (ATCC 19585), 0X174 (13706-B1) was assayed using Escherichia co// C (ATCC 13706). Indigenous bacterial viruses were isolated from raw sewage, grown, and assayed using the bacterial hosts Escherichia coli 3000 (ATCC15597), Escherichia co// C (ATCC 13706), Salmonella typhimurium (ATCC 19585), Proteus vulgaris, Citrobacterfreundii, Enterobacter aerogenes, and Klebsiella pneumoniae were obtained from the Microbiology and Cell Science culture collection, UF. Preparation of phage stocks An isolated plaque was excised and added to 3% Tryptic Soy Broth (Difco) with the appropriate host and incubated overnight at 37°C. Phages solutions were plated using the standard agar overlay procedure (Snustad and Dean 1971). The next day the lysates plates were scraped and centrifuged at 12,100xg (Beckman J2-HS centrifuge) for 10 minutes (Sambrook et al. 1989). Deionized water was used to scrape lysates for the electron microscopy studies of adsorption. The supernatant was filtered with 0.45pm pore sized filters (Millipore Corp, Bedford Mass.) to create a stock solution. Phage stocks were concentrated by a 10% PEG 8000 (polyethylene glycol) procedure (Sambrook et

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33 al. 1989). Phage were plated and assayed as plaque-forming units (PFU/mL) using the standard agar overlay procedure (Snustad and Dean 1971). Poliovirus-1 LSc strain (ATCC VR-59) was assayed as PFU on BGM cells using an agar overlay method (Smith and Gerba 1982). Isolation of Bacteriophage Using Nitrocellulose Filters Procedure 1 A 10-mL filtered raw sewage sample with the addition of imidazole glycine buffer to a final concentration of 0.02 M or the addition of buffer and NaCI, MgClz, or Na3C6H507-2H20 to a final concentration of 0.01 M was passed through a series of three 0.45 pm HA filters (Millipore) at pH 7. Viruses that adsorbed to the first in the series of three filters were eluted with 3% Beef Extract at pH 9 and isolated. Viruses that were not adsorbed after passage through three filters were also isolated. The samples were then plaque assayed using bacterial hosts previously mentioned according to standard procedure. Procedure 2 A 10-mL filtered raw sewage sample in imidazole glycine buffer to a final concentration of 0.02 M, or buffer with the addition of 0.01 % Tween 80 was passed through a series of three 0.45 pm HA filter (Millipore) at pH 3.5. Viruses that were not adsorbed after passage through three filters were isolated, and viruses that adsorbed to the last of the series of three filters were eluted with 3% Beef Extract at pH 9 and isolated. The samples were then plaque assayed using bacterial hosts previously mentioned according to standard procedure.

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34 Isolation of Bacteriophage Resistant to High Temperature Initially, raw sewage was placed in a 40°C or SOX water bath for 4 weeks, and assayed weekly. Bacteriophages were isolated after 4 weeks and plaque assayed using bacterial hosts previously mentioned according to standard procedure. Selection of Bacteriophage for Additional Studies Procedure 1 The isolated bacteriophage at a 10^ PFU/ mL concentration in 0.02 M imidazole glycine buffer, or buffer with the addition of 0.1 M MgCl2 was passed through a 0.45 micron HA filter (Millipore) at pH 7 to select phage based on hydrophobic adsorption properties. Bacteriophage were selected that adsorbed to the filter in buffer or buffer and salt solutions as predominately hydrophobic. Bacteriophages were also selected that did not adsorb to the filter in the presence of buffer and salt solutions as weakly hydrophobic. The samples were then plaque assayed using standard procedure. Procedure 2 Isolated bacteriophage at a lO^PFU/ mL concentration in 0.02 M imidazole glycine buffer, or buffer with the addition of 0.1% Tween 80 was passed through a 0.45 |im HA filter (Millipore) at pH 3.5 to select bacteriophage based on electrostatic adsorption properties. Bacteriophages were selected that did adsorb to the filter in the presence of buffer and detergents as predominately electrostatic. Bacteriophages were also selected that did not adsorb to the filter in buffer solutions as weakly electrostatic. The samples were then plaque assayed using standard procedure.

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35 Procedure 3 The isolated bacteriophage at a concentration of lO^PFU/ mL were placed In UV dechlorinated water and, incubated in a 40°C or SOX water bath for extended time periods and assayed weekly. The absence of residual chlorine was ascertained by the addition of orthotoulidine (Clesceri et al. 1998). Characterization of Bacteriophage Adsorption to Filters under Varying Conditions Procedure 1 The selected isolates at a 10^ PFU/mL concentration in 0.02M imidazole glycine buffer at pH 7, with the addition of 0.001 M MgCl2, 0.01 M MgClz, or 0.1 M MgCIa was passed through 0.45 HA filter (Millipore). Elution of the filters was done with 3% Beef Extract at pH 9. The samples were then plaque assayed using standard procedure. Procedure 2 The selected isolates at a 10^ PFU/mL concentration in 0.02M imidazole glycine buffer at pH 7 were passed through a 0.45 ^m 1 MDS filter. The filter was serial eluted with 10 mL 0.02M imidazole glycine buffer, and then varying concentrations of 0.001 M MgCl2, 0.01 M MgCl2, orO.IM MgCl2. The samples were then plaque assayed using standard procedure. Procedure 3 The selected isolates at a 10^ PFU/mL concentration in 0.02M imidazole glycine were also passed through a 0.45 ^im HA filter (Millipore) at pH 3.5 and the filter was serial eluted with 10 mL 0.02M imidazole glycine buffer, and then varying concentrations of a neutral detergent, 0.001% TweenSO, 0.01%

PAGE 48

36 TweenSO, and 0.1% TweenSO. Final elution was with 3% Beef Extract or 0.1% Tween 80 and 1M iVIgClj. The resultant samples were then plaque assayed as previously mentioned. Bacteriophage Interactions to Particles Diatomaceous earth coating Diatomaceous earth was coated according to a two-step procedure (Lukasik et al. 1996). Diatomaceous earth was mixed with 0.25M FeCb for 30 minutes, drained and air-dried. Then the solid was soaked in 3M ammonium hydroxide for 10 minutes, rinsed and dried. The second part of coating consisted of soaking the solid in 0.5M AICI3 for 30 minutes, drained and dried, soaked in 3M ammonium hydroxide for 10 minutes, rinsed and dried. Adsorption procedure A O.lg sample of Argonide (Sanford, PL) aluminum hydroxide coated nanoparticles (filter fibers) or coated diatomaceous earth (Lukasik et al. 1996) were added to a concentration of 10^ PFU/ mL bacteriophage in deionized water, agitated on shaker (Reliable Scientific) for ISminutes, 30 minutes, and 1 hour. The samples were centrifuged at 1200xg (Dynac II centrifuge) for 15 minutes. A sample of the supernatant was plaque assayed using standard procedure. Electron microscopy procedure A stock solution of 10^^ PFU/ mL bacteriophage and O.lg of coated filter fibers were agitated on shaker (Reliable Scientific) for 1 hour. The samples were centrifuged at 1200xg (Dynac II centrifuge) for 15 minutes. A supernatant sample was removed and studied using electron microscopy (Ackermann and Nguyen 1983, Yanko et al. 1999). One drop of sample was placed on a 300 mesh

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37 formvar coated grid for 1 minute. The excess was removed using filter paper. The grid was rinsed with two drops distilled water and the excess was removed using filter paper. Two drops of 1% Uranyl acetate was applied to the grid for 1 minute and the excess was removed using filter paper. The grid was air dried and examined on a Zeiss electron microscope operating at 80kV or 100 kV (Bozzola and Russell 1999). Results Isolation of Bacteriophage from Raw Sewage Samples We used the standard method of adsorption-elution using negatively charged microporous filters under the two conditions described in the Materials and Methods section to isolate indigenous bacteriophage from raw sewage. This procedure resulted in the isolation of 86 bacteriophages (Table A-1). We also isolated other phages based on their survival in raw sewage sample at elevated temperatures over a 4-week period. 1^^ set of conditions At pH 7, viruses suspended in buffer or buffer and salts solutions that adsorbed to the filters were eluted with 3% beef extract at pH 9 and exhibit strong hydrophobic characteristics. Viruses from the resulting salt solutions that did not adsorb were isolated and considered to exhibit weak hydrophobic characteristics (Table 2-1). The data indicates that MgCb and Na3C6H507-2H20 enhance the adsorption of indigenous bacteriophage to the filters at a pH 7 by approximately 34%. Based on the T-test there is no significant difference between adsorption in the presence of MgCl2 and Na3C6H507-2H20 (sodium citrate) (Table 2-1).

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38 2"'' set of conditions At pH 3.5, viruses in buffer solutions that did not adsorb to the filters were isolated and considered to exhibit weak electrostatic characteristics. Viruses in the Tween 80 solution that adsorbed to the third filter were eluted with 3% Beef Extract at pH 9 and considered to exhibit strong electrostatic characteristics (Table 2-1). The data indicates that adsorption of indigenous bacteriophage to the filters at pH 3.5 is influenced by both hydrophobic and electrostatic forces with phage adsorption at approximately 94% with a standard deviation of 2%. Although there is a decrease in adsorption with the addition of neutral detergent to approximately 54% with a standard deviation of 13%, a significant portion of indigenous phage remain adsorbed in the presence of a neutral detergent. Survival at elevated temperatures The survival of indigenous phages in raw sewage was studied over a period of four weeks. There was a steady significant decrease in the population of indigenous bacteriophage (using the hosts E. coli C3000, E. coli KC, S. typhimurium, and K. pneumoniae) at the elevated temperatures of 40°C and 50°C. The survival of phages at 4°C indicates that a significant decrease in the phage population is due to the increase in temperature and not the composition of the raw sewage solution. At the end of the four-week period, phages were isolated as being more resistant to environmental factors (Figure 2-1). Selection of Bacteriophage The isolated bacteriophages were then selected based on the adsorption characteristics, either strong or weak hydrophobic and either strong or weak electrostatic interactions. Adsorption of the phages 60KCK2 (80%) and 61KCK10

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39 Table 2-1 . Adsorption of bacteriophage in filtered raw sewage at pH 7 and pH 3.5' SOLUTIONS % ADSORPTION ^ Buffer pH 7 61 +/5 C 0.01M NaCI pH 7 82 +/7 B 0.01 M MgCl2pH 7 97 +/3 A 0.01 M Na3C6H507-2H20 pH 7 94 +/4 A Buffer pH 3.5 94 +/2 A 0.01%Tween 80 pH 3.5 56 +/13 B ^ Prefiltered raw sewage with an initial phage concentration of approximately 5x10^ PFU/mL was passed through a series of three 0.45[jm microporous filters in various solutions. The hosts used include Escherichia coli 3000, Escherichia coliC, Salmonella typhi murium, Proteus vulgaris, Citrobacter freundii, Enterobacter aerogenes, and Klebsiella pneumoniae. ''Average of adsorption to the three-0.45ijm Millipore HA filters. ^Values at the same pH followed by the same letter are not significantly different at P=0.05. (82%) to 0.45|jm HA millipore filters at pH 7 in 0.02M imidazole glycine buffer suggests strong hydrophobic interactions (Table 2-2). However, the minimal adsorption of the phage 60STP2 (46%) to the filters at pH 7 with the addition of 0.1 M MgCl2 salts indicates weak hydrophobic interactions (Table 2-2). At pH 3.5, we found that hydrophobic and electrostatic interactions play a role in adsorption. At pH 3.5, the poor adsorption of the bacteriophage 60STP2 (3%) in the presence of 0.02M imidazole glycine buffer suggests weak electrostatic interactions (Table 2-2). The adsorption of the bacteriophage 61KCK10 (54%) in the presence of 0.1% Tween 80, which disrupts hydrophobic interactions at pH 3.5 indicates predominate electrostatic interactions (Table 2-2). We selected bacteriophage that exhibited weak or strong hydrophobic characteristics at pH 7,

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40 1 ^ 1 ^ ^ • 0 12 3 4 TIME (Week) 'Phage 4°C --^-PHA^ PHAGE SOX Figure 2-1 . Raw sewage bacteriophage survival at 40°C and 50°C over time^ ^Bacteriophage in raw sewage at various temperatures were assayed, and isolated at 4 weeks using the hosts Escherichia coli 3000, Escherichia coli C, Salmonella typhimurium, Proteus vulgaris, Citrobacterfreundii, Enterobacter aerogenes, and Klebsiella pneumoniae. and phage that exhibited weak or strong electrostatic characteristics at pH 3.5. Some bacteriophage such as 61STP7 and 61KCK10 were also selected based on their resemblance to poliovirusi adsorption properties (Table 2-2). The isolated bacteriophage at a concentration of 10 PFU/mL were placed in UV dechlorinated water and, incubated in a 40°C or 50°C water bath for extended time periods. At 40°C, all the isolated phage but 60STP2 had a 2

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41 Table 2-2. Adsorption of selected bacteriophage to microporous filters at pH 7 and pH 3.5' SOURCE OF PHAGE HDCIT 1 1 V_y O 1 %ADSORPTION OF VIRUSES AT pH7: % ADSORPTION OF VIRUSES AT pH 3.5: Buffer^ 0.1M MgCl2 " Buffer^ 0.1% Tween 80" ISOLATED FROM SEWAGE (Our study) E. Coli C3000 60C3F2 36+10 91+6 93 + 10 33 + 8 E. Coli C 60KCK2 80 + 4 100 + 2 92 + 6 37 + 8 61KCK9 31 + 10 91 +.7 90 + 10 9 + 12 61KCK10 82 + 6 99 + 3 59 + 6 54 + 9 s. Typhimurium 60STP2 0 + 3 46 + 6 3 + 2 3 + 6 61STP7 0 + 2 69 + 4 29 + 6 14 + 4 CULTURE COLLECTION E. Coli C3000 MS2 18 + 8 75 + 8 100 + 8 27 + 5 £. Coli C 0X174 0 + 5 87 + 5 82 + 6 45 + 5 S. Typhimurium PRD1 22 + 6 62 + 6 99 + 3 33 + 9 BGM Cell line POLIO 1 0 + 3 72 + 3 98 + 4 60 + 12 ^The solutions containing 10^ PFU/mL viruses were passed through 0.45 pm filter. "All solutions are in 0.02M imidazole-glycine at pH 7 or pH 3.5. log or less reduction over a 12-week time span indicating an insignificant reduction in the phage population (Figure 2-2). The laboratory cultures and 60STP2 had a more significant reduction in population over the six week time period (Figure 2-2). At 50°C, the results show the only phage not to show a significant reduction in population is 61KCK10 (Figure 2-3). The laboratory cultures and other selected phages showed a substantial decrease in numbers over the six week time period (Figure 2-3).

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42 7 1 2 — 1 J , 1 ; 0 4 8 12 TIME(Week) 60STP2 )( 61STP7 ™60KCK2 61KCK10 -^ '^MS2 — -PRD1 — ^ -PHIX Figure 2-2. Survival of bacteriophage at 40°C in UV dechlorinated water^ ^Bacteriophage at a concentration of 10^ PFU/mL were placed in tubes of 9 mL UV dechlorinated water and placed in 40°C water bath. Characterization of Bacteriophage The characterization of selected bacteriophages at a 10^ PFU/mL concentration were carried out using 0.45 pm HA millipore filters and 0.02M imidazole-glycine buffer and various concentrations of MgCb at pH 7. At neutral pH, the filter and bacteriophage exhibit negative surface charges. Due to the charges, bacteriophages tend to adsorb poorly in the presence of buffer. The results show that the previous results obtained in our study are accurate, the bacteriophages 60KCK2 and 61KCK10 adsorbed in buffer alone indicating strong hydrophobic interactions. The electrostatic repulsion decreases

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43 2 4 TIME(Week) •60STP2 MS2 "61STP7 'PRD1 •60KCK2 PHIX '61KCK10 Figure 2-3. Survival of bacteriophage at SOX in UV dechlorinated water^ ^Bacteriophage at a concentration of 10^ PFU/mL were placed in tubes of 9 mL UV dechlorinated water and placed in SOX water bath. as the ionic strength increases due to screening of the surfaces charges, which generally promotes adsorption. The bacteriophage 60STP2 adsorbed poorly in the presence of salts indicating that it exhibits weak hydrophobic interactions (Figure 2-4). Characterization of selected phages by adsorption to microporous filters were carried out using 0.4S pm HA millipore filters and subsequent rinses with 0.02M imidazoleglycine buffer and increasing concentrations of Tween 80 (a neutral detergent) at pH 3.S. At a low pH, the filter exhibits a negative charge and generally bacteriophages exhibit positive surface charges. Due to the charges, bacteriophages tend to adsorb to the filter in the presence of buffer at

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44 0.02 0.0225 0.045 0.27 Ionic Strength (MgClj) 60KCK2 61KCK9 — 61KCK10 — 60STP2 61STP7 — ^ "MS2 — ^ -POLIO Figure 2-4. Bacteriophage adsorption to 0.45 pm HA nitrocellulose filters at pH 7 in the presence of MgCb^ ^All solutions contain 10^PFU/mL viruses in 0.02M imidazole glycine buffer and increasing concentrations of MgCl2 were passed through a 0.45 |jm filter. All solutions were adjusted to pH 7. low pH due to electrostatic and hydrophobic charges. The bacteriophage 60STP2 adsorbed initially at 3 % and did not elute off with increasing detergent concentration indicating weak electrostatic interactions (Figure 2-5). As the concentration of Tween 80 increases; hydrophobic interactions are disrupted. The phage 61KCK10 adsorbed initially at 59 % and was not eluted from the filter in the presence of high Tween 80 concentrations. This indicates that electrostatic interactions predominate (Figure 2-5).

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45 Figure 2-5. Bacteriophage elution from 0.45 pm HA nitrocellulose filters at pH 3.5 in the presence of Tween 80^ concentration of 10 PFU/ mL bacteriophage in 0.02M imidazole glycine buffer was passed through a 0.45 pm filter and subsequently rinsed with 0.02M buffer and increasing concentrations of Tween 80. All solutions are in 0.02M imidazole glycine buffer and were adjusted to pH 3.5. Further characterization of the selected bacteriophages was done using 1MDS electropositive filters at a neutral pH. At pH 7, bacteriophages exhibit negative surface charges and readily adsorb to 1MDS filters. Subsequent rinses with 0.02M imidazole glycine buffer and increasing concentrations of MgCb indicated that the antichaotropic agent, MgCIa is capable of eluting adsorbed viruses. Based on the results, the bacteriophage 61KCK10 and poliovirus-1 exhibit predominate electrostatic interactions and the bacteriophage 60STP2 and 61STP7 exhibit weak electrostatic interactions (Figure 2-6).

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46 0.020 0.0225 0.033 0.045 0.270 Ionic Strength (MgCl2) )!<-' 60STP2 -61STP7 60KCK2 — ^^61KCK9 — ^61KCK10 • 'MS2 — =-f = POLIO 1 Figure 2-6. Bacteriophage elution from 0.45 |jm 1MDS charged modified filters at pH 7 in the presence of MgCl2^ ^All solutions are in 0.02M imidazole glycine buffer and were adjusted to pH 7. A concentration of lO^PFU/ mL bacteriophage in 0.02M imidazole glycine buffer was passed through a 0.45 [jm filter and subsequently rinsed with 0.02M buffer and increasing concentrations of MgC^. The diatomaceous earth was modified with a mixture of 0.25MFeCl3 and 0.5M AICI3 and rinsed with 3N ammonium hydroxide to give a metallic hydroxide coating (Lukasik et al. 1996). The modified nanosized filter fibers from Argonide (Sanford, FL) have an aluminum hydroxide coating. Batch adsorption results show that most adsorption occurs within the first 15 minutes (Table 2-3). The bacteriophage MS2 adsorbs poorly to unmodified diatomaceous earth, and over 80% to the other modified materials (Table2-3). The phage 60STP2 adsorbed poorly to all the materials (Table 2-3). The electron micrograph of MS2 to the

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47 Table 2-3. Adsorption of bacteriophage to defined media^ ADSORPTION TO DEFINED MEDIA MEDIA PHAGE Percent Adsorption 1 Ol 1 III 1. yjKJl Mill. finmln Wwl 1 1 II 1 . Diatomaceous Earth MS2 0 10 27 60STP2 0 0 2 Modified Diatomaceous Earth MS2 87 93 100 60STP2 0 13 32 Modified Filter Fibers MS2 81 92 99 60STP2 27 38 44 ^A O.lg sample of diatomaceous earth, modified diatomaceous earth, or coated filter fibers (Argonide) was suspended in deionized water with an initial concentration of 10^ PFU/mL bacteriophage and mixed for 15, 30, or 60minutes. Argonide coated filter fibers show that the MS2 phage adsorbs to the material (Figure 2-7). The electron micrograph of 60STP2 to the Argonide coated filter fibers indicates minimal adsorption to the coated filter fibers by the head (capsid) portion of the phage at 50K (125,000x) (Figure 2-8). Discussion Bacteriophages were isolated using membrane adsorption-elution filtration methodology (Farrah et al. 1976, Rao and Labzoffsky 1969, Wallis and Melnick 1967). The results have shown that bacteriophages can be isolated, selected, and characterized based on their adsorption properties. It is thought that the different types of viruses adsorb differently due to, the differences in the surface proteins of the capsid (Hatefi and Hanstein 1 969, Shields and Farrah 2002). Viruses contain a protein coat therefore they may exhibit charged or hydrophobic groups on the surface, and thus factors such as pH, ionic strength, and chaotropic agents have been shown to affect adsorption to solids. By using

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48 100nm Figure 2-7. MS-2 adsorption to nanosized filter fibers^ stock solution of 10^^ PFU/mL bacteriophage and 0.1g of coated filter fibers were agitated for 1 hour in deionized water, centrifuged, and a sample of supernatant was removed and studied using electron microscopy. Electron micrographs were taken at 50K (125,000x). defined media we were able to alter the properties of the solution to change viral characteristics. At pH 7, nitrocellulose filters exhibit a negative charge and viruses exhibit a negative charge so adsorption by electrostatic interactions should be minimal. We were able to show the extent of viral hydrophobic interactions by increasing the concentration of an antichaotropic agent such as MgCl2. Antichaotropic agents have been shown to disrupt electrostatic interactions by screening the charges, which would promote hydrophobic and van der Waals interactions (Farrah et al. 1981). Our results show that there was

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49 Figure 2-8. 60STP2 adsorption to nanosized filter fibers^ stock solution of 10^^ PFU/mL bacteriophage and 0.1g of coated filter fibers were agitated for 1 hour in deionized water, centrifuged, and a sample of supernatant was removed and studied using electron microscopy. Electron micrographs were taken at 50K (125,000x). a significant difference in adsorption of bacteriophage by filters in the presence of monovalent and multivalent salts are in agreement with previous studies (Lukasik et al. 2000). However, the results also show that there was no significant difference in adsorption of bacteriophage by filters in the presence of divalent or trivalent salts, which is in opposition to previous reports (Gerba 1984b, Mix 1974, Wallis et al. 1972, Wallis et al. 1979). At pH 3.5, nitrocellulose filters exhibit a negative charge and viruses exhibit a positive charge therefore electrostatic and hydrophobic interactions are possible. We were able to show the extent of viral

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50 electrostatic interactions by increasing the concentration of a neutral detergent such as TweenSO. The addition of a chaotropic agent or neutral detergent promote elution of phages by making the solution more lipophilic which allows for the interactions of apoiar groups and the solution (Farrah et al. 1981 , Farrah 1982). Our results show that Tween 80 (a neutral detergent) does promote elution of viruses to an extent by disrupting hydrophobic interactions. However, the amount of elution is dependent on the viral properties. Further elution can be accomplished by using a combination of a chaotropic and antichaotropic agents, or a protein solution such as beef extract, which disrupts both electrostatic and hydrophobic interactions. Adsorption of select viruses such as MS2, a predominantly hydrophobic bacteriophage to modified defined media has been previously studied (Bales et al. 1993. Farrah et al. 1991, Lukasik et al. 1996, Lukasik et al. 1999, Penrod et al. 1996, Shields and Farrah 2002, Zerda et al. 1985). In the current study we used hydroxide coated diatomaceous earth and filter fibers to examine the difference in adsorption between MS2 a frequently used model, and a phage 60STP2. Our studies were in agreement with previous studies that MS2 adsorbed well to the modified material (Farrah et al. 1991, Lukasik et al. 1996, Lukasik et al. 1999). However, our studies showed that the phage 60STP2 adsorbed poorly to these modified materials. When the phage 60STP2 did adsorb it was by the head (protein capsid). Environmental influences have also been shown to influence adsorption and survival. These factors include; dissolved organic compounds (Sobsey and

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51 Hickey 1985b), suspended solids (Sobsey and Cromeans 1985a), temperature, and biological predators. The raw sewage used for isolation based on survival at elevated temperatures was not prefiltered to remove debris. However, our results of bacteriophage survival at 4°C, 40°C, and 50°C show that the decrease in the phage population was primarily due to an increase in temperature and not the composition of the raw sewage samples. The results show that bacteriophage can be selected as models for human pathogenic viruses based on their adsorption characteristics. We have isolated a range of bacteriophage that can be used as models for other enteric organisms for various applications based on their adsorption and survival properties. The results show that understanding the properties of the virus, solid, and environment are important parameters for identifying the mechanism of adsorption.

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CHAPTER 3 VIRAL INTERACTIONS WITH NATURAL MEDIA Introduction The presence of enteric viruses in groundwater has been documented (Table 3-1) (Griffin et al. 1999, Keswick and Gerba 1980, Vaughn et al. 1978, Yates et al. 1985). As previously stated contamination of groundwater occurs through a variety of sources including septic tanks, landfills, artificial recharge, inefficient water treatment processes, polluted surface waters, runoff, and drainfields (Yates et al. 1985). Viruses have been known to travel several meters in a variety of systems (Bales et al. 1989, Schaub and Sorber 1977, Skilton and Wheeler 1988, Vaughn et al. 1978, Vaughn et al. 1983, Yanko et al. 1999). Since water treatment processes do not completely remove enteric viruses; understanding the adsorption properties of the soil system is important. Table 3-1 . Detection of viruses in groundwater ENTERIC VIRUSES TYPES References Polio 1,2,3 Griffin et al. 1999, Vaughn et al. 1978 Coxsackie A16, B3, 84 Griffin et al. 1999, Vaughn et al. 1978 Echo 6, 7, 11, 12,21,23, 24, 25 Griffin et al. 1999, Vaughn et al. 1978 Hepatitis A Griffin et al. 1999 Nonrt/alk Griffin etal. 1999 Unknown Vaughn etal. 1978 52

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53 Adsorption Viral adsorption in natural media has been well studied (Bales et ai. 1989, Bitton 1975, Bitton 1980, Dizeret al. 1984, Dowd et al. 1998, Fuhs 1987, Funderburg et al.1981, Gerba et al. 1975, Gerba et al. 1981, Gerba 1984b, Goyal and Gerba 1979, Ketratanakul et al. 1991, Lance and Gerba 1984b, Powelson et al. 1991, Powelson and Gerba 1994, Schaub and Sorber 1977, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995). The behavior of bacteriophage adsorption to natural media such as mixed-liquor suspended solids and soils are complex. The results show that adsorption to natural media seems to be due to various forces such as van der Waals forces, hydrophobic, and electrostatic interactions (Bitton 1975, Gerba 1984b, Dizeret al. 1984, Dowd et al. 1998, Lance and Gerba 1984a, Powelson et al. 1991, and Taylor et al. 1981). At a neutral pH, soils generally have a net negative charge and many viruses have a net negative charge. Numerous studies have shown that there is less adsorption at higher pH values (Burge and Enkiri 1978, Sobsey et al. 1980a, Taylor et al. 1981). This can be explained according to the DLVO theory that at a higher pH there is an increased electrostatic repulsion between the solid and virus particles. Research has shown that the addition of antichaotropic agents such as MgS04 promotes adsorption primarily by strengthening hydrophobic interactions (Farrah 1982, Hatefi and Hanstein 1969). According to the DLVO theory an increase in ionic strength compresses the double layer causing adsorption by van der Waals and hydrophobic forces. Hydrophobic interactions were studied using octyl sepharose columns, which contain hydrophobic groups with varying concentrations of NaCI (Shields and Farrah 2002). As the

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54 . concentration of salts decrease the double layer is less compressed and allows for electrostatic repulsion. This allows viruses with the least hydrophobicity to be eluted first (Shields and Farrah 2002). It has also been found in various studies that chaotropic agents, urea, ethanol, and neutral detergents tend to solubilize hydrophobic molecules and disrupt hydrophobic interactions (Farrah et al. 1981, Hatefi and Hanstein 1969). However, it is also important to note that natural media are heterogeneous surfaces composed of patches of positive charges on negatively charged particles. These sites are often composed of oxides that carry a positive charge and can adsorb negatively charged viruses at near neutral pH. The activation thermodynamics of viral adsorption to various filter media have also been studied (Preston and Farrah 1988a). They studied kinetics as a function of temperature, and the results indicated that adsorption was a physical process and describes the initial stages of adsorption to microporous filters (Preston and Farrah 1988a). They also have shown that correlation between the percent of viral adsorption and the energy of activation were dependent on the solids, and that these differences may influence the mechanism of adsorption (Preston and Farrah 1988a). Tracers Theoretically under certain conditions groundwater contamination is possible by viral transport through the soils. Many investigators have studied transport by using tracers (Tables 1-2 and 1-3) (Bales et al. 1989, Gerba 1984a, Griffin et al. 1999, Harvey 1997, Paul et al. 1995, Paul et al. 1997, Schaub and Sorber 1977, Skilton and Wheeler 1988, Vaughn et al. 1978, Vaughn et al. 1983, Yanko et al. 1999). Bacteriophage tracers are frequently used in place of enteric

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55 viruses since they are easier and safer to work with. Models that have been used frequently include MS-2, PRD-1, other F+ specific bacteriophage, and 0X174 (Schijven 2000). F+ specific phage such as MS-2, and PRD-1 are hydrophobic bacteriophage that do not adsorb well to solids and are relatively stable are used as worst-case models for possible contamination (Goyal and Gerba 1979, Havelaar et al. 1993, Schijven 2000). Biological tracers can better predict colloidal transport in a system than solutes (Bales et al. 1989). The prediction of the adsorption, survival and subsequent elution of colloidal virus particles is improved by using biological tracers consisting of similar properties. Survival Considering viral contamination is possible, understanding the survival mechanisms of viruses in groundwater systems is of importance. There are numerous factors involved with the inactivation of viruses in the environment; such as temperature, ionic strength, moisture content, pH, soil type, sunlight, and biological predators as previously stated (Table 1-5) (Gerba et al. 1991 , Hurst et al. 1980a, Hurst et al. 1980b, Powelson and Gerba 1994, Schaub and Sorber 1977, Sobsey et al. 1980a, Sobsey et al. 1986, Sobsey et al. 1995, Ward and Ashley 1978, Ward and Ashley 1979, Yates et al. 1985, Yeager and O'Brien 1979). At low temperatures, viruses survive for a longer period of time than at higher temperatures. Other components such as detergents, and pH changes can affect the stability of the viruses (Ward and Ashley 1979). It is important to note that inactivation is generally negligible within the time scale of the

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56 experiments. However for field experiments viral inactivation may be significant, especially under some conditions such as unsaturated, low flow rate, high adsorption. Materials and Methods Media Samples of mixed-liquor suspended solids were obtained from the University of Florida Wastewater Reclamation Facility in Gainesville, Florida. Soils were obtained from the University of Florida Natural Area Teaching Lab (NATL), Gainesville, Florida. Soil cores were obtained from Joan Rose at the University of South Florida Tampa, Florida. The soils have been previously characterized and marked (Table 3-2). Solutions The buffer solution used in our study was 0.02M imidazole and 0.02M glycine from Sigma Co. made in deionized water. The solutions used in our study were as previously described. Viruses The following phages with their hosts were used in these studies: MS2 (ATCC 15597-B1) assayed using Escherichia co// 3000 (ATCC15597), PRD1 assayed using Salmonella typhimurium (ATCC 19585), 0X174 (13706-B1) assayed using Escherichia coliC (ATCC 13706). Bacterial viruses isolated in our study and their hosts are: 60C3F2 assayed using Escherichia coli 3000 (ATCC 15597), 60KCK2, 61KCK9, and 61KCK10 assayed using Escherichia coli C {ATCC 13706), 60STP2 and 61STP7 assayed using Salmonella typhimuhum (ATCC 19585).

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57 Table 3-2. Soil characteristics TYPE r^omnn^ition dH Depth Comments 1 Silty sandy 6.23 53cm upland pine sandy UF NATL 2 Silty sand organics 7.0 54cm hammock, UF NATL 3 Clayey silty sand organics 6.1 60cm Sandy Hammock, UF NATL 4 Clayey silty sand organics 6.32 57cm hammock forest, UF NATL 5' Silty pale orange to yellow brown sand 6.8 17-1 9ft Top 6' 5.41 17-1 9ft Bottom T Fine grain clayey sand 6.10 28-30ft Top Upper clayplastic, stiff to medium stiff 8" 5.72 28-30ft Bottom 9^ Soft to medium stiff sandy clay 4.16 42-44ft Top Lower clayPrimary confining unit 10" 4.77 42-44ft Bottom " Consists of 3 undisturbed (S lelby tube) soil samples from boring SB-27 at depths of 17 ft. bis, 28 ft. bis, 42ft. bis (bis-below land surface) These core were 2 ft. in length that were divided in half (a top half and a bottom half). Phage stocks were prepared and assayed as previously described. Preparation of phage stocks for groundwater and survival studies was done using deionized water to avoid nutrient contamination. Adsorption of Viruses to IMixed-Liquor Suspended Solids Procedure 1 Concentrations of 10^ PFU/ mL of selected bacteriophages, or poliovirusi were placed in mixed-liquor suspended solids to a final volume of 50mL. The suspension was agitated on a shaker (Reliable Scientific) at low speed for 1, 5,

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58 15, 30, or 60 minutes at room temperature, centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed. Samples were then plaque assayed according to standard procedure. Procedure 2 Approximately 10^ PFU/mL phage were added to biosolids to a final volume of 50mL. The pH was adjusted with HCI or NaOH solutions to pH 3.5 or pH 7. The suspension was agitated on a shaker (Reliable Scientific) at low speed for sixty minutes, centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed. The supernatant was removed and the solids left were then serial treated at low speed for 10 minutes with 0.1% Tween 80, 0.1% Tween 80 and 1M MgCIa, and 10% Beef extract pH 7. The samples were centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed for each elution. The samples were then plaque assayed according to standard procedure. Adsorption of Viruses to Soils Procedure 1 A two gram sample of material was sieved to 25 mesh, shaken (Reliable Scientific) for 30 minutes with 2 mL deionized water to breakup aggregates and stabilize the temperature. The sample was centrifuged at 1200xg for 10 minutes (Dynac II centrifuge) and the solution removed. Then a 2 mL solution of deionized water; which contains 10^ PFU/ mL bacteriophage or virus, was added. The pH was taken, then the suspension was agitated (Reliable Scientific) at low speed for thirty minutes, centrifuged at 1200xg for 10 minutes (Dynac II centrifuge), and a sample of supernatant was removed. The samples were then plaque assayed.

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59 Procedure 2 A two gram sample of material sieved to 25 mesh, was added to deionized water and shaken (Reliable Scientific) for 30 minutes to breakup aggregates and stabilize the temperature. The sample was centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes and the solution removed. Then a 2 mL solution of deionized water; which contains 10^ PFU/mL bacteriophage, or polio 1 virus, was added. The pH was adjusted with HCI or NaOH solutions to pH 3.5 or 7. Then the suspension was agitated at low speed for sixty minutes, centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes, and a sample of supernatant was removed. The samples were then serial treated at low speed on a shaker (Reliable Scientific) for 10 minutes with 0.1% Tween 80, 0.1% Tween 80 and 1M MgCl2, and 10% Beef extract pH 7. The samples were centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes, and a sample of supernatant was removed for each elution. The samples were then plaque assayed. Kinetic Adsorption Studies The adsorption of selected bacteriophage to natural media was examined to identify adsorption rates at varying temperatures. Approximately 10® PFU/ mL phage were added to mixed-liquor suspended solids at a final volume of 50mL, a 2 mL solution of deionized water and 2 grams soil, or a 10 mL solution of deionized water at various temperatures of 4°C, 25°C, and 37°C. The pH was taken, then the suspension was agitated at low speed on a shaker (Reliable Scientific) for specified times, centrifuged at 1200xg (Dynac II centrifuge) for 10 minutes, and a sample of supernatant was removed and plaque assayed.

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60 Column Adsorption Experiments Miniature columns One set of column soil adsorption experiments were carried out using columns of 2.5 cm diameter X 13 cm length in a 60 mL syringe with the tip packed with glass beads and poly propylene fibers at room temperature (Figure 3-1). Dry soil was sieved with a 25 mesh sieve (710 pm), made into a slurry and poured into columns at a depth of 10 cm and stirred to prevent layering and stabilized. The packed columns were rinsed with 5 equal bed volumes of deionized water at pH 7 to reach equilibrium. An initial sample was taken and a solution of deionized water containing 10^ PFU/mL bacteriophage or polio 1 virus was passed through the column by gravity flow. The effluent samples were collected until 4 pore volumes had passed and plaque assayed. Large columns Another set of column soil adsorption experiments were carried out using larger columns of 5 cm diameter X 30 cm length in PVC pipe with screen and poly propylene fibers at room temperature (Figure 3-1). Dry soil was sieved with a 25 mesh sieve (710 pm), made into a slurry and poured into columns at a depth of 20 cm and stirred to prevent layering and stabilized. The packed columns were rinsed with 10 equal bed volumes of deionized water at pH 7 until at equilibrium. An initial sample was taken and then a solution of deionized water containing 10^ PFU/mL bacteriophage or virus was passed through the column by gravity flow. The effluent samples were collected until 8 pore volumes had passed and plaque assayed.

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Figure 3-1. Soil Column Setup A. The miniature column 2.5 cm diameter X 13 cm length on the left is packed at the tip with polypropylene fibers and glass beads. The column has a soil depth of 10 cm. B. The large column 5 cm diameter X 30 cm length PVC pipe with screen and polypropylene on the right. The column has a soil depth of 20 cm. Groundwater Tracer Studies Six observation wells were augured to reach groundwater and into the saturated zone for sampling (Table 3-3). The wells consist of 5ft. PVC pipe (2") coupled with a 2" coupler to a 3ft. screen (2") with tip for flow through. Well water samples were taken with a bailer, which has a one way flow valve was used in sampling. Background samples were taken and plaque assayed at 0.1 mL, and ImL with

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62 Table 3-3. Well Characteristics V Velio Depth dH 1 1 in ft lull 6ft 9in 5.51 2 12ft 7ft. lin. 6.39 3 27 ft 6ft. 8 in. 5.74 4 49 ft 7ft. 5.64 5 71 ft 6ft. 11 in. 5.6 6 73 ft 6ft. 9in. 5.82 ^Distance from septic tank around the soil adsorption field. the appropriate hosts. Then lOOmL 10^ PFU/mL 60STP2, and lOOmL 10^^ PFU/mL MS2 were flushed down (poured into) the septic system. Well water samples were taken every 4-8 hours. The samples were pooled as a composite of each day for initial readings and plaque assayed. Plaque assays of well water samples were corrected for background and individual samples were assayed further if counts in the composite were high. Wells 1 and 3 were used for a repeat (second) study using the same techniques Groundwater Survival Studies The groundwater characteristics were measured and shown in Table 3-4. Then duplicate samples of 10 mL deionized water with 10^ PFU/mL 60STP2, and 10 mL deionized water with 10^ PFU/mL MS2 were placed in polypropylene tubes in monitoring wells 1 and 3. Initial samples were taken and plaque assayed. Then samples were taken every 2 days and plaque assayed.

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63 Table 3-4. Weather conditions Groundwater Characteristics Water Temp. (°C)^ Rain inches^ Max. Temp.^ (°C) Mm. Temp. ( ^) DAYS OF EXPE RIMENTS 3/9/2003 21 1.55 19.4 14.4 3/10/2003 22 0 26.1 12.8 3/11/2003 22 0 26.7 11.7 3/12/2003 0 26.1 12.8 3/1 3/2003 22 fafa 1.27 26.7 15.6 3/14/2003 T 1 23.9 15 3/1 5/2003 22 0 05 17.8 13.9 3/16/2003 22 0 18.9 13.9 3/23/2003 22 T 22.8 14.4 3/24/2003 22 0 25 11.1 3/25/2003 22 0 24.4 9.4 3/26/2003 22 0 27.2 13.3 3/27/2003 22 0 22.8 17.2 3/28/2003 22 0.33 26.1 15.6 3/29/2003 22 0 28.3 13.3 3/30/2003 22 0.20 19.4 8.3 3/31/2003 21 0 14.4 2.8 4/1/2003 21 0 20.6 1.7 4/2/2003 21 0 23.9 5.6 4/3/2003 21 0 25.6 8.3 4/4/2003 22 0 27.8 13.3 4/5/2003 22 0 28.9 13.9 4/6/2003 22 0 29.4 16.1 ^Obtained from the NOAA_NWS Jacksonville, FL ( www.srh.noaa.qov/iax ) ''Measured experimentally and is an average of the 6 wells Results Batch Adsorption Studies The results of the batch adsorption studies using model viruses shows that significant adsorption with various bacteriophage occur over an extended period of time (Figure 3-2). The results of thirty minute batch adsorption of selected bacteriophage to mixed-liquor suspended solids at pH 7 show that the phage 60KCK2 (91%) and 61 KCK10 (85%) show significant adsorption while the

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64 5 15 30 45 60 TIME (MIN.) --MS2 -B-PRDI -A-PHIX PV Figure 3-2. Adsorption of model bacteriophage to mixed-liquor suspended solids^ « • 1 ^The experiments were carried out mixing 10^ PFU/mL bactenophage in 50mL mixed-liquor suspended solids at pH 7 for specified times on shaker (Reliable Scientific). phage 60STP2 (0%) has poor adsorption (Table 3-5). The frequently used model phages MS-2, PRD-1, poliovirus-1 and the select phage 61STP7 adsorbed to the mixed-liquor suspended solids (Table 3-5). The thirty minute batch adsorption results of selected bacteriophages to the ten soils, four from NATL Natural Area Teaching Lab at UP Gainesville, FL and six obtained from J. Rose at USF Tampa, FL at pH 7 show that the bacteriophage 60KCK2, 61KCK10, 0X174 adsorbed well and the phage 60STP2 had the poorest adsorption to all the soils (Tables 3-6 and 3-7). There was a significant increase in adsorption as the depth of the soil samples increased, which was due to the

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65 Table 3-5. Adsorption to mixed-liquor suspended solids by select bacteriophage^ PERCENT ADSORPTION TO MIXED LIQUOR SUSPENDED SOLIDS BACTERIOPHAGE % ADSORPTION 60STP2 0 + 0 61STP7 49 + 2 D" 60KCK2 91 +3 A 61KCK9 RQ + 1 A 61KCK10 85 + 2B MS2 63 + 6 C PRD1 56 + 5C OX174 92 + 7 A POLIO 60 + 6 C . — c . , : : 7~~~T ! cr\ 1 "The experiments were carried out mixing 10^ PFU/mL bacteriophage in 50mL mixed-liquor suspended solids at pH 7 for thirty minutes. "Values followed by the same letter are not significantly different at P-0.05. increase in the content of day. The batch adsorption experiments for 60 minutes of MS2 and 60STP2 at pH 3.5 and pH 7 using mixed-liquor suspended solids or soils show that at pH 3.5 the bacteriophage MS-2 adsorbs well to all media, and the phage 60STP2 adsorbs poorly except to the mixed-liquor suspended solids (Table 3-8). At pH 7, the phage 60STP2 adsorbs poorly and MS-2 adsorption varies depending on the media (Table 3-8). The detergent disrupts hydrophobic interactions and elutes 60STP2 from most media except sludge (Table 3-8). The detergent solutions elute MS-2 from the forest soil samples at pH 3.5 and 7 (Table 3-8). There is some elution of MS-2 from other media with the neutral detergent. Solutions of 1% Tween 80 detergent and 1M MgCb eluted MS-2 from most media types (Table 3-8). However MS-2 remains adsorbed to sandy soil at pH 3.5, clay soils at pH 3.5 and 7, and mixed-liquor suspended solids at pH 3.5 and 7 (Table 3-8). The phage 60STP2 is significantly eluted from most materials

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66 Table 3-6. Adsorption to natural area teaching lab soils by select bacteriophage' Phage PERCENT ADSORPTION TO NATL" SOILS SANDY FOREST SANDY-CLAY FOREST-CLAY 60KCK2 51 + 3 70 + 3 B 55 + 4 B 48 + 7 B 61KCK10 33 + 7 C 73 + 8 B 35 + 4 D 52 + 5 B 60STP2 0 + 0 0 + 0 r\ J. r\ U + u n + n 61STP7 30 +4 C 27 + 5 D 23 + 7E 27 + 2 D MS-2 5 + 9 D 36 + 3 D 22 + 3 E 0 + 0 PRD1 70 + 3A 57 + 4 C 42 + 2 C 61 + 5 A 0X174 70 + 6 A 84 + 6 A 70 + 7 A 64 + 8 A Polio 0 + 6 E 24 + 4 D 49 + 3 B 35 + 2 C ^The experiments were carried out mixing 10^ PFU/mL bacteriophage in 2mL deionized water and 2 gram soil solution at pH 7 for thirty minutes. ''NATLNatural Area Teaching Lab at the University of Florida Gainesville, FL Values in the same column followed by the same letter are not significantly different at P=0.05. using a detergent. However, the phage was eluted from all other media using a solution containing 1% Tween 80 detergent and 1M MgCb (Table 3-8). The results indicate that most of the interactions are due to hydrophobic and electrostatic interactions. The results of the batch adsorption experiments show that an expected trend of adsorption occurs based on the viral adsorption properties the phages were selected for. Variation in the adsorption pattern are most likely due to the effects of interfering particles such as organics and particulates in the media such as clays and oxides. The factors found to be significant in the adsorption

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67 Table 3-7. Adsorption to soils by select bacteriophage^ PHAGE PERCENT ADSORPTION TO SOILS'' 6*= r 8" 9^ 10^ 60KCK2 97 + 3 92 + 3 A f\r\ 1 o 100 + 2 yy + o \ uu + u inn + n 1 uu 3 61KCK10 ft7 + R Of ' u QQ + 1 99 + 1 99 + 0 100 + 0 100 + 0 60STP2 32 + 2 17 + 4 93 + 9 77 + 1 95 + 1 95 + 2 61STP7 0 + 0 69 + 5 93 + 7 48 + 2 100 + 0 100 + 0 MS-2 60 + 9 82 + 3 74 + 2 76+1 99 + 1 99 + 1 PRD-1 45 + 4 34 + 2 44 + 5 69 + 6 100 + 0 100 + 0 0X174 99+1 100 + 0 100 + 0 98+1 100 + 0 100 + 0 Polio 49 + 4 66 + 6 56 + 5 \ — 1 54 + 4 94 + 0 75 + 3 deionized water and 2 gram soil solution at pH 7 for thirty minutes. "Joan Rose, University of South Florida, Tampa, FL See Table 3-2 for specific soil characteristics of the samples. There is an increase in clay content as the sample number increases. phenomena of the batch adsorption experiments are time (Figure 3-2), viral adsorption properties (Figure 3-2 and Tables 3-5, 3-6, 3-7, 3-8), composition of the solutions (Table 3-8), pH (Table 3-8), and composition of the solids (Tables 3-5, 3-6, 3-7, 3-8) are significant factors in the adsorption phenomena. Kinetic Adsorption Studies The kinetics of adsorption of the selected bacteriophage to natural media was determined as a function of temperature to study the thermodynamic parameters. The percent of adsorption reported here is at 2 hours, the maximum amount of time we allowed for adsorption to occur (Table 3-9). At 4°C, MS-2

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68 Table 3-8. Adsorption to natural media at pH 7 and pH 3.5 by select phage^ PERCENT ADSORPTION TO NATURAL MEDIA BACTERIOPHAGE SANDY SOIL FOREST SOIL CLAY SOIL MLSS" pH 3.5 7 3.5 7 3.5 7 3.5 7 MS2-Buffer 100 73 5 21 100 100 95 21 60STP2-Buffer 1 62 32 35 27 35 86 27 MS-2-Tween 79 1 0 0 79 74 89 21 60STP2-Tween 0 20 0 0 0 3 79 27 MS-2-Tween&MgCI 59 0 0 0 76 47 24 13 60STP2-Tween& MgCb 0 0 0 0 0 0 0 0 bacteriophage in 50 mL mixed-liquor suspended solids or 2mL deionized water and 2 gram soil solutions at pH 3.5 or 7 for sixty minutes. "MLSS is an abbreviation for mixed liquor suspended solids. (87%) adsorbed well and the phage 60STP2 (58%) adsorbed to the mixed-liquor suspended solids media (Figure 3-3). However, there was a significant difference in adsorption to the forest soil. The bacteriophage MS-2 (72%) adsorbed well but the phage 60STP2 adsorbed minimally (2%)(Figure 3-3). At 25°C, MS-2 (91%) and 60STP2 (64%) phages adsorbed to the sludge, MS-2 (75%) adsorbed to the forest soil, but the phage 60STP2 (7%) adsorbed poorly to forest soils (Figure 34). At 37°C, MS-2 adsorbed to the mixed-liquor suspended solids and soils at 99% and 97% respectively (Figure 3-5). There was increased adsorption of MS-2 to the forest soil. The bacteriophage 60STP2 adsorbed well to sludge (99%) and there was increased adsorption to the forest soils (40%) (Figure 3-5). The Arrhenius plot uses the adsorption rate constant (-k) versus the inverse of

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69 Table 3-9. Adsorption of bacteriophage to natural media^ PERCENT ADSORPTION TO NATURAL MEDIA AT VARIOUS TEMPERATURE TEMPERATURE C° NATURAL MEDIA MS-2 60STP2 4 MLSS" 87 + 2 A' 58 + 7 B 4 Forest soil 63 + 6 A 2 + 2 B 25 MLSS 91 + 4 A 64 + 9 B 25 Forest soil 64 + 8A 5 + 5 B 37 MLSS' 99 + 2 A 99 + OA 37 Forest soil 89 + 3A 36 + 5 B solids at a final volume of 50mL, or a 2 mL solution of deionized water and 2grams soil at various temperatures and agitated for 2 hours. ''MLSS is an abbreviation for mixed liquor suspended solids. 'Values in the same row followed by the same letter are not significantly different at P=0.05. temperature in Kelvin. The adsorption rate constant is the slope of the rate constant plot, which uses the natural log of viruses in the supernatant versus time in seconds (Figures 3-3, 3-4, 3-5). Activation thermodynamic comparisons show that phage adsorption to natural medias at various temperatures is a physical process with a low energy of activation of <40 Kcal/mol (Table 3-10). The results also suggest that adsorption of the phage 60STP2 is minimal to the forest soil. The Arrhenius plot of phage to natural media shows that 60STP2 adsorbed poorer to natural media than the model MS-2 (Figure 3-6).

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70 100 1 TIME (min.) |—irSludge MS-2 —4— Sludge 60STP2 1Soil MS-2 » # Soil 60STP2 Figure 3-3. Adsorption at 4°C of select bacteriophage to natural media ^ 10^ PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL or 2mL solution of deionized water and forest soil at 4°C and agitated for 2 hours. Column Adsorption Experiments The experiments were carried out at room temperature using 10 cm soil depth X 2.5 cm diameter in a 60 cm syringe or a 20 cm soil depth x 5 cm diameter in a 50 cm PVC column with gravitational flow. Breakthrough plot is concentration supernatant/ initial concentration versus time. Effluent samples were collected, and assayed. The results show that a significant difference in soil column breakthrough requires an optimal distance for adsorption and soil retardation factors to affect the curves. At 10 cm soil depth, there is a minimal

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71 TIME (min.) A Sludge MS-2 -•—Sludge 60STP2 m Soil MS-2 t Soil60STP2 Figure 3-4. Adsorption at 25°C of select bacteriophage to natural media 10^ PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL or 2mL solution of deionized water and forest soil at 25°C and agitated for 2 hours. difference in the passage of column effluent between MS2 and 60STP2 (Figures 3-7 and 3-8). The bacteriophage 60KCK2 adsorbed well to both soils but was found in the column effluent of sandy soil after time (Figures 3-7 and 3-8). However, if the distance is increased to a 20 cm soil depth there is a significant difference in breakthrough, possibly due to an increase in length that increases contact time and binding sites. The phage 60STP2 had a significant breakthrough as compared to MS-2 (Figure 3-9). In sandy soil columns (5 cm diameter X 37 cm depth) with a flow rate of 1 .SmL/min., both phages were found

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72 TIME (min.) 4 Sludge MS-2 -^Sludge 60STP2 Soil MS-2 ® Soil60STP2 Figure 3-5. Adsorption at 37°C of select bacteriophage to natural media 10^ PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL or 2mL solution of deionized water and forest soil at 37°C and agitated for 2 hours. in column effluent in 15 minutes (Figure 3-10). In forest soil columns with a flow rate of 0.98 mL/min the results show that approximately a 60% of phage 60STP2 were found in the column effluent in 1 and 1/2 hours, whereas 2 and 1/2 hours were required for significant numbers of MS-2 to be found in the column effluent (Figure 3-11). In a column of forest soil (5 cm diameter X 35 cm depth) with a flow rate of 1 .2 mUmin. approximately 50% of the phage 60STP2 was found in the column effluent in an hour, whereas only 10% of MS-2 was found in the effluent in 2 hours (Figure 3-12). MS-2 required a longer amount of time for

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73 Table 3-10. Activation thermodynamics of select bacteriophage adsorption to natural media^ ACTIVATION THERMODYNAMICS OF SELECT BACTERIOPHAGE 25°C (298K) EA (kcal/mol) AG (kcal/mol) AH (kcal/mol) AS (cal/mol per K) Sludge MS2 3.34 22.24 2.75 -65.4 Sludge 60STP2 9.13 22.74 8.54 -47.65 Soil MS2 4.03 22.32 3.44 -63.36 Soil 60STP2 18.31 24.04 17.72 -21.21 Comparative activation thermodynamics at 25°C for phage adsorption to solids. A 10® PFU/mL phage concentration was added to mixed-liquor suspended solids at a final volume of 50mL, or a 2 mL solution of deionized water and 2grams soil at various temperatures and agitated for 2 hours. passage through the column, and poliovirus-1 adsorbed well to the columns (Figure 3-12). Groundwater Studies Six observation wells were sampled to monitor the transport of tracer bacteriophage through soils. The results were assayed and corrected for background counts (Table 3-11). Our results show that the first observation of 60STP2 and MS-2 in well water occurred within the first 8 hours in well 1 (Figure 3-13). It is also obvious from the results that 60STP2 shows up first followed closely by MS-2, and that a greater amount of 60STP2 was found (Figures 3-13, 3-14, 3-15, 3-16, 3-17, 3-18). MS-2 and 60STP2 were found in the same wells at approximately the same time (Figures 3-13, 3-14, 3-15, 3-16, 3-17, 3-18). The results observed show that the phage traveled in similar paths through the field, and to a horizontal distance of at least 73 feet from the tank through the field in a

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74 -5 -6 -7 -13 -14 -15 3.23E-03 3.36E-03 3.61 E-03 TEMP (1/K) Sludge MS2 Soil MS2 — -Sludge 60STP2 — Soil60STP2 Figure 3-6. Arrhenius plot of select bacteriophage adsorption to natural nnedia^ ^The Arrhenius plot uses the adsorption rate constant (-k) versus the inverse of temperature in Kelvin. A 10^ PFU/mL phage concentration was added to mixedliquor suspended solids at a final volume of 50mL, or a 2 mL solution of deionized water and 2grams soil at various temperatures and agitated. minimal amount of time (Figure 3-18). However, our study was carried out under rainy conditions where the soil was saturated. The results of the second study under dryer conditions using wells 1 , 3, and 5 have shown similar results. The survival study has shown that the phage 60STP2, and MS2 were inactivated between 8 and 10 days (Figure 3-19). The viruses we selected based on specific adsorption characteristics primarily adsorbed as we expected. The bacteriophages 60KCK2 was selected based on strong hydrophobic interactions; 60STP2 was selected for its weak Discussion

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75 0 15 30 45 60 75 90 120 TIME (minutes) 60STP2 IVIS2 60KCK2 Figure 3-7. Transport in (10 cm) sandy soil column of bacteriophage^ ^The experiment was carried out at room temperature using a 10 cm sandy soil depth column in a 60 cm syringe with gravitational flow and 10^ PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. hydrophobic and electrostatic characteristics, and 61STP7 was selected based on moderate hydrophobic and electrostatic characteristics, and 61KCK10 was selected based on strong hydrophobic and electrostatic properties. However, the entire system including the virus, solution, and solid defines the adsorption pattern. Due to the numerous factors involved in these complex systems, it is necessary to define the entire system each time to determine the mechanism of adsorption. The parameters that have been found to affect adsorption have been previously stated in the introduction (Bitton 1980, Blanc and Nasser 1996, Gerba et al. 1981, Gerba 1984b, Goya! and Gerba 1979, Lance and Gerba 1984a,

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76 0 15 30 45 60 75 90 120 Time (minutes) ---60STP2 MS-2 -e-60KCK2 Figure 3-8. Transport in (10 cm) forest soil column of bacteriophage^ ^The experiment was carried out at room temperature using a 10 cm forest soil depth column in a 60 cm syringe with gravitational flow and 10^ PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. Lance and Gerba 1984b, Nasser et al. 1993, Sobsey et al. 1995, Yahya et al. 1993, Yates et al. 1985). As seen in Figures 3-2 to 3-8 and Tables 3-5 to 3-9, variation in temperature, pH, solids, and phages affect adsorption of phages to the solids. Nevertheless, knowledge of the significant parameters can give a predictive adsorption model. At a neutral pH, soils generally have a net negative charge and many viruses have a net negative charge. Most studies have found enhanced adsorption with the addition of a high concentration of salts or a decrease in pH, large soil surface area, or a high cation exchange capacity (Dowd et al. 1998, Lance and Gerba 1984a, Taylor et al. 1981).

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77 Figure 3-9. Transport in (20 cm) sandy soil column of bacteriophage ^The experiment was carried out at room temperature using a 20 cm soil depth in a 50 cm PVC column with gravitational flow and 10^ PFU/mL phage concentration. The abbreviations Cs =concentration supernatant and Co =initial concentration. Previous studies have shown that an increase in the clay content of soils, increases adsorption of the viruses, and that organic materials interfere with viral adsorption to soils (Gerba et al. 1981 , Goyal and Gerba 1979, Sobsey et al. 1980a). Our results were in agreement that an increase in adsorption is mainly due to the increase in clay content as the depth of the sample increases. Other studies have shown that the length of the column, and that the flow rate can affect viral adsorption (Lance and Gerba 1984b). We also found high

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78 TIME (min.) "»-60STP2 — MS2 -i^PV Figure 3-1 0. Transport of viruses in (37 cm) sandy soil column^ ^The experiment was carried out at room temperature using a 37cm sandy soil depth in a 50 cm PVC column with gravitational flow and 10^ PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. variability in adsorption to mixed-liquor suspended solid (MLSS) samples, which we believe occurs due to interfering particles such as organics in the media. The batch adsorption studies with the natural media have shown that as the time allowed for viral adsorption increased there was an increase in adsorption at pH 7, and that an increase in column size from a 10 cm soil depth to 20 cm soil depth allowed for a difference in transport time between viruses. Another factor minimally affecting time dependent viral adsorption was flow rate.

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79 o O o 30 60 105 120 TIME (min.) 180 240 •60STP2 'MS2 Figure 3-1 1 . Transport in (20 cm) forest soil column of bacteriophage^ ^The experiment was carried out at room temperature using a 20 cm soil depth in a 50 cm PVC column with gravitational flow and 10^ PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. Several studies have shown that an increase in ionic strength of a solution was found to enhance adsorption and detergents interfered with adsorption of several enteroviruses and rotavirus SA1 1 (Dizer et al. 1984, Lance and Gerba 1984a, Lytle and Routson 1995, Sobsey et al. 1980a, and Taylor et al. 1981). These observations are in agreement with what we observed in various natural media systems (Tables 3-8 and 3-9).

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Figure 3-12. Transport of viruses in (35 cm) forest soil column ^The experiment was carried out at room temperature using a 35 cm forest soil depth in a 50 cm PVC column with gravitational flow and 10^ PFU/mL phage. The abbreviations Cs =concentration supernatant and Co =initial concentration. Previous studies and our results also show that viral adsorption to natural media at a neutral pH is dependent on the viral surface composition (Dowd et al 1998, Penrod et al. 1996, Schijven 2000). Studies have shown that most viruses may behave differently under similar conditions (Burge and Enkiri 1978, Goyal and Gerba 1979, Shields and Farrah 2002). It is believed that their differences in

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81 Table 3-1 1 . Bacteriophage in monitoring wells Well# PFU/mL' £. co/i C-3000 S. Typhimurium 1 12 ^Before the addition of bacteriophage, a sample of well water was removed and a ImL sample was assayed as PFU/mL using the hosts Escherichia coli C-3000 and Salmonella typhimurium. adsorption are due to differences in their hydrophobicity and electrical charges on the capsid surfaces (Shields and Farrah 2002). The results for the most part follow the DLVO theory so an increase in ionic strength screens electrostatic charges and promotes hydrophobic and van der Waals interactions. Our results show that the pH had a significant effect on adsorption of viruses to media. Previous studies show that adsorption varies based on pH dependent adsorption properties related to the isoelectric point of the viruses and solids (Dowd et al. 1998, Gerba et al. 1981, Gerba 1984b, Mix 1974, Mix 1987, Sobsey et al. 1980a, Taylor et al. 1981). A virus particle is colloidal in nature so it must be physically adsorbed to a surface in order to be removed. The kinetics of adsorption of the selected bacteriophage to natural media was determined as a function of temperature to study the thermodynamic parameters. Results of previous studies and our study show that phage adsorption to natural medias at various temperatures is a physical process with a low energy of activation of <40 Kcal/mol (Table 3-10) (Adamson 1982, Preston and Farrah 1988a). The

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82 75 70 65 60 55 50 _. 45 E 40 £ 35 30 25 20 15 10 5 0 1 1 1 " ^ 1 8 16 24 32 40 48 56 64 72 80 88 96 TIME (Hour) •MS-2 '60STP2] Figure 3-13. Tracer study in well 1 at a 10 ft distance from the system ^ ^Bacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). Arrhenius plot uses the adsorption rate constant (-k) versus the inverse of temperature in Kelvin. The adsorption rate constant is the slope of the rate constant plot, which uses the natural log of viruses in the supernatant versus time in seconds (Figures 3-3 to 3-5). The Arrhenius plot of the phage to natural media indicates that the phage 60STP2 adsorbed poorer to natural media than the model MS-2 (Figure 3-6). The results show that adsorption was dependent on temperature, and solid composition. Adsorption of MS-2 and 60STP2 increased significantly at 37°C to all media. This phenomenon was confirmed by

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83 75 ^ 70 65 60 55 50 8 16 24 32 40 48 56 64 72 80 88 96 TIME (Hour) MS-2 — 60STP2 Figure 3-14. Tracer study in well 2 at a 12 ft distance from the systenn ^Bacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). controls of bacteriophage in solution without the biosolids that inactivation was not a significant factor over the time frame of the experiments. Due to the presence of enteric viruses in groundwater many investigators have studied the transport phenomena by using tracers (Bales et al. 1989, Gerba 1984a, Griffin et al. 1999, Harvey 1997, Paul et al. 1995, Paul et ai. 1997, Schaub and Sorber 1977, Skilton and Wheeler 1988, Vaughn et al. 1978, Vaughn et al. 1983, Yanko et al. 1999). The bacteriophage MS-2, and PRD-1 are hydrophobic bacteriophage that have been found to adsorb poorly to various

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84 75 70 65 60 55 50 E 40 8 16 24 32 40 48 56 64 72 80 88 96 TIME (Hour) MS-2 — 60STP2 Figure 3-1 5. Tracer study in well 3 at a 27 ft distance from the system ^ ^Bacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). solids so they are frequently used as worst-case models for possible contamination (Goyal and Gerba 1979, Schijven 2000). However, we found that MS-2 and PRD-1 do not adsorb that poorly to all media, and that adsorption was dependent on the various factors in the systems. Our results also show that by selecting bacteriophage based on their adsorption properties, and the system parameters we can predict better models for transport systems. The bacteriophage 60STP2 selected based on its weak hydrophobic and electrostatic properties was shown to adsorb poorly to virtually all media and in transport

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85 75 1 70 65 60 55 50 ^ 45 E 40 8 16 24 32 40 48 56 64 72 80 88 96 TIME (Hour) — -MS-2 — HI— 60STP2! Rgure 3-16. Tracer study in well 4 at a 49 ft distance from the system^ ^Bacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). studies had increased flow with less reduction than the previously used MS-2 phage. It is well known that viruses can flow through the soils from waste disposal systems into the water supplies. Studies have found that viruses survive better at temperatures of 10°C or below and that there is significant inactivation at temperatures of 23°C and higher (Alvarez et al. 2000, Blanc and Nasser 1996, Nasser et al. 1993). Our results indicated that it took between eight and ten days for complete inactivation of the bacteriophage at an average temperature of 22°C. These results are in

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86 8 16 24 32 40 48 56 64 72 80 88 96 TIME (Hour) -MS-2 '60STP2 Figure 3-17. Tracer study in well 5 at a 71 ft distance from the system^ ^Bacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). agreement with previous studies (Alvarez et al. 2000). It is important to note that inactivation is generally negligible within the time scale of the experiments. However for field experiments viral inactivation may be significant, especially under some conditions such as unsaturated, low flow rate, high adsorption. The results also show that there was approximately a 5 logio reduction in the phage tracers. The reduction occurred mainly due to adsorption since the results of the survival study show a low rate of inactivation.

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87 75 70 65 60 55 50 ^ 45 E 40 £ 35 ^ 30 25 20 15 10 5 0 1 1 . 1 — 8 16 24 32 40 48 56 64 TIME (Hour) 72 80 88 96 MS-2 '60STP2 Figure 3-18. Tracer study in well 6 at a 73 ft distance from the system^ ^Bacteriophage were added to a septic system and monitoring well samples were taken and assayed every four hours around the soil adsorption field (SAF). In summary, the results show that viral adsorption to media is based on specific adsorption properties that for the most part follow the DLVO theory. The results also show that adsorption or transport is dependent on specific factors such as, retention time, composition of the solutions, ionic strength, pH, viral coat composition, viral type, the composition of the solids, and temperature. It is fundamental to understand the interactions involved in the adsorption, survival and transport of viruses. Using our results and results from other studies we have

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88 Figure 3-19. Bacteriophage inactivation in wells^ ^Bacteriophage 60STP2 and MS-2 at 10^PFU/mL in deionized water were placed in polypropylene tubes in the monitoring wells and assayed daily for inactivation. compared the selected indigenous bacteriophage to enteric viruses (Table 3-12) (Gerba et al. 1981, Goyal and Gerba 1979, Shields and Farrah 2002).

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89 Table 3-12. Summary of select bacteriophage iviuuci Viruses Interactions Possible Model Uses'' Enteric Viruses^ 60STP2 Weak hydrophobic/electrostatic Groundwater tracer, barrier materials, WWT^ processes Echo-1, CoxB4 61STP7 Hydrophobic, slightly electrostatic Groundwater tracer, WWT processes, barrier materials Cox 83 60KCK2 Hydrophobic Adsorption, WWT processes 61KCK9 Hydrophobic WWT processes 61KCK10 Hydrophobic/electrostatic Adsorption Echo-5 MS-2 Hydrophobic Groundwater tracer, WWT processes PRD1 Hydrophobic/electrostatic Groundwater tracer, WWT processes 0X174 Weak hydrophobic/electrostatic Groundwater tracer. Barrier materials POLIO 1 Electrostatic WWT processes ^ Enteric viruses with similar adsorption as the selected bacteriophage and virus models. ''The select bacteriophage could possible be used as models for the enteric viruses listed in the processes (uses) identified. "VWNTr is an abbreviation for wastewater treatment

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CHAPTER 4 IDENTIFICATION AND PROTEIN COAT CHARCTERIZATION OF SELECTED BACTERIOPHAGE Introduction To be a representative, the model organism should have similar characteristics to the virus or viruses being studied. As previously stated several organisms have been studied as models for enteric viruses to evaluate resistance to processes and fate in treatment processes. Model organisms have also been used as tracers in water supplies. Frequently these organisms were chosen based on size, shape, and nucleic acid. They were not chosen based on their adsorption characteristics. Previous research has shown that the adsorption mechanisms of some models are not comparable to those of the pathogenic viruses they are supposed to represent (Shields and Farrah 1983, Sobsey et al. 1986). The results of various studies demonstrate that the adsorptive differences of viruses with similar isoelectric points or characteristics are most likely due to localized groups of negative and positive charges, and possible spans of hydrophobic groups exist on viral surfaces (Mix 1974, Penrod et al. 1996, Schijven 2000). So to understand the mechanisms involved in adsorption; it is necessary to identify the surface amino acid residues. To understand the differences in adsorption it is also important to identify the bacteriophage and its lineage. Current research has identified other methods of classification that are 90

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91 based on genome organization using protein, molecular techniques and physical parameters using cryo-electron microscopy and x-ray diffraction to obtain atomic detail (Baker et al 1999, Bamford et al. 2002, Clark et al. 2001, Ford et al. 1998, Juhala et al. 2000, Hendrix et al. 1999, Rohwer and Edwards 2002). The use of cryo-electron microscopy and x-ray diffraction have lead to advances in the development of programs such as roadmaps or the VIPER website to illustrate the surfaces of viruses at the atomic level (Reddy et al. 2001 , Rossman and Palmenberg 1988, http://mmtsb.scripps.edu/viper/ ). However, the examination of the surface coat amino acid residues of the capsid can be done using a variety of programs found on the ExPASy molecular server and include BLAST (Altschul et al. 1990, Gish and States 1993, Madden et al. 1996, Zhang and Madden 1997) for sequence homology, Rasmol (Sayle and Milner-White 1995) and Chime/ Protein Explorer (MDI informations system plug-in) for structural analysis, ClustalW and Bioedit (Hall 1999) for the multiple sequence alignment, Swiss-Prot for protein data, SAPS (Brendel et al. 1992) for the statistics on protein sequences, Protscale and ProtoPram (Bjellqvist et al. 1993, Gill and von Hippel 1989, Guruprasad et al. 1990, Ikai 1980, Kyte and Doolittle 1982). Materials and Methods Viruses The following phages with their hosts were used in these studies: MS2 (ATCC 15597-B1) assayed using Escherichia co// C3000 (ATCC15597), PRD1 assayed using Salmonella typhimurium (ATCC 19585), 0X174 (13706-B1) assayed using Escherichia coli C {ATCC 13706), Lambda (ATCC 23724-B2) assayed using Escherichia co// C600 (ATCC 23724), T1 (ATCC 11303-B1), T4 (ATCC 11303-

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92 B4), and T5 (ATCC 1 1303-B5) assayed using Escherichia coli B (ATCC 1 1303). Bacterial viruses isolated in our study and their hosts are: 60C3F2 assayed using Escherichia coli 3000 (ATCC15597), 60KCK2, 61KCK9, and 61KCK10 assayed using Escherichia coliC (ATCC 13706), 60STP2 and 61STP7 assayed using Salmonella typhimurium (ATCC 19585). Phage stocks were prepared and assayed as previously described. Preparation of phage stocks for electron microscopy studies was done using deionized water. Cross Reactivity (Host Range) The isolated bacteriophage 60KCK2, 61KCK10, 60STP2, 61STP7, and the reference phages MS-2, PRD1, 0X174 were plated with the hosts Escherichia coli 3000, Escherichia coliC, Proteus vulgaris, Citrobacter freundii, Enterobacter aerogenes. Salmonella typhimunum, and Klebsiella pneumoniae and assayed using standard procedure (Snustad and Dean 1971). The host range was determined by the phage's ability to replicate. Plaque IMorphology The isolated bacteriophage and the reference phages were plated with their respective hosts, and viral morphology characteristics such as size and clarity of the plaques were studied using standard procedure (Snustad and Dean 1971). Electron Microscopy Electron microscopy was used to examine structural characteristics of the isolated phage 60KCK2, 61KCK10, 60STP2, and 61STP7. A drop of high titer phage stock was placed on 300 mesh formvar grids for 1 minute and excess was removed using filter paper. The grid was then rinsed with two drops distilled water and the excess was removed using filter paper. Two drops of 1% Uranyl

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93 acetate was applied to the grid for 1 minute and the excess was removed using filter paper. The grid was air dried and examined on a Zeiss electron microscope operating at 80kV or 100kV (Bozzola and Russell 1999). Electron microscopy was used to examine the adsorption of bacteriophage 60STP2 to Salmonella typhimurium. A drop of high titer bacterial host was placed on 300 mesh formvar grids, and then a drop of high titer bacteriophage stock was placed on the grid and allowed to set for five minutes. The excess was removed using filter paper. The grid was then rinsed with two drops distilled water and the excess was removed using filter paper. Two drops of 1 % Uranyl acetate was applied to the grid for 1 minute and the excess was removed using filter paper. The grid was air dried and examined on a Zeiss electron microscope operating at 80kV or 100kV (Bozzola and Russell 1999). Nucleic Acid Determination The nucleic acid of the isolated bacteriophage and the reference phages were identified by an enzymatic reaction using RNase (Promega Madison, Wl). Two drops of bacteriophage and two drops host were added to 10 mL 1% Tryptone (Difco) and incubated overnight at 37°C. A 0.5 mL sample of each culture was taken in duplicate. To one set lOuL (5ug/uL) RNase was added and to the other set nothing was added. The samples were incubated for 30 minutes at 37°C. A lawn of each host was spread on a Tryptic Soy Agar plate and 10ul of each sample were spotted on the lawn. The plates were allowed to air dry. Top agar with 37uL (5ug/uL) RNase added. The plates were inverted and incubated overnight.

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94 Protein Analysis Protein analysis was done using the isolated phage 60KCK2, 61KCK10, 60STP2, 61STP7, and reference bacteriophage from the Siphoviridae family Lambda (A), T1, T5; and T4 from the Myoviridae family. A 30uL sample of phage stock was diluted with 30uL of 2 X SDS sample buffer and the solution boiled at 100°C for 10 minutes (Sambrook et al. 1989). The samples were loaded into wells 2-8 at 15uL per sample. Broad Range Molecular weight markers were obtained from Promega (Madison, Wl) and loaded onto the gels according to manufactures instructions at the end wells. The precasted 4-20% SDSpolyacrylamide gradient gels (BioRad Hercules, CA) were ran at 40mA. The bands were stained according to the Silver Stain procedure (Sambrook et al. 1989). Restriction Enzyme Analysis DNA extraction was accomplished using the phenol-chlorofonn procedure (Sambrook et al. 1989). The samples were concentrated according to the ethanol precipitation techniques (Sambrook et al. 1989). The pellet was resuspended in 30 uL TE (Tris-CI and 0.1M EDTA) at pH 8. Restriction enzyme analysis was done using the isolated phage 60KCK2, 61KCK10, 60STP2, 61STP7, and reference bacteriophage from the Siphoviridae family Lambda (A), T1 , T5, and the Myoviridae family T4. The selected bacteriophage were enzymatically digested with EcoRV, EcoRI and Hindi in combination, EcoRI, and Hindi purchased from Promega Madison, Wl or no enzyme according to supplier's recommendations (Echols and Murialdo 1978, Hoess et al. 1980). A 20uL sample of the generated fragments were loaded into wells 2-8 and Bench Top

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95 1 Kb DNA Ladder was used as a molecular weight marker and loaded into the end wells according to manufacturers instructions (Promega Madison, Wl). The samples were electrophoretically separated on a 1% agarose gels in TBE buffer at 75V (Sambrook et al. 1989). The gels were selectively post stained in Ethidium Bromide (0.5ug/ mL) and viewed in a UV box. Sequencing Gel Excision Based on the knowledge of Siphoviridae family, the unique fragments for the major capsid genes 1.6Kbp and integrase gene 2.24Kbp for bacteriophage 60STP2 were excised and eluted off the gel containing the fragments from the digestion with the enzymes Hindi and EcoRI according to manufacturers directions (Qiagen QIAquick gel extraction). Cloning The extracted fragments were washed and ligated into PUC19 vector digested with Hindi and EcoRI and transformed into competent DH5a cells (Clark et al. 2001). The samples were plated for screening on Luria-Bertani agar supplemented with 100 pg/mL ampicillin, isopropyl-P-D-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-Beta-D-galactoside (X-gal). Screening PUC19 cloning vector is a lacZampR based system and analysis of positive clones was performed using blue/white screening. The white colonies are selected and inoculated in 5mL LBamp media, incubated overnight at 37°C and plasmid purified according to the Qiagen miniprep kit directions (Qiagen). The resultant samples were digested with EcoRI and Hindi in combination according

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96 to manufacturers directions (Promega Madison, Wl). A 20uL sample of the generated fragments were loaded into wells 2-8 and Bench Top 1 Kb DNA Ladder was used as a molecular weight marker and loaded into the end wells according to manufacturers instructions (Promega Madison, Wl). The samples were electrophoretically separated on a 1% agarose gels in TBE buffer at 75V(Sambrook et al. 1989). The gels were post stained in Ethidium Bromide (0.5 |jg/mL) and viewed in a UV box. Plasmid Isolation and DNA Sequencing Plasmids were extracted using a plasmid miniprep kit (Qiagen, Inc.) according to manufacturer's instructions. Sequencing reactions were performed using fluorescently labeled dideoxy terminator chemistry with PUC universal primers and a Perkin Elmer GeneAmp PGR System 9600 (Perkin Elmer-Cetus, Norwalk, Conn.). Extension products were separated and read with a LI-COR DNA Sequencer model 4000L at the Microbiology and Cell Science department at the University of Florida Gainesville, FL. Sequence Analysis of the capsid and INT Gene Bacteriophage sequences were identified in our study and were compared to previously published sequences of bacteriophage from the Siphoviridae and Myoviridae family (GENBANK). The sequences were aligned and compared using Bioedit software and ClustalW. Protein Coat Analysis The bacteriophage protein coat sequences were analyzed using a variety of programs. Sequence homology was examined using programs found on the ExPASy server and BLAST for sequence homology Structural analysis was

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97 determined if possible utilizing the Brookhaven protein database bank ( ww^w.rcsb.org/pdb/index.html ). Rastop, and Viper (http://mmtsb.scripps.edu/viper) (Berman et al. 2000, Sayle and Milner-White 1995, Reddy et al. 2001) for structural analysis. The programs ClustalW and Bioedit were used for the multiple sequence alignment. Characteristic of capsid residues were analyzed using programs on the ExPASy server such as SwissProt for protein data, SAPS, Protscale and ProtoPram. Results Cross Reactivity (Host Range) The selected bacteriophage 60KCK2 and 61 KCK10 formed plaques on the hosts Escherichia coli 3000 and Escherichia coliC. Bacteriophages 60STP2 and 61STP7 formed plaques on the host Salmonella typhimurium exclusively, MS-2 formed plaques on the host Escherichia coli 3000, PRD-1 formed plaques on the host Salmonella typhimurium, and 0x174 formed plaques on the host Escherichia coli C (Table 4-1). Plaque Morphology and Electron Microscopy The viral morphology characteristics such as size and clarity of the plaques, and the structural characteristics of the selected phage are presented in Table 4-1. The results show that phage 60KCK2, 61KCK10, and 60STP2 were found to be approximately 50-60nm, icosahedral, tailed phage (Figures 4-1, 4-2, 4-3). The bacteriophage 61STP7 was found to be a >70nm, icosahedral tailed phage (Figure 4-4). Electron microscopy was used to examine the adsorption of the bacteriophage 60STP2 to Salmonella typhimurium (Figure 4-5). The results indicated that initial adsorption of 60STP2 to the host occurs by the tail.

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98 Table 4-1 . Identification characteristics of select bacteriophage Isolated phage (our study) Host range Plaque Morphology RNase^ EM FAMILY 60KCK2 E.coli KC, C3000 clear, large DNA 50-60nm, tail Siphoviridae 61KCK10 E.coli KC, C3000 clear, large DNA 50-60nm, tail Siphoviridae 60STP2 s. typhimurium cloudv pinpoint DNA 50-60nm tail Siphoviridae 61STP7 S. typhimurium cloudy/clear, small DNA >70nm, tail Myoviridae Reference phage Host range Plaque Morphology RNase* EM FAMILY MS-2 E.coli C3000 clear, small RNA 30nm Leviviridae PRD1 o o. typhimurium clear, small DNA 58-60nm Tectiviridae 0X174 E. coliC clear, large DNA 25nm Microvirldae A'' E. coli C600 cloudy/clear, small DNA Siphoviridae T1^ E. coli C clear, small DNA Siphoviridae 14" E. coli C clear, large DNA Myoviridae E. coli C clear, large DNA Siphoviridae ^RNase used for nucleic acid detennination ''Reference bacteriophage for families Nucleic Acid Determination The nucleic acid of the phage was identified by an enzymatic reaction using RNase (Table 4-1). The bacteriophage 61KCK10, 60KCK2, 60STP2, 61STP7, PRD-1, and 0X174 were found to have a DNA nucleic core. The phage MS-2 was found to have a RNA nucleic acid.

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99 50nm Figure 4-1 . Electron micrograph of 60KCK2^ ^ The image was taken at 80K and the magnification is approximately 200,000X. Protein Analysis and Restriction Enzyme Analysis The molecular identification of isolated bacteriophages was done using protein analysis and restriction enzyme analysis. The profiles of isolated phage were compared to the profiles of the reference bacteriophage (Table 4-2). Protein profiles were generated by protein analysis of the phages (Figure 4-6). Restriction enzyme profiles were generated using the enzymes EcoRV (Figure 47), and EcoRI and Hindi in combination (Figures4-8). The bacteriophage 60KCK2 and T5 had similar profiles, and the phage 61KCK10 and Lambda (A) had similar profiles (Figures 4-6 to 4-8). The bacteriophage 60STP2 and T1 had similar profiles, and the phage 61STP7and T4 had similar profiles (Figures 4-6 to 4-8).

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100 50nm Figure 4-2. Electron micrograph of 61 KCK10^ ^ The image was taken at 80K and the magnification is approximately 200,000X. Sequencing The integrase and capsid genes were isolated (Figures 4-9 and 4-10). The Integrase gene did not have any significant sequence similarities to any known sequence in Genbank after a blast search. However, the capsid gene had significant similarities to a Podoviridae phage, ST64B (Figure 4-11). It also had similarities to the Siphoviridae bacteriophage such as HK97and D3 (Figure 4-11). There was strong similarity between the capsid sequences of bacteriophage from the different families. Protein Coat Analysis The capsid amino acid residues were analyzed to compare the residue characteristics versus viral characteristics of other phage (Figure 4-12).

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101 Figure 4-3. Electron micrograph of 60STP2^ ^ The image was taken at 40K and the magnification is approximately 100,000X. The results show that we were able to predict the adsorption of a virus based on the hydrophobic and charged residues of the capsid to some extent (Table 4-3 and 4-4). It is our belief that the residues at the surface are significant. However, the atomic details of most viruses have not yet been studied ( http://mmtsb.scripps.edu/viper/ and wv\/w.rcsb.org/pdb/index.html) (Berman et al. 2000, Reddy et al. 2001). Our results would assist in predicting viral adsorption to different types of surfaces that are charge dependent. Discussion Our study used several techniques for identification of indigenous bacteriophage. For our studies we wanted to properly classify the indigenous bacteriophage into the perspective families and study the differences between

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102 'op]. Figure 4-4. Electron micrograph of 61STP7^ ^The image was taken at 63K and the magnification is approximately 158,700X. the members as it relates to the adsorption characteristics. Physical parameters were identified by such methods as cross reactivity to hosts, plaque morphology, nucleic acid determination, and electron microscopy. From the results obtained we were able to classify the phage into perspective families. However, it has been found that this type of identification may be completely different than classification based on the genome (Haggard-Ljungquist et al. 1992, Monod et al. 1997, Tetart et al. 1996). So we used other methods of identification that included protein and restriction enzyme analysis. The results indicated that we have properly placed the selected bacteriophage in the proper families. However, we also found that the bacteriophage families are similar in gene organization, and that there is significant homology between families as previous reported 100 nm

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103 Figure 4-5. Electron micrograph of the bacteriophage 60STP2 with Salmonella typhimuhum ^ ^ The image was taken at 63K and the magnification is approximately 158,700X. (Bamford et al. 2002, Hendrix et al. 1999, Rohwer and Edwards 2002). We also found a significant amount of diversity within the families, especially Siphoviridae, this is in agreement with previous studies (Bamford et al. 2002, Clark et al. 2001, Ford et al. 1998, Juhala et al. 2000). Due to these limitations there are numerous bacteriophage that remain unclassified in the ICTV taxonomy database (Van Regenmortel et al. 1999 Virus Database Web Site http://www.ncbi.nlm.nih.gov/ICTVdb/index.htm ). When we examined the bacteriophage 60STP2 by electron microscopy we found that the structure was conserved as Siphoviridae. However, the genomic sequence of the capsid indicated a significant similarity to the phage ST64B in the Podoviridae family. It

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104 Table 4-2. Similarities of isolated and reference bacteriophage ISOLATED PHAGE (Our study) REFERENCE PHAGE FAMILY Protein Analysis Restriction Enzyme Analysis EcoRV 60KCK2 T5 T5 Siphoviridae 61KCK1G A A Siphoviridae 60STP2 T1 T1 Siphoviridae 61STP7 T4 T4 Myoviridae also showed some strong similarity to the Siphoviridae family members. We did not receive any significant similarities for the Integrase gene indicating that it is probably an unsequenced bacteriophage. Previous studies have shown that even though structures of the capsid are conserved the sequence is not (Baker et al. 1999, Bamford et al. 2002). Viruses are composed of an outer coat of proteins. It is believed that viral adsorption behavior is due to differences in their hydrophobicity and electrical charges on the capsid surfaces (Shields and Farrah 2002). The results of various studies demonstrate that the adsorptive differences of viruses with similar isoelectric points or characteristics are most likely due to localized groups of negative and positive charges, and possible spans of hydrophobic groups exist on viral surfaces (Mix 1974, Penrod et al. 1996, Schijven 2000). The surface charge on the virus originates from the ionization of amino acid side chains, and

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Figure 4-6. 4-20% SDS-PAGE of bacteriophage ^ ^ Lanes 1-4 are of indigenous phage 60KCK2, 61KCK10, 60STP2, AND 61STP7. Lanes 5-8 are of standard phage A, T1 , T4, and T5. The molecular weight marker is Broad Range Protein Molecular Weight Marker 10-15-25-35-50-75-100-150225 KDa. that this charge is a function of pH. When suspended in solutions there may be basic or negatively charged residues that become ionized and give a net surface charge that is pH dependent. The results show it is the surface charges of viruses that detemiine adsorption (Gerba et al. 1981, Penrod et al. 1996, Shields and Farrah 2002). Our results of the amino acid analysis of the viral capsid proteins show that this is probable. However, our study cannot identify the distribution of charges or possible spans of hydrophobic groups that are on the surface of the virus. This can only be accomplished by using molecular methods, cryo-electron microscopy, and x-ray diffraction to obtain atomic detail of the

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106 Figure 4-7. Restriction enzyme EcoRV analysis of bacteriophage ^ ^ Digestion was done with the enzyme EcoRV. Lanes 1 -4 are of isolated phage 60KCK2, 61KCK10, 60STP2, and 61STP7. Lanes 5-8 are of reference phage A, T1 , T4, and T5. The molecular weight marker is a Bench Top 1 Kb DNA Ladder (Promega). surfaces (Baker et al 1999). The use of cryo-electron microscopy and x-ray diffraction have lead to advances into the development of programs such as roadmaps or the VIPER website to illustrate the surfaces of viruses at the atomic level (Berman et al. 2000, Reddy et al. 2001, Rossman and Palmenberg 1988,

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107 Figure 4-8. Restriction enzyme EcoRI and Hindi analysis of bacteriophage ^ ^ Digestion was done with the enzymes EcoRI and Hindi in combination. Lanes 1-4 are of isolated phage 60KCK2, 61KCK10, 60STP2, and 61STP7. Lanes 5-8 are of reference phage A, T1 , T4, and T5. The molecular weight marker is a Bench Top 1 Kb DNA Ladder (Promega). http://mmtsb.scripps.edu/viper/ and www.rcsb.orq/pdb/index.html ). Currently these methods have only been used for a few well-known viruses (Baker et al 1999, Penrodetal. 1996).

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108 Figure 4-9. Restriction enzyme EcoRI and Hindi analysis of 60STP2^ ^ Digestion was done with the enzymes EcoRI and Hindi in combination. Lanes land 2 are of indigenous phage 60STP2. The molecular weight marker is a Bench Top 1Kb DNA Ladder (Promega).

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109 Figure 4-10. Screening plasmid restriction enzyme EcoRi and Hincll analysis of the capsid and integrase genes of 60STP2^ ^ Digestion was done with EcoRI and Hindi in combination. Lanes 1,4,5 are of capsid gene. Lanes 2,3,6,7,8 are of Integrase gene. The molecular weight marker is a Bench Top 1Kb DNA Ladder (Promega).

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110 Sequence ST64B HK97 D3CP N15 Lambda Consensus 1 1 1 1 1 1 1 10 20 ... I I 30 40 50 I.... I.... I. ...I.... I.... I 1 1 1 TGGCGCTGTCGCTCCTGACCTCTCCGCTGCACTACGGGCAGCACAAGACA 50 1 1 1 Sequence ST64B HK97 D3CP N15 Lambda Consensus 1 1 1 51 1 1 1 60 70 80 90 100 I I I .... I .... I I I I I GCTT|cHGcHGCAGHcGH||TgGgGGAgBc 34 1 26 100 1 gJg 3 TCACCAAATTCCTCCAAGG. aBB:g|aHaBcBci9B^4^^^^84 Sequence 35 C| ST64B HK97 D3CP N15 Lambda Consensus 1 Sequence ST64B HK97 D3CP N15 Lambda Consensus 1 0 Sequence 134 ST64B HK97 D3CP N15 Lajnbda Consensus 3 6 210 220 230 240 250

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111 Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Lambda Consensus 460 470 480 490 500

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112 510 520 530 540 550 Seq-305 398 ST64B 358 HK97 398 D3CP 4 96 N15 329 Lambda 392 Consensus 18 6 560 570 580 590 600 Consensus 211 H 9 naHaB jBBB moH B w 188 wB 234 610 620 Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Consensus

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113 Seq-305 ST64B HK97 D3CP N15 Lambda Consensus Seq-305 ST64B HK97 D3CP N15 Lainbda Consensus 760 770 780 790 800 ^^^^^^^^^^^^^^^^^^^^^^^^^ Seq-305 ST64B HK97 D3CP N15 T. amH Ha Consensus 860 870 880 890 900 Seq-305 ST64B HK97 D3CP N15 Lambda Consensus 910 920 930 .1 I I I I . . 940 .1 I 950 960 Seq-305 ST64B HK97 D3CP N15 Lambda Consensus 970 980 990 1000

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114 1030 1040 1050 Seq-305 846 1 ST64B 806 I HK97 885 ( D3CP 98 9 N15 808 1 Lambda 873 Consensus 432 1 Seq-305 889 1 ST64B 849 I HK97 935 ( D3CP 1039 1 N15 855 Lambda 919 1 Consensus 456 Seq-305 932 ST64B 892 HK97 984 ' D3CP 1088 1 N15 900 Lambda 966 1 Consensus 485 1160 1170 1180 1190 .1 I I I I I I . . . 1200 . I Seq-305 980 ST64B 940 HK97 1031 D3CP 1135 N15 947 Lambda 1016 Consensus 510 Seq-305 1022 ST64B 982 HK97 1073 D3CP 1177 N15 995 Lambda 1066 Consensus 529

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115 1260 Seq-305 1040 ST64B 1032 HK97 1122 D3CP 1226 N15 1045 Lambda 1115 Consensus 549 Seq-305 1040 ST64B 1079 HK97 1172 D3CP 1274 N15 1084 Lambda 1165 Consensus 557 Seq-305 1040 ST64B 1129 HK97 1222 D3CP 1322 N15 1084 Lambda 1170 Consensus 558 1310 . I . . . 1320 1330 1340 1350 gACi 1040 1128 1221 1321 1084 1170 558 i 1360 . I . . . -CTl 1370 . I . . . 1380 . I . . , 1390 . I . . . 1400 ICGgATCgc l T T cic tB9c tItItIagIag c aHt tIaItHtIaGAjAAgc|^BTc|cgCgTAflT T GScGgCgGgc|c( 1140 1260 ICGTGAGAAAG 1370 558 1410 Seq-305 1040 1040 ST64B 1140 1140 HK97 1260 1260 D3CP 1371 GCCGGCGGTG 1380 N15 1084 1084 Lambda 1170 1170 Consensus 558 558 Figure 4-1 1 . Sequence analysis of the capsid gene of 60STP2^ ^ The capsid gene was compared against Blast similarities of bacteriophage from Genbank. The sequence is our unknown sequence; ST64B is a Podoviridae capsid sequence, and the capsid sequences of Siphoviridae members D3, HK97, N15, and Lambda.

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116 m 6 k " " 1 e a ^^^^^^^^^^^^ * ^ * ^"^^ * a ^^-'^^^^^^//[^ ^^^^^^ *^ 340^ * _ 360 *_ 3 80 * 400 * 420 * Seq-305 : S^JIsSHMST og — . 355 STe4B : ^|[^KTTHTG(^H|sQ^HiH^^ : ^01 D3 : i^HEsjj^^^^lTgslTls : 395 Figure 4-12. Amino acid residue analysis of the capsid gene of 60STP2^ ^ The capsid amino acid residues of the unknown sequence (Seq-305) were compared against similar capsid genes. The phage ST64B is a Podoviridae, and bacteriophage HK97 and D3 are bacteriophage of Siphoviridae.

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117 Table 4-3. Capsid sequence amino acid hydrophobic residues and adsorption characterization of various viruses FAMILY VIRUS Pl a GRAVY b %LVIFM c % Hydrophobic Residues Adsorption Char(s)'^ Based on char(s) -viridae Prev. Current PodoT3 6.5 -0.108 27.1 54.5 s s T7 6.7 -0.059 27.2 54.2 s s ST64B 5 4 -0 433 26 4 48.4 w-m iviyoTA o. o n A AO m III o MLI IVI W 5 5 -0 337 28.9 48.9 w-m P2 5.0 -0.399 28.3 45.7 w-m SiphoLambda 5.3 -0.417 27.3 47.5 w D3 4.8 -0.269 26.6 50.1 m HK97 5.1 -0.367 26.5 46.8 w P21 4.0 -0.354 29.2 48 m N15 6.1 -0.286 28.4 49.3 m-s LeviMS-2 8 0.006 27.7 49.6 s s s F2 8.7 0.021 27.1 49.6 s s Micro
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118 Table 4-4. Capsid sequence amino acid charged residues and adsorption characterization of various viruses rAMlLY \/IDI lO P" a oKAVY b 1/ r~) [— KKtU /O KK-bU % /O Adsorption Char(s)'' Based on viridae Prev. Current PodoT3 6.5 -0.108 21.3 -0.6 m m T7 6.7 -0.059 21.7 -0.3 m m ST64B 5.4 -0.433 29.2 -1.7 s MyoT4 5.3 -0.142 20.3 -1.5 s m-s MU 5.5 -0.337 23.9 -1.6 m-s P2 5.0 -0.399 27.2 -2.5 s SiphoLambda 5.3 -0.417 24.9 -1.5 m-s D3 4.8 -0.269 22.5 -3.8 s HK97 5.1 -0.367 23.6 -2.9 s P21 4.0 -0.354 26.3 -1.2 m-s N15 6.1 -0.286 24.9 -0.3 m LeviMS-2 8 0.006 14.6 0.8 w w w F2 8.7 0.021 14 1.6 s m Micro0X174 6.4 -0.421 19.2 -0.9 w-m m w-m TectiPRD1 5.2 -0.197 14.9 -1.8 s m m-s PicornaPV1 6.1 -0.224 17.5 -1.2 s m-s m-s CB3 6.1 -0.201 14.8 -1 m CB4 5.8 -0.213 15.4 -1.7 w m-s CBS 5.8 -0.207 13.9 -1.4 m m El 5.8 -0.223 14.9 -1.8 w m-s E5 5.6 -0.187 15.6 -2.3 s s ^Isoelectric point (pi) is based on the capsid protein only ''GRAVY=Grand Average hydropathicity ''KRED= Charged residues (Lys, Asp, Arg, Glu) '^Previous adsorption based on Shields and Farrah 2002; m=moderate, s=strong, and w=weak

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CHAPTER 5 SUMMARY AND CONCLUSIONS Overall, our study was done to develop a method of selecting models for human pathogenic viruses for use in monitoring and assessing various materials, or as tracers in order to better predict the vulnerability of groundwater sources, and in treatment units. Previously, most models have been chosen based on similarities shared with human pathogenic viruses such as size, shape, and nucleic acid type. These studies have found that no one model is sufficient to examine the adsorption, fate, and transport of human pathogenic viruses (Dizer et al. 1984, Gerba et al. 1981, Goyal and Gerba 1979, Havelaar et al. 1993, lAWPRC 1 991 , Yates et al. 1 985). In the first part of our study we decided to select and characterize a range of bacteriophage for use as models based on their adsorption characteristics using defined media such as filters, and natural media such as mixed-liquor suspended solids and soils. In the second part of our study we identified the selected bacteriophages and characterized the phages and viruses from previous studies and our study based on the amino acid residues of the capsid to facilitate in explaining the mechanism(s) of adsorption. In the first part of our study, bacteriophages were selected and characterized from filtered raw sewage isolates based on specific adsorption properties using membrane filtration with variations in pH, ionic strength of the solution, and concentration of detergents to identify a range of phage based on 119

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120 properties such as their hydrophobic or electrostatic nature, the strength of the interactions, and temperature resistance. At pH 7, using nitrocellulose filters that are negatively charged and negatively charged viruses, we were able to show the extent adsorption was increased by increasing the concentration of an antichaotropic agent such as MgCb in solution (Farrah et al. 1981). Antichaotropic agents have been shown to disrupt electrostatic interactions by screening the charges, which would promote hydrophobic and van der Waals interactions (Farrah et al. 1981). We were also able to demonstrate the importance of the valence of salts with multivalent salts being better than monovalent salts for adsorption. At pH 3.5, nitrocellulose filters exhibit a negative charge and viruses exhibit a positive charge therefore electrostatic and hydrophobic interactions are possible. We were able to demonstrate the extent of viral electrostatic interactions by increasing the concentration of a neutral detergent such as Tween 80. The addition of a chaotropic agent or neutral detergent promote elution of phages by making the solution more lipophilic which allows for the interactions of apolar groups and the solution (Farrah et al. 1981 , Farrah 1982). We further characterized the bacteriophage adsorptive properties by using hydroxide coated diatomaceous earth and filter fibers to examine the difference in adsorption between MS2 a frequently used model, and a phage 60STP2 that exhibits weak hydrophobic and electrostatic interactions. Our study has shown that the bacteriophage adsorbed as expected in a controlled setting. Next we investigated the adsorptive interactions between the selected bacteriophage interactions and natural media such as mixed-liquor suspended

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121 solids and a variety of soils. Variations in pH, salt concentrations, and detergent concentrations were also used with the natural media to define significant factors involved in the adsorption and transport phenomena. The viruses we selected based on specific adsorption characteristics basically adsorbed as we had expected. However, the entire system defines the adsorption pattern. We found that the significant factors that affect adsorption were the composition of the solid, temperature, virus type, viral composition, pH, and characteristics of the solution. We also found that an increase in soil depth, and a slower flow rate allowed for increased retention time that increased viral adsorption. The results of adsorption to solids by select phages at varying temperatures show adsorption was dependent on temperature, and solid composition as previously shown. A virus particle is colloidal in nature so it must be physically adsorbed to a surface in order to be removed. Our results show that phage adsorption to natural medias at various temperatures is a physical process with a low energy of activation of <40 Kcal/mol (Table 3-10). Our results also have shown that the bacteriophage 60STP2 could be a better worst-case model than MS-2 considering that it adsorbed poorer to defined and natural media. Our study shows that the worst-case models MS-2 and PRD-1 do not adsorb that poorly to all media, and that adsorption was dependent on the various factors in the systems. The bacteriophage 60STP2 selected based on its weak adsorption to nitrocellulose filters. Adsorption experiments indicated that it has weak hydrophobic and electrostatic properties, and has been shown to adsorb poorly to virtually all media under various conditions. In the tracer studies performed

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122 the phage 60STP2 was found in the wells approximately 8 hours before MS-2. Our results have shown that it took between eight and ten days for complete inactivation of both MS-2 and 60STP2. In the final part of our study we used several techniques for family identification of the selected bacteriophages. For our studies we wanted to properly classify the bacteriophage into the perspective families and study the differences between the members as it relates to the adsorption characteristics. Viruses contain a protein coat, therefore, they may exhibit charged or hydrophobic groups on the surface, and thus factors such as pH, ionic strength, and chaotropic agents have been shown to affect adsorption to solids. The techniques used were to identify the selected bacteriophage were electron microscopy, host range, plaque formation, enzymatic, protein, and molecular procedures. From the results obtained we were able to classify the phage into perspective families. However, we also found that some of the bacteriophage families are similar in gene organization, and that there is significant homology between families. We also found a significant amount of diversity within the families, especially Siphoviridae. When we examined the phage 60STP2 by electron microscopy we found that the structure was conserved as Siphoviridae. However, the genomic sequence of the capsid indicated a significant similarity to the phage ST64B in the Podoviridae family. It also showed some strong similarity to the Siphoviridae family members. We did not observe any significant similarities for the integrase gene indicating that it is probably an unsequenced bacteriophage. Our results of the amino acid analysis of the viral capsid proteins

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123 show that there is significant differences in the composition that correspond to the adsorption properties of the viruses. However, our study can not identify the distribution of charges or possible spans of hydrophobic groups that are on the surface of the virus. This can only be accomplished by using molecular methods, cryo-electron microscopy, and x-ray diffraction to obtain atomic detail of the surfaces (Baker et al 1999). Currently these methods have only been used for a few well-known viruses. In summary, we have isolated a range of phage that can be used as models for other enteric organisms based on their adsorption properties. These bacteriophages can be used to identify the adsorptive patterns and properties of similar enteric viruses to various media. Our results show that viral adsorption to media is based on specific adsorption properties that for the most part follow the DLVO theory. Due to the numerous factors involved in these complex systems, the results show that it is necessary to define the entire system each time to detemnine the mechanism of adsorption. The results also show that adsorption or transport is dependent on specific factors such as, retention time, composition of the solutions, ionic strength, pH, viral coat composition, and the composition of the solids. Nevertheless, knowledge of the significant parameters can give a predictive adsorption model. Our results also show that by selecting bacteriophage based on their adsorption properties, and the system parameters we can predict better models for transport systems.

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APPENDIX A BACTERIOPHAGE ADSORPTION Table A-1. Phage Adsorption to 0.45 |jm Nitrocellulose Filters at pH 7 and 3.5 HOST PHAGES % ADSORBED at pH 7 to 0.45mM HA MILLIPORE FILTERS % ADSORBED at pH 3.5 to 0.45mM ha MILLIPORE FILTERS Ri ipFFR 0 1M MaCU BUFFER 0 1 % TWEEN-80 60PW1 74 +21 91 +4 75 +4 35 +1 1 60PW2 45 +7 82 +2 95 +3 57 +13 60PW3 72 +17 86 +12 97 +1 46 +9 P. vulgaris 60PW4 37 +10 94 +5 97 +3 82 +6 1 00PVTV1 0 +0 90 +8 90 +5 10+5 1 00PVTV2 10 +6 52 +10 78 +8 48 +20 100PVTV3 23 +5 39+14 51 +20 8+2 100P\/TV4 84 +4 97 +3 97 +0 21 +5 60KCK1 85 +5 93+6 94 +5 35+12 ROKCK? vj \j IN w r\.^ 80 +4 100 +2 92 +6 37 +8 60KCK3 89+11 100 +0 98 +2 51 +9 60KCK4 98 +2 100 +2 98+1 39+14 100KCTK1 31+21 75+17 72 +9 45 +3 100KCTK2 98 +3 100 +0 80 +4 72 +5 100KCTK3 38+15 70 +8 82 +5 51 +7 E. coli KC 100KCTK4 20 +3 80 +8 100 +5 75 +9 61KCK5 15 +4 83 +6 92 +7 54+10 61KCK6 1 +3 58+14 99+1 48 +6 61KCK7 18+5 69+10 77 +5 31 +17 61KCK8 39 +9 81 +6 87 +6 43 +4 61KCK9 31 +10 91 +7 90+10 9+12 61KCK10 82 +6 99 +3 59 +6 54+9 61KCK11 72+15 94 +2 90 +4 54+3 61KCK12 48+4 57 +2 87+2 67 +8 The bacteriophage concentrations used were 10 PFU/mL, and the solutions used were 0.02M imidazole Glycine at pH 7, 0.02M imidazole Glycine and 0.1M MgCb at pH 7, 0.02M imidazole Glycine at pH 3.5, and 0.02M imidazole Glycine and 0.1% TweenSO at pH 3.5. 124

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125 Table A-1 . Continued % ADSORBED at pH 7 % ADSORBED at pH 3.5 HOST PHAGES to 0.45mM HA MILLIPORE FILTERS to 0.45|jM ha MILLIPORE FILTERS BUFFER 0.1M MgCl2 BUFFER 0.1% TWEEN-80 10C3A 20+1 76 +2 75 +3 52 +3 10C3B 21 +4 74 +6 87 +7 51 +11 10C3C 20 +2 81 +5 95+1 19+4 10C3D 29 +6 93 +3 100 +0 46+11 10C3Di 40 +6 75 +6 98 +2 0+0 10C3Dp 40+1 87 +2 100 +2 55+4 10C3Ei 38 +9 82 +8 90 +2 73+15 10C3Ep 10+2 49 +10 100 +0 75 +3 10C3F 60+18 89 +2 99 +1 70+10 10C3G 25+16 67+13 95 +2 63+15 10C3H 18+9 92 +4 98 +4 65+12 60C3B3 28 +2 78 +10 98 +2 52+18 60C3B4 43 +17 88 +4 78 +10 51 +13 60C3B5 18 +13 69 +7 75 +4 0+2 60C3B6 29 +5 99 +2 81 +5 11 +6 60C3F1 18+8 85 +6 91 +8 29 +7 E.coli 60C3F2 36+10 91 +6 93+10 33 +8 C3000 60C3F3 18+5 88 +5 98 +6 55 +4 60C3F4 19+4 81 +1 82 +2 41 +6 60C3F5 21 +5 72 +9 87 +7 68 +2 60C3FX 15+3 59 +5 90 +5 38+14 60C3H1 18+9 47+11 82 +4 0+1 60C3H2 61 +5 79 +7 81 +3 9+9 60C3M 33 +4 84 +8 87 +4 27 +5 100C3TC1 24+17 88 +3 80 +2 61 +9 100C3TC2 14 +7 89+4 91 +3 72 +4 100C3TC3 0 +3 100 +2 99+1 32 +6 100C3TC4 49 +6 100 +2 98 +2 47 +7 61C3C5 0+0 42 +21 87 +4 18+9 61C3C6 11 +4 71 +5 81 +2 5+2 61C3C7 19+3 83 +7 93 +7 21 +2 61C3C8 17 +9 85 +8 97 +2 28 +4 61C3C9 10 +4 54 +8 97 +1 35 +4 61C3C10 15+12 66+11 92 +10 21 +7 The bacteriophage concentrations used were 10^PFU/mL, and the solutions used were 0.02M imidazole Glycine at pH 7, 0.02M imidazole Glycine and 0.1M MgCl2 at pH 7, 0.02M imidazole Glycine at pH 3.5, and 0.02M imidazole Glycine and 0.1% TweenBO at pH 3.5.

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126 Table A-1. Continued HOST PHAGES % ADSORBED at pH 7 to 0.45|ji\/l HA IVIILLIPORE FILTERS % ADSORBED at pH 3.5 to 0.45mm ha MILLIPORE FILTERS BUFFER 0.1M MgCl2 BUFFER 0.1%TWEEN-80 HOST PHAGES % ADSORBED pH 7 % ADSORBED pH 3.5 BUFFER 0.1M MgCl2 BUFFER 0.1% TWEEN-80 S. typhimurium 60STS1 76 +5 85 +4 96 +2 57 +4 60STS2 65 +2 97 +2 99+1 46 +3 60STP2 0+3 46 +6 3+2 3+6 D1STS5 49 +3 99+1 100 +2 43 +6 61STP5 31 +2 81 +2 80 +2 21 +9 61STS6 35 +7 79 +6 93 +3 29 +5 61STS7 0+5 37+16 77 +4 51 +4 dISTSo 41 +5 55 +6 51 +4 5 +2 61STP7 0 +2 69 +4 29 +6 14+4 aerogenes 60EAE1 68 +8 96 +3 68+18 16+5 60EAE2 78 +13 100 +0 87 +10 27 +9 60EAE3 46 +9 92 +6 18+2 15+5 67 +5 100 +2 100 +0 0+3 10+2 55 +9 93 +8 74 +20 93 +7 100+1 100 +2 25+14 80 +4 100 +0 100+1 51 +5 innFATF4 41 +0 no _i_0 93 +3 91 +5 17+9 C. freundii 60CFF1 62 +12 81 +10 67 +10 0+0 60CFF2 62 +18 93 +6 79 +13 25+10 K. pneumoniae 60KPP1 39 +6 78 +7 68 +12 7 +2 60KPP3 43 +9 57 +4 27 +3 0+1 DUI\rr4 48 +7 83+12 46 +9 4+2 61KPP5 10+4 92 +6 98 +2 65+10 61KPP6 38 +7 73 +8 46 +5 0+2 61KPP7 26 +6 84 +4 94 +6 29 +7 61KPP8 9+9 71 +9 91 +5 11 +7 61KPP9 0+2 59 +5 71 +7 42+15 61KPP10 51 +5 98 +2 100 +2 42 +8 he bacteriophage concentrations used were 10^FU/ mL, and the solutions used were 0.02M imidazole Glycine at pH 7, 0.02M imidazole Glycine and 0.1M MgCl2 at pH 7, 0.02M imidazole Glycine at pH 3.5, and 0.02M imidazole Glycine and 0.1% TweenSO at pH 3.5.

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APPENDIX B AMINO ACID SEQUENCES OF VIRAL CAPSIDS Caudovirales Leviviridae >sp|P0361 1 |C0AT_BPF2 Coat protein Bacteriophage f2. ASNFTQFVLVNDGGTGNVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRK YTIKVEVPKVATQTVGGVELPVAAWRSYLNLELTIPIFATNSDCELIVKAMQGLLKDGNPI PSAIAANSGIY >sp|P03612|COAT_BPMS2 Coat protein Bacteriophage MS2. ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRK YTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNP IPSAIAANSGIY Microviridae >sp|P03641|VGF_BPPHX Capsid protein (F protein) (GPF) phage phi-X174. SNIQTGAERMPHDLSHLGFLAGQIGRLITISTTPVIAGDSFEMDAVGALRLSPLRRGLAID STVDIFTFYVPHRHVYGEQWIKFMKDGVNATPLPTVNTTGYIDHAAFLGTINPDTNKIPK HLFQGYLNIYNNYFKAPWMPDRTEANPNELNQDDARYGFRCCHLKNIWTAPLPPETEL SRQMTTSTTSIDIMGLQAAYANLHTDQERDYFMQRYHDVISSFGGKTSYDADNRPLLV MRSNLWASGYDVDGTDQTSLGQFSGRVQQTYKHSVPRFFVPEHGTMFTLALVRFPPT ATKEIQYLNAKGALTYTDIAGDPVLYGNLPPREISMKDVFRSGDSSKKFKIAEGQWYRY APSYVSPAYHLLEGFPFIQEPPSGDLQERVLIRHHDYDQCFQSVQLLQWNSQVKFNVT VYRNLPTTRDSIMTS Tectiviridae sp|P22535|COA3_BPPRD Major capsid protein (Protein P3) phage PRD1. AQVQQLTPAQQAALRNQQAMAANLQARQIVLQQSYPVIQQVETQTFDPANRSVFDVT PANVGIVKGFLVKVTAAITNNHATEAVALTDFGPANLVQRVIYYDPDNQRHTETSGWHL HFVNTAKQGAPFLSSMVTDSPIKYGDVMNVIDAPATIAAGATGELTMYYWVPLAYSETD LTGAVLANVPQSKQRLKLEFANNNTAFAAVGANPLEAIYQGAGAADCEFEEISYTVYQS YLDQLPVGQNGYILPLIDLSTLYNLENSAQAGLTPNVDFWQYANLYRYLSTIAVFDNGG SFNAGTDINYLSQRTANFSDTRKLDPKTWAAQTRRRIATDFPKGVYYCDNRDKPIYTLQ YGNVGFWNPKTVNQNARLLMGYEYFTSRTELVNAGTISTT 127

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128 Myoviridae >sp|Q9T1W1|VPT_BPMU Major head subunit (gpT) Bacteriophage Mu. MIVTPASIKALMTSWRKDFQGGLEDAPSQYNKIAMWNSSTRSNTYGWLGKFPTLKEW VGKRTIQQMEAHGYSIANKTFEGTVGISRDDFEDDNLGIYAPIFQEMGRSAAVQPDELIF KLLKDGFTQPCYDGQNFFDKEHPVYPNVDGTGSAVNTSNIVEQDSFSGLPFYLLDCSR AVKPLIFQERRKPELVARTRIDDDHVFMDNEFLFGASTRRAAGYGFWQMAVAVKGDLT LDNLWKGWQLMRSFEGDGGKKLGLKPTHIWPVGLEKAAEQLLNRELFADGNTTVSN EMKGKLQLWADYL >sp|P25477|VPN_BPP2 Major capsid protein precursor (GPN) Bacteriophage P2. MRQETRFKFNAYLSRVAELNGIDAGDVSKKFTVEPSVTQTLMNTMQESSDFLTRINIVP VSEMKGEKIGIGVTGSIASTTDTAGGTERQPKDFSKLASNKYECDQINFDFYIRYKTLDL WARYQDFQLRIRNAIIKRQSLDFIMAGFNGVKRAETSDRSSNPMLQDVAVGWLQKYRN EAPARVMSKVTDEEGRTTSEVIRVGKGGDYASLDALVMDATNNLIEPWYQEDPDLWI VGRQLLADKYFPIVNKEQDNSEMLAADVIISQKRIGNLPAVRVPYFPADAMLITKLENLSI YYMDDSHRRVIEENPKLDRVENYESMNIDYWEDYAAGCLVEKIKVGDFSTPAKATAEP GA >sp|P04535|COAT_BPT4 Major capsid protein (Protein Gp23) Bacteriophage T4. MTIKTKAELLNKWKPLLEGEGLPEIANSKQAIIAKIFENQEKDFQTAPEYKDEKIAQAFGS FLTEAEIGGDHGYNATNIAAGQTSGAVTQIGPAVMGMVRRAIPNLIAFDICGVQPMNSP TGQVFALRAWGKDPVAAGAKEAFHPMYGPDAMFSGQGAAKKFPALAASTQTTVGDI YTHFFQETGTVYLQASVQVTIDAGATDAAKLDAEIKKQMEAGALVEIAEGMATSIAELQE GFNGSTDNPWNEMGFRIDKQVIEAKSRQLKAAYSIELAQDLRAVHGMDADAELSGILAT EIMLEINREWDWINYSAQVGKSGMTLTPGSKAGVFDFQDPIDIRGARWAGESFKALLF QIDKEAVEIARQTGRGEGNFIIASRNWNVLASVDTGISYAAQGLATGFSTDTTKSVFAG VLGGKYRVYIDQYAKQDYFTVGYKGPNEMDAGIYYAPYVALTPLRGSDPKNFQPVMGF KTRYGIGINPFAESAAQAPASRIQSGMPSILNSLGKNAYFRRVYVKGI Podoviridae >tr|Q8HAD2 Sb6 Salmonella typhimurium phage ST64B. MAVDIKDVEQVAQELQQKFDDFKAKNDKRVEAIEQEKGKU\GQVETLNGKLSELENLK SDLEKELLELKRPARGAQNKVAAEHKDAFVGFLRKGREDGLRDLERKALQVGTDEDG GYAVPEELDRSILSLLKDEWMRQEATVITVGGSDYKKLVNLGGTASGWVGETDTRSQ TATSRLGLIEPFMGEIYGNPQATQKMLDDAFFNVEAWINSEU\TEFAEQEEIAFTTGDGT KKPKGFLAYESTEESDKARAFGKLQHIVSGEATAVTADAIIKLIYTLRKAHRTGAKFMMN NNSLFAIRLLKDTEGNYLWRPGLELGQPSSLAGYGIAENEQMPDIAADAKAIAFGNFKR GYTIVDRIGTRILRDPYTNKPFVGFYTTKRTGGMLVDSQAIKLLKIAAA

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129 >sp|P26747|VG05_BPP22 Coat protein (Protein gp5) Bacteriophage P22. ALNEGQIVTLAVDEIIETISAITPMAQKAKKYTPPAASMQRSSNTIWMPVEQESPTQEGW DLTDKATGLLELNVAVNMGEPDNDFFQLRADDLRDETAYRRRIQSAARKLANNVELKV ANMAAEMGSLVITSPDAIGTNTADAWNFVADAEEIMFSRELNRDMGTSYFFNPQDYKK AGYDLTKRDIFGRIPEEAYRDGTIQRQVAGFDDVLRSPKLPVLTKSTATGITVSGAQSFK PVAWQLDNDGNKVNVDNRFATVTLSATTGMKRGDKISFAGVKFLGQMAKNVLAQDAT FSWRWDGTHVEITPKPVALDDVSLSPEQRAYANVNTSLADAMAVNILNVKDARTNVF WADDAIRIVSQPIPANHELFAGMKTTSFSIPDVGLNGIFATQGDISTLSGLCRIALWYGVN ATRPEAIGVGLPGQTA >sp|P19693|VCAA_BPT3 Major capsid protein 10A Bacteriophage T3. MANIQGGQQIGTNQGKGQSAADKLALFLKVFGGEVLTAFARTSVTMPRHMLRSIASGK SAQFPVIGRTKAAYLKPGENLDDKRKDIKHTEKVIHIDGLLTADVLIYDIEDAMNHYDVRA EYTAQLGESLAMAADGAVLAELAGLVNLPDGSNENIEGLGKPTVLTLVKPTTGSLTDPV ELGKAIIAQLTIARASLTKNYVPAADRTFYTTPDNYSAILAALMPNAANYQALLDPERGTI RNVMGFEWEVPHLTAGGAGDTREDAPADQKHAFPATSSTTVKVALDNWGLFQHRS AVGTVKLKDU\LERARRANYQADQIIAKYAMGHGGLRPEAAGAIVLPKVSE >sp|P19726|VCAA_BPT7 Major capsid protein 10A Bacteriophage T7. MASMTGGQQMGTNQGKGWAAGDKLMFLKVFGGEVLTAFARTSVTTSRHMVRSISS GKSAQFPVLGRTQAAYLAPGENLDDKRKDIKHTEKVITIDGLLTADVLIYDIEDAMNHYD VRSEYTSQLGESLAMAADGAVLAEIAGLCNVESKYNEN I EGLGTATVI ETTQN KAALTD QVALGKEIIAALTKARAALTKNYVPAADRVFYCDPDSYSAILAALMPNAANYAALIDPEK GSIRNVMGFEWEVPHLTAGGAGTAREGTTGQKHVFPANKGEGNVKVAKDNVIGLFM HRSAVGTVKLRDLALERARRANFQADQIIAKYAMGHGGLRPEAAGAWFKVE Siphoviridae >tr|Q9XJT3 Major head protein Bacteriophage D3. MSDFEKQIGELNASLKQVGDQIKSQAEQVNTQIANFGEMNKETRAKVDELLTAQGELQ ARLSAAEQAMLANEKRDGGEEAPKTAGQMVAESLKEQGVTSSLRGSHRVSMPRSAIT SIDGSGGALVAPDRRPGWAAPQRRLTIRDLVAPGTTESNSVEYVRETGFVNNAAPVS EGTQKPYSDLTFELENAPVRTIAHLFKASRQILDDASALQSYIDARARYGLMLVEECQLL YGNGTGANLHGIIPQAQAYAPPSGVWTAEQRIDRIRLAILQAQLAEFPASGIVLNPIDWA LIELNKDAENRYIIGSPQNGTTPTLWRLPWETQAITQDEFLTGAFSLGAQIFDRMDIEVL VSTENDKDFENNMVTIRAEERU\FAVYRPEAFVTGSLTAS >sp|P49861|COAT_BPHK7 Major capsid protein precursor (GPS) -phage HK97. MSELALIQKAIEESQQKMTQLFDAQKAEIESTGQVSKQLQSDLMKVQEELTKSGTRLFD LEQKLASGAENPGEKKSFSERAAEELIKSWDGKQGTFGAKTFNKSLGSDADSAGSLIQ PMQIPGIIMPGLRRLTIRDLU\QGRTSSNALEYVREEVFTNNADWAEKALKPESDITFSK QTANVKTIAHWVQASRQVMDDAPMLQSYINNRLMYGLALKEEGQLLNGDGTGDNLEG LNKVATAYDTSLNATGDTRADIIAHAIYQVTESEFSASGIVLNPRDWHNIALLKDNEGRYI FGGPQAFTSNIMWGLPWPTKAQAAGTFTVGGFDMASQVWDRMDATVEVSREDRDN FVKNMLTILCEERLALAHYRPTAIIKGTFSSGS

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130 >sp|P03713|HEAD_l_AMBD Major head protein (GPE) Bacteriophage lambda. MSMYTTAQLLAANEQKFKFDPLFLRLFFRESYPFTTEKVYLSQIPGLVNMALYVSPIVSG EV1RSRGGSTSEFTPGYVKPKHEVNPQMTLRRLPDEDPQNU\DPAYRRRRI1MQNMRD EELAIAQVEEMQAVSAVLKGKYTMTGEAFDPVEVDMGRSEENNITQSGGTEWSKRDK STYDPTDDIEAYALNASGWNIIVFDPKGWALFRSFKAVKEKLDTRRGSNSELETAVKDL GKAVSYKGMYGDVAIWYSGQYVENGVKKNFLPDNTMVLGNTQARGLRTYGCIQDAD AQREGINASARYPKNWVTTGDPAREFTMIQSAPLMLLADPDEFVSVQLA >tr|064322 Gp8 Bacteriophage N15. MSVYTTAQLLAVNEKKFKFDPLFLRIFFRETYPFSTEKVYLSQIPGLVNMALYVSPIVSGK VIRSRGGSTSEFTPGYVKPKHEVNPLMTLRRLPDEDPQNLADPVYRRRRIILQNMKDEE LAIAQVEEKQAVAAVLSGKYTMTGEAFEPVEVDMGRSAGNNIVQAGAAAWSSRDKET YDPTDDIEAYALNASGWNIIVFDPKGWALFRSFKAVKEKLDTRRGSNSELETALKDLGK AVSYKGMYGDVAIWYSGQYIENDVKKNYLPDLTMVLGNTQARGLRTYGCILDADAQR EGINASTRYPKNWVQTGDPAREFTMIQSAPLMLLADPDEFVSVKLA >sp|P36270|HEAD_BPP21 (Head protein GP7) Bacteriophage P21 (phage 21). MGLFTTRQLLGYTEQKVKFRALFLELFFRRTVNFHTEEVMLDKITGKTPVAAYVSPWE GKVLRHRGGETRVLRPGYVKPKHEFNYQQAVERLPGEDPSQLNDPAYRRLRIITDNLK QEEHAIVQVEEMQAVNAVLYGKYTMEGDQFEKIEVDFGRSTKNNITQGSGKEWSKQD RDTFDPTHDIDLYCDLASGLVNIAIMDGTVWRLLNGFKLFREKLDTRRGSNSQLETAVK DLGAWSFKGYYGDLMWAKTSYIAEDGIEKRYLPDGMLVLGNTAADGIRCYGAIQDAQ ALSEGWASSRYPKHWLTVGDPAREFTMTQSAPLMVLPDPDEFVWQVK Picornaviridae Poliovirus >sp|P0LIO1] SPNIEACGYSDRVLQLTLGNSTITTQEAANSWAYGRWPEYLRDSEANPVDQPTEPDV AACRFYTLDTVSWTKESRGWWWKLPDALRDMGLFGQNMYYHYLGRSGYTVHVQCN ASKFHQGALGVFAVPEMCLAGDSNTTTMHTSYQNANPGEKGGTFTGTFTPDNNQTSP ARRFCPVDYLLGNGTLLGNAFVFPHQIINLRTNNCATLVLPYVNSLSIDSMVKHNNWGIA ILPLAPLNFASESSPEIPITLTIAPMCCEFNGLRNITLPRLQGLPVMNTPGSNQYLTADNF QSPCALPEFDVTPPIDIPGEVKNMMELAEIDTMIPFDLSATKKNTMEMYRVRLSDKPHT DDPILGLSLSPASDPRLSHTMLGEILNYYTHWAGSLKFTFLFCGFMMATGKLLVSYAPP GADPPKKRKEAMLGTHVIWDIGLQSSCTMWPWISNTTYRQTIDDSFTEGGYISVFYQT RIWPLSTPREMDILGFVSACNDFSVRLLRDTTHIEQKAI_AQGLGQMLESMIDNTVRETV GAATSRDALPNTEASGPTHSKEIPALTAVETGATNPLVPSDTVQTRHWQHRSRSESSI ESFFARGACVTIMTVDNPASTTNKDKLFAVWKITYKDTVQLRRKLEFFTYSRFDMELTF WTANFTETNNGHALNQVYQIMYVPPGAPVPEKWDDYTWQTSSNPSIFYTYGTAPARI SVPYVGISNAYSHFYDGFSKVPLKDQSAALGDSLYGAASLNDFGILAVRWNDHNPTKV TSKIRVYLKPKHIRVWCPRPPRAVAYYGPGVDYKDGTLTPLSTKDLTTY

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131 Echovirus >sp|ECHOVIRUS 1] SPTVEECGYSDRVRSITLGNSTITTQECAN\AA/GYGEWPEYLSDNEATAEDQPTQPDV ATCRFYTLDSVQWENGSPGWWWKFPDALRDMGLFGQNM YYHYLG RAGYTIHVQCN ASKFHQGCILWCVPEAEMGSAQTSGWNYEHISKGEIASRFTTTTTAEDHGVQAAVW NAGMGVGVGNLTIFPHQWINLRTNNSATIVMPYVNSVPMDNMYRHHNFTLMIIPFVPLD FSAGASTYVPITVTVAPMCAEYNGLRLAGHQGLPTMNTPGSNQFLTSDDFQSPSAMP QFDVTPEMHIPGEVRNLMEIAEVDSVMPINNDSAAKVSSMEAYRVELSTNTNAGTQVF GFQLNPGAESVMNRTLMGEILNYYAHWSGSIKITFVFCGSAMTTGKFLLSYAPPGAGA PKTRKDAMLGTHWWDVGLQSSCVLCIPWISQTHYRFVEKDPYTNAGFVTCWYQTSV VSPASNQPKCYMMCMVSACNDFSVRMLRDTKFIEQTSFYQGDVQNAVEGAMVRVAD TVQTSATNSERVPNLTAVETGHTSQAVPGDTMQTRHVINNHVRSESTIENFLARSACVF YLEYKTGTKEDSNSFNNWVITTRRVAQLRRKLEMFTYLRFDMEITWITSSQDQSTSQN QNAPVLTHQIMYVPPGGPIPVSVDDYSWQTSTNPSIFWTEGNAPARMSIPFISIGNAYS NFYDGWSHFSQAGVYGFTTLNNMGQLFFRHVNKPNPAAITSVARIYFKPKHVRAWVP RPPRLCPYI NSTNVNFEPKPVTEVRTN I ITT >sp|ECHOVIRUS 5] SPSAEECGFSDRVRSLTLGNSTITTQESANVWGYGRWPDYLADDQATAEDQPTQPD VATCRFYTLESVSWQSGSAGWWWKFPEALKDMGLFGQNMYYHYLGRAGYTIHVQCN ASKFHQGCLLWCVPEAEMGCADVTSWTALNLINGEDAHTFSPSEATAEAGKVQTAV CNAGMGVAVGNLTIFPHQWINLRTNNCATIVMPYINSVPMDNMFRHYNFTLMVIPFAPL ASQGGSTYVPITITIAPMCAEYNGLRLSTQPQGLPVMNTPGSNQFLTSDDFQSPCAMP EFDVTPPMDIPGEVRNIMEIAEVDSWPVNNMSSKVKTIEAYQIPVSVGTTVRGDAIFSF QLNPGNSPVLNRTLLGEIINYYAHWSGSIKLTFLFCGSAMATGKLLLAYSPPGASVPTSR KDAMLGTHIIWDLGLQSSCVLCVPWISQTHYRMVQQDEYSAAGYITCWYQTNIIVPPDT PTDCIVLCFVSACNDFSVRMLKDTPFVEQEADLQGDSEHAVESAVSRVADTIMSGPSN SQQVPALTAVETGHTSQWPSDTIQTRHVQNFHSRSESTIENFLSRSACVHIANYNAKG DKTDVNRFDRWEINIREMVQLRRKCEMFTYLRFDIEVTFVITSKQDQGPKLNQDMPVLT HQIMYVPPGGSVPSTVESYAWQTSTNPSVFWTEGNAPARMSIPFISIGNAYSSFYDGW SHFTQKGVYGYNTLNKMGQLFVRHVNKETPTPVTSTIRVYFKPKHIRAWVPRPPRLCP YVNKTNVNFITTQVTEPRNDLNDVPKSEHNMHTY Coxsackie virus >sp|COXSACKIE B3| SPTVEECGYSDRARSITLGNSTITTQECANWVGYGVWPDYLKDSEATAEDQPTQPDV ATCRFYTLDSVQWQKTSPGWWWKLPDALSNLGLFGQNMQYHYLGRTGYTVHVQCN ASKFHQGCLLWCVPEAEMGCATLDNTPSSAELLGGDTAKEFADKPVASGSNKLVQRV VYNAGMGVGVGNLTIFPHQWINLRTNNSATIVMPYTNSVPMDNMFRHNNVTLMVIPFV PLDYCPGSTTYVPITVTIAPMCAEYNGLRLAGHQGLPTMNTPGSCQFLTSDDFQSPSA MPQYDVTPEMRIPGEVKNLMEIAEVDSWPVQNVGEKVNSMEAYQIPVRSNEGSGTQ VFGFPLQPGYSSVFSRTLLGEILNYYTHWSGSIKLTFMFCGSAMATGKFLLAYSPPGAG APTKRVDAMLGTHVIWDVGLQSSCVLCIPWISQTHYRFVASDEYTAGGFITCWYQTNIV VPADAQSSCYIMCFVSACNDFSVRLLKDTPFISQQNFFQGPVEDAITAAIGRVADTVGT GPTNSEAIPALTAAETGHTSQWPGDTMQTRHVKNYHSRSESTIENFLCRSACVYFTEY KNSGAKRYAEWVLTPRQAAQLRRKLEFFTYVRFDLELTFVITSTQQPSTTQNQDAQILT HQIMYVPPGGPVPDKVDSYVWQTSTNPSVFWTEGNAPPRMSIPFLSIGNAYSNFYDG

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132 WSEFSRNGVYGI NTLNN MGTLYARHVNAGSTGPI KSTI Rl YFKPKHVKAWI PRPPRLCQ YEKAKNVNFQPSGVTTTRQSITTMTN T >sp|COXSACKIE B4| MGAQVSTQKTGAHETSLSASGNSIIHYTNINYYKDAASNSANRQDFTQDPSKFTEPVKD VMIKSLPALNSPTVEECGYSDRVRSITLGNSTITTQECANVWGYGVWPDYLSDEEATA EDQPTQPDVATCRFYTLNSVKWEMQSAGWWWKFPDALSEMGLFGQNMQYHYLGRS GYTIHVQCNASKFHQGCLLWCVPEAEMGCTNAENAPAYGDLCGGETAKSFEQNAAT GKTAVQTAVCNAGMGVGVGNLTIYPHQWINLRTNNSATIVMPYINSVPMDNMFRHNNF TLMIIPFAPLDYVTGASSYIPITVTVAPMSAEYNGLRLAGHQGLPTMLTPGSTQFLTSDD FQSPSAMPQFDVTPEMNIPGQVRNLMEIAEVDSWPINNLKANLMTMEAYRVQVRSTD EMGGQIFGFPLQPGASSVLQRTLLGEILNYYTHWSGSLKLTFVFCGSAMATGKFLLAYS PPGAGAPDSRKNAMLGTHVIWDVGLQSSCVLCVPWISQTHYRYWDDKYTASGFISC WYQTNVIVPAEAQKSCYIMCFVSACNDFSVRMLRDTQFIKQTNFYQGPTEESVERAMG RVADTIARGPSNSEQIPALTAVETGHTSQVDPSDTMQTRHVHNYHSRSESSIENFLCRS ACVIYIKYSSAESNNLKRYAEWVINTRQVAQLRRKMEMFTYIRCDMELTFVITSHQEMS TATNSDVPVQTHQIMYVPPGGPVPTSVNDYVWQTSTNPSIFWTEGNAPPRMSIPFMSI GNAYTMFYDGWSNFSRDGIYGYNSLNNMGTIYARHVNDSSPGGLTSTIRIYFKPKHVK AYVPRPPRLCQYKKAKSVNFDVEAVTAERASLITT >sp|COXSACKIE B5] SPSAEECGYSDRVRSITLGNSTITTQECANVWGYGTWPTYLKDEEATAEDQPTQPDV ATCRFYTLESVMWQQSSPGWWWKFPDALSNMGLFGQNMQYHYLGRAGYTVHVQCN ASKFHQGCLLWCVPEAEMGCATLANKPDQKSLSNGETANTFDSQNTTGQTAVQANVI NAGMGVGVGNLTIFPHQWINLRTNNSATIVMPYINSVPMDNMFRHNNFTLMIIPFAPLSY STGATTYVPITVTVAPMCAEYNGLRLAGKQGLPTMLTPGSNQFLTSDDFQSPSAMPQF DVTPEMAIPGQVNNLMEIAEVDSWPVNNTEGKVSSIEAYQIPVQSNSTNGSQVFGFPLI PGASSVLNRTLLGEILNYYTHWSGSIKLTFMFCGSAMATGKFLLAYSPPGAGAPTTRKE AMLGTHVIWDVGLQSSCVLCIPWISQTHYRYWVDEYTAGGYITCWYQTNIWPADTQS DCKILCFVSACNDFSVRMLKDTPFIKQDSFYQGPPGEAVERAIARVADTISSGPVNSESI PALTAAETGHTSQWPADTMQTRHVKNYHSRSESTVENFLCRSACVYYTTYKNHGTD GDNFAYWVINTRQVAQLRRKLEMFTYARFDLELTFVITSTQEQSTIQGQDSPVLTHQIM YVPPGGPVPTKINSYSWQTSTNPSVFWTEGSAPPRISIPFISIGNAYSMFYDGWAKFDK QGTYGINTLNNMGTLYMRHVNDGSPGPIVSTVRIYFKPKHVKTVWPRPPRLCQYQKAG NVNFEPTGVTESRTEITAMQTT

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BIOGRAPHICAL SKETCH Cheryl M. Boice was born In Cortland, New York on January 11, 1972. In 1990, she graduated from Cortland Sr. High School in Cortland, New York. After graduation, she attended Columbia College in Columbia, South Carolina. In 1996, Cheryl transferred to the University of Florida and graduated with a Bachelor of Science degree in April 1998; and a Master of Science degree in 2000. 146

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Samuel R. Farrah, Chair Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it confomns to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Thomas A. Bobik, Assistant Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Madeline E. Rasche, Assistant Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. L. Koopmar _ Professor of Environmental^ngineering Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Richard B. Dickinson, Associate Professor of Chemical Engineering

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This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the deg ree of Doctor o f Philosophy. August 2003 ' ^ (\a:^o Dean, tlofleqe Dean, College of Agric4ltura\and Life Sciences rici|ttura\ar Dean, Graduate School

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This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the deg ree of Doctor o f Philosophy. August 2003 ' ^ (\a:^o Dean, tlofleqe Dean, College of Agric4ltura\and Life Sciences rici|ttura\ar Dean, Graduate School


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