Techniques for virus detection in the marine environment

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Techniques for virus detection in the marine environment
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Bitton, Gabriel
Farrah, Samuel R.
Hoffmann, Edward M.
Feldberg, Bruce N.
Chou, Y. J.
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Abstract:
This study dealt with the development of methodology for virus recovery from the marine environment. We have explored a variety of methods for the recovery of enteroviruses from seawater which has been inoculated with high liters of poliovirus 1, 2, and 3, coxsackie B3 and echovirus 1 and 4. We have developed a technique based on the adsorption of viruses to Filterite filters, elution with 1% non-fat dry milk (NFDM), and organic flocculation of the eluates. This method resulted in the efficient recovery of most enteroviruses tested except for echovirus type 1. The method was also suitable for virus recovery from tap water. Various eluents were tested for their ability to desorb viruses from marine sediments. In laboratory experiments 4M urea-0.05M lysine was the most efficient in virus recovery from sediment. Although the method is time consuming, it results in a small volume of concentrate. The methods developed in the course of this study were used to recover indigenous viruses from estuarine water and sediments. Enteroviruses were found in both water and sediment in a section of Matanzas River which was closed with regard to shellfish harvesting.

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Publication No. 53


TECHNIQUES FOR VIRUS DETECTION
IN THE MARINE ENVIRONMENT

By

Gabriel Bitton, Samuel R. Farrah, Edward M. Hoffmann,
Bruce N. Feldberg and Y. J. Chou


Department of Environmental Engineering Sciences
Department of Microbiology and Cell Sciences
University of Florida
Gainesville








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TECHNIQUES FOR VIRUS DETECTION
IN THE MARINE ENVIRONMENT


By


Gabriel Bitton, Samuel R. Farrah, Edward M. Hoffmann,
Bruce N. Feldberg and Y. J. Chou


PUBLICATION NO. 53


FLORIDA WATER RESOURCES RESEARCH CENTER



RESEARCH PROJECT TECHNICAL COMPLETION REPORT


OWRT Project Number A-036-FLA



Annual Allotment Agreement Numbers

14-34-0001-9010
14-34-0001-0110



Report Submitted January, 1981


The work upon which this report is based was supported in part
by funds provided by the United States Department of the
Interior, Office of Water Research and Technology
as Authorized under the Water Resources
Research Act of 1964 as amended.













TABLE OF CONTENTS

LIST OF TABLES .................................................. Page

LIST OF TABLES ........................................ ........... iii

LIST OF FIGURES.................................................. v

ABSTRACT ................................................. ....... vi

PUBLICATIONS .................................................. vii

THESIS ........................................................ vii

INTRODUCTION .................................................... 1

LITERATURE REVIEW...................... ......................... 2

Occurrence of Viruses in Marine Waters ...................... 2
-Virus Survival in Marine Waters............................. 2
Potential Health Hazards Due to the Presence of Enteric Virus-
es in Marine Waters......................................... 3
Methodology For Virus Detection in the Marine Environment... 3
Methodology For Virus Detection in Sediment ................. 4

MAIERIALS AND METHODS........................................... 8

Virus Used and their Assay.................................. 8
Water Samples.............................................. 8
Concentration Step: Viral Adsorption and Elution............ 9
Reconcentration of Filter Eluates ........................... 9
Virus Recovery from Sediments .............................. 10
Concentration of Indigenous Viruses from Field Samples...... 14

RESULTS AND DISCUSSION............................. ............. 19

Comparison of Existing Methods for the Recovery of Poliovirus
from Seawater....... ..... ... .T............................ 19
The Use of Technical, Purified and Isoelectric Casein in the
Concentration of Poliovirus from Seawater................... 19
Use of Non-Fat Dry Milk for the Concentration of Poliovirus
from Seawater............................................... 21
Concentration of Poliovirus from Large Volumes of Seawater
and Tapwater Using the NFDM TechniQue....................... 21
Concentration of Other Enteroviruses From Seawater Using the
NFDM Technique.............................................. 29







1










Tb
TABLE OF CONTENTS -- Continued

Page
Virus Recovery From Marine Sediments........................ 31
Recovery of Indigenous Enteroviruses From Seawater and
Marine Sediments ........................................... 35-

CONCLUSIONS..................................................... 38

REFERENCES .................. ................................... 39



























1

1i
1ii






LIST OF TABLES

Table Page

1 Virus concentration from large volumes of seawater and sewage
effluents .................................................. 6,7

S 2 The effect of Antifoam A Spray on the recovery of poliovirus
type 1 using AV3 host cells .................................. 12

3 The effect of Antifoam A Spray on the recovery of poliovirus
type 1 using MA104 host cells................................ 13
4 Water quality parameters of City of Tampa sewage treatment
plant effluents and Tampa Bay water.......................... 16
5 Comparison of two existing methods for the concentration of
poliovirus type 1 from seawater ............................. 20
6 Use of technical and purified casein in the concentration of
poliovirus type 1 from seawater............................. 21

1 IfTect of p1H on elution of poliovirus type I from membrane
fi ILers using 0.5% purified casein .......................... 23

8 Use of isoelectric casein in the concentration of poliovirus
type 1 from seawater ............................. ......... 24

9 Effect of pH on elution of poliovirus type 1 from Filterite
filters using 1% (w/v) non-fat dry milk (NFDM)............. 25

10 Concentration of poliovirus type 1 from seawater using var-
ious concentrations of non-fat dry milk (NFDM).............. 26

11 Concentration of poliovirus from 20 gallons (76 liters) of
seawater using the NFDM technique........................... 27

12 Concentration of poliovirus type 1 from 50 gallons (189
liters) of tapwater by the non-fat dry milk (NFDM) technique 28

13 Concentration of six enterovirusj'. from ',(rawia-jr by t.hr
non-fat dry milk (NFUM) technique.......................
| 14 Adsorption of Poliovirus type 1 to a marine sandy sediment.. 32

15 Elution of Poliovirus type 1 from a marine sandy sediment... 33

16 Recovery of Poliovirus type 1 from intercoastal waterway
sediment................................................... 34

!i

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List of Tables (continued):
Table Pa rq
17 Use of non-fat dry milk (NFDM) technique for the recovery
of naturally occurring enterovirus from sewage treatment
plant effluents and Tampa Bay water ......................... 36

18 Detection of indigenous enteroviruses in estuarine water
and sediment in St. Augustine, Fla. (June 1980)............. 37













LIST OF FIGURES


Page


Fijyre


,


Page I



36



37










*4
,*;

.i
2,


i
,


1 Location of wastewater treatment plants and virus samp-
ling stations in St. Augustine, Fla......................








ABSTRACT


This study dealt with the development of methodology for virus
recovery from the marine environment.

We have explored a variety of methods for the recovery of entero-
viruses from seawater which has been inoculated with high liters of
poliovirus 1, 2, and 3, coxsackie B3 and echovirus 1 and 4. We have deve-
loped a technique based on the adsorption of viruses to Filterite filters,
elution with 1% non-fat dry milk (NFDM), and organic flocculation of the
eluates. This method resulted in the efficient recovery of most entero-
viruses tested except for echovirus type 1. The method was also suitable
for virus recovery from tap water.

Various eluents were tested for their ability to desorb viruses
from marine sediments. In laboratory experiments 4M urea-O.O5M lysine
was the most efficient in virus recovery from sediment. Although the
method is time consuming, it results in a small volume of concentrate.

The methods developed in the course of this study were used to re-
cover indigenous viruses from estuarine water and sediments. Enteroviruses
were found in both water and sediment in a section of Matanzas River which
was closed with regard to shellfish harvesting.
















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PUBLICATIONS


G. Bitton, B.N. Feldberg and S.R. Farrah. 1979. Concentration of entero-
viruses from seawater and tapwater by organic flocculation using
non-fat dry milk and casein. Water Air and Soil Pollution. 12:
187-195.


G. Bitton, P. Chou and S.R. Farrah. Virus recovery from marine sediments.
(In Preparation).

THESIS


Feldberg, B.N. 1979. The use of non-fat dry milk for the concentration
of enLeroviruses from seawater, tapwater, and sewage treatment
plant effluents. M.S. thesis, 75 pp. Department of Environmental
engineering Sciences. University of Florida, Gainesville, Florida.
32611 .








INTRODUCTION

Wastes resulting from man's activities have been traditionally (and
are still) discharged into the ocean. These include liquid wastewater,
sludge, oil, industrial and thermal wastes. With ever increasing popula-
tions along the coastlines around the world, it is by now realized that
the ocean can no more be considered as the "infinite sink" for the dis-
posal of fecal and other wastes. The coastal waters of the USA receive
more than 8 billion gallons of municipal sewage in a single day. Approxi-
mately half of this domestic sewage receives secondary treatment (McNulty,
1977). Ocean outfalls are commonly used for the disposal of non treated
or partially treated sewage. Their main advantages are low cost as
compared to tertiary treatment of wastewater. Their design is based on
location, water depth, distance from shore, and mixing regime.

Over the past 25 years much information has been gathered concerning
the fate of enteric bacteria in marine waters.. These pathogens are
generally eliminated by physical dilution and by die-off mechanisms, which
include temperature, predation, nutrient deficiency, algal and bacterial
antibiotics or toxins, solar radiation, heavy metals and salinity. (Mitchell,
1968; Mitchell and Chamberlin, 1975). Other factors, such as sedimentation,
lead to the accumulation of the bacterial pathogens in marine sediments.
(Gerba et al., 1978; Rittenberg et al., 1958). With regard to viruses more
information is being gathered about their occurrence and fate in marine
and estuarine waters (Edmond et al., 1978; Goyal et al., 1978; 1979;
LaBelle et al. 1980). However, more efforts must be spent on the improvement
or development of methodology for virus detection in marine and estuarine
waters as well as sediments.
This report explores the methodological aspects of virus occurrence in
the marine environment.





-2-


LITERATURE REVIEW

We will briefly review the occurrence of viruses in the marine/estuarine
environment, the potential public health hazards resulting from their entry
in ocean waters, and the existing methodology for their detection in that
particular environment.


Occurrence of Viruses in Marine Waters

The contamination of seawater by viruses mainly occurs through the
disposal of sewage or sewage effluents into estuarine waters or offshore dis- .
posal via sewage outfalls.

A survey carried out in the Mediterranean sea by the Environmental
Health Laboratory of Hebrew University led to the detection of several types
of enteric viruses (Poliovirus, Echovirus, Coxsackievirus) as far as 1500 m
from the discharge point. Coliform monitoring showed that these bacteria
were less stable than viruses in seawater. (Shuval et al., 1971). Another
virus survey was carried out along the Houston ship channel and in Galveston
Bay, Texas. (Metcalf et al., 1974). This study showed that sewage treat-
ment plants may discharge from 70 x 106 to 1746 x 106 viruses per day.
Examination of the Houston ship channel for viruses led to the detection of
enteroviruses as far as 8 miles (=13 kilometers) from the contamination
source. Ruiter and Fujioka (1978) have shown that up to 8x1010 viruses are
discharged daily into Mamala Bay via a sewage outfall. More recently human
viruses were detected in coastal waters and sediments (Edmond et al. 1978;
Goyal et al., 1978; 1979; LaBelle et al., 1980). These studies have essenti-
ally shown that marine/estuarine waters are frequently contaminated with
viruses and that classical indicator bacteria are not suitable for demon-
strating the presence or absence of viruses in the marine environment. These
findings prompted world-wide research on the factors which control the
fate of viruses in marine waters.


Virus Survival in Marine Waters

Laboratory studies have shown that virus survival is primarily controlled
by 'physical, chemical, and biological factors (Bitton,1978). Some of these
laboratory experiments were confirmed by "in situ" studies. However, "the
virucidal agent" remains yet to be identified.-- It was, however,- repeatedly
demonstrated that temperature is the most decisive factor controlling virus
survival in seawater and other coastal waters as well.

Viruses display an affinity for suspended solids in the aquatic environ-
ment (Bitton,1980a). These solids may be silts, clay minerals, cell debris
and particulate organic matter. The solid-associated viruses tend to
settle into bottom sediments where they reach higher concentrations than
in the overlying waters (Gerba et al., 1978; Goyal et al., 1977). Therefore,
sediments may serve as reservoirs for viruses as well as for enteric bacteria.
This has a public health significance since resuspension of the upper layers
of sediments may increase the virus concentration in the water column. Sediment
resuspension is particularly significant in shallow waters and is due to
motor boat activity, currents, swimming and changes in water quality.







;J: Potential Health Hazards Due to the Presence of Enteric Viruses
g in Marine Waters

In the last decade, water has been recognized as a possible route for
the transmission of enteric viral diseases. (Berg, 1967). We have now
evidence that the infectious hepatitis virus may be transmitted via the
water route. However there is a lack of epidemiological data concerning
the association between swimming in water and viral diseases. Recently,
it-has been demonstrated that there was a statistically significant associa-
tion between swimming and shigellosis. Shigella sonnei was isolated from
the Mississippi River near Dubuque, Iowa and was shown to be responsible
for disease in individuals who had water in their mouth while swimming
(Rosenberg et al., 1976). These are interesting data since Shigella has
a low infectious dose (10 to 100 bacterial cells) as compared to other patho-
gens. This infectious dose is not very far from the infectious dose of enteric
viruses.

Viral diseases may also be contracted following the consumption of shell-
fish which have been harvested from beds contaminated with sewage effluents
(Metcalf, 1976). Shellfish (oysters, clams, mussels) have the ability to
concentrate viruses within their gastrointestinal tract. Viruses may later
diffuse from the alimentary tract to other tissues. Although infectious
hepatitis and gastroenteritis are the only diseases shown to be associated
with shellfish consumption, it is possible that other viral diseases are
transmitted via this route.


Methodology for virus detection in the marine environment

Microporous filters have been mostly used for virus concentration from
marine and estuarine waters (Goyal and Gerba, 1981). The virus concentra-
tor developed by Wallis et al., (1972) was used by Metcalf et al., (1974)
to concentrate viruses from estuarine and surface waters. Clarifying filters
(1-jim orlon filters) were used to remove large particles that tended to
clog the Millipore filters that were used as virus adsorbents. It was noted
that the clarifying filters tended to adsorb viruses as larger volumes of
water were processed. Metcalf and co-workers (1974) used a modified version
of the previous method to detect viruses in Houston ship channel and
Galveston Bay. A procedure for concentration of enteroviruses from 15 to 100
gallons of turbid estuarine water using a filter aid (Celite) and Millipore
membranes was described by Hill, et al., (1974). However, these procedures
permitted recovery of less- then 3- of added virus when low numbers: of viruses
were used to test the procedures.

Sobsey et al., (1977) used asbestos-fiberglass (Cox) filters in place
of the nitrocellulose (Millipore) filters previously used. Since these
filters were more resistant to clogging than Millipore filters, the clarifying
filters were eliminated. The virus concentrator used by Sobsey et al., (1977)
employed a single prefilter along with two flat sheets of membrane filters
as the virus adsorbents. Virus in estuarine water at pH 3.5 and 0.0005 M
aluminum chloride adsorbed to the filters and could be recovered by treating
the filters with glycine buffer at pH=11.5. However, virus in this eluate
could not be reconcentrated in a smaller volume using a second membrane







adsorption-elution step as was used when tapwater was sampled for virus
(Sobsey et al., 1973). Adjusting eluates to pH 3.5 resulted in the for-
mation of large amounts of floc that rapidly clogged filters used for re-
concentration. However, the authors (Sobsey et al., 1977) observed that
flocculation could be increased by addition of ferric chloride. The flocs
that formed were efficient virus adsorbents and could be used to recon-
centrate the virus to a final volume of 20 ml. The flat filters used by
Sobsey et al., (1977) permitted relatively low initial flow rates and also
clogged rapidly. Frequently, 1-2 hours were required to process 50 gallons
of estuarine water. Since pleated fiber-glass filters were shown to per-
mit processing of large volumes of tapwater at high flow rates (Farrah,
et al., 1976), these filters were tested for their ability to concentrate
virus from estuarine water by Payment et al., (1977). The use of pleated
fiberglass filters permitted the concentration of virus from 400 to 1,000
liters of estuarine water. Virus was recovered by treating the filters
with high pH glycine. A two-step flocculation procedure using aluminum
chloride was used to concentrate virus to a final volume of 20-40 ml. .
This reconcentration procedure was found to give inconsistent recoveries
when estuarine water with different amounts of organic compounds and
from different sites were tested. Therefore, this reconcentration proce-
dure was replaced by a procedure using aluminum hydroxide flocculation and
hydroextraction. (Farrah et al., 1977). The completed procedure using
pleated membrane filters as the virus adsorbent, glycine buffer to elute *
adsorbed virus, and aluminum hydroxide flocculation along with hydroextrac-
tion to reconcentrate eluted virus, was used in virus monitoring in
Galveston Bay (Goyal et al., 1978) and in seawater near a deep sea sewage
outfall off Miami, Florida (Edmond et al., 1978). It thus appears that
the pleated Filterite microporous filters offer large surface areas for
virus adsorption and are relatively resistant to clogging. The optimal
virus adsorption to these filters requires an AIC13 concentration of
0.0005M to 0.0015M and a pH of 3.5. However, elution of adsorbed viruses
with glycine buffer at high pH (pH=11.5) may be detrimental to some pH-
sensitive viruses such as adenoviruses or rotaviruses. Therefore one needs
to look for eluents which work efficiently at pH=9.0-9.5. In 1976, Katzenelson
and his collaborators described a method which responded to this need. These
investigators showed that beef extract could be used as an eluent in virus
concentration from tapwater. After elution at pH=9.0, viruses could be
concentrated via organic flocculation of the beef extract at pH=3.5. The
sediment obtained from flocculation could be resolubilized with 0.15M
Na2HPO4 at alkaline pH. It is therefore useful to explore virus detection
methodology which uses eluents that work well at pH<10. The methods discussed
above are summarized in Table 1.

Methodology for virus detection in sediments

Viruses as well as enteric bacteria may become associated with organic
and inorganic particulates present in water. The solid-associated viruses
tend to settle into the bottom sediments where they reach high concentrations
(Edmond et al., 1978; Gerba et al., 1977; Goyal et. al., 1977; Matson eli. (0.
1978). It has been shown that sediment-associated viruses may survive longer
than in the water column (DeFlora et al., 1975; Gerba and McLeod, 1976;
Smith et al., 1978). The mechanism of the protective effect displayed by
sediment is not clear(Bitton, 1980b). Sediments may therefore serve as
reservoirs for enteric pathogens, particularly viruses. Resuspension of the
upper layer of sediments through motor boat activity, swimming activity,







currents, dredging or changes in water quality, may lead to an increase
of virus concentration in the water column.

Few studies have been undertaken on the development of methods for
virus detection in sediments. De Flora et al. (1975) showed an accumula-
tion of viruses in marine sediments in the vicinity of sewage outfalls in
Italy. They used sterilized seawater for eluting viruses from sediments.
Glycine buffer (0.05M; pH=8.5) and nutrient broth (pH=8.5) have also been
used for virus recovery from marine sediments (Metcalf et al. 1974;
Vaughn and Metcalf, 1975). However, none of the above investigators de-
termined the efficiency of the eluents used. Gerba et al. (1977b) described
a method for virus recovery from marine sediments. The procedure consists
of eluting the sediment with 0.25M glycine buffer, pH=11.5, containing
0.05M EDTA. Following a 10-minute contact time, the sediment was spun
down and the supernatant was concentrated via membrane filtration. Virus
was recovered from sediment with an efficiency of 50%. This method has
two major drawbacks: The pH of the eluent is too high and may lead to
inactivation of pH-sensitive viruses as was discussed previously. The
second drawback is the clogging of microporous filters used in the recon-
centration step. It is therefore necessary to evaluate a wide range of
eluents for virus recovery from marine and estuarine sediments. These
eluents should be used at a pH lower than 11 .5. Moreover, some other means
of eluate reconcentration (other than membrane filtration) should be
explored. Since no study has dealt with methodology for virus recovery
from freshwater sediments, some of the findings may be helpful in the
development of methodology for virus recovery from these types of sediments.








Table 1 : Virus concentration from large volumes of seawater and sewage effluents.


Concentration
system


Adsorption
conditions


2 step adsorption
el ution



Adsorption, elu-
tion, inorganic
flocculation


Adsorption, elu-
tion, inorganic
flocculation


Adsorption, elu-
tion, inorganic
flocculation


Adsorption, elu-
tion, inorganic
flocculation


0.00o5 M Aid3
pH = 3.5



0.0015 M A1Cl3
pH = 3.5



0.0015 M A1Cl3
pH = 3.5


0.0015 M AICl3
pH = 3.5



0.0005 M A1Cl 3
pH = 3.5


63 Coastal



53 Seawater


10-40 60-80


20-100


15-20


Estuarine


50 Seawater




41 Seawater


4. -: I~. .*d..


Seawater


Initial
volume
(1 )


Final
vol ume
(ml )


Final
percent
recovery


4Wate r
type


Reference


Metcal f
et al .
(1974)


Gerba
et al.
(1978)

Farrah
et al.
(1977)


Payment
et al.
(1976)


Sobsey
et al.
(1977)











Table 1 ------continued.


Concentration
system,


Adsorption
conditions


Adsorption, elu-
tion, inorganic
flocculation


Adsorption, beef
extract elution,
organic
flocculation


AdsorptionW,
elution,
inorganic
flocculation


0.0005 M AlC13
pH = 3.5


0.0005 M A1Cl3
pH = 3.5



0.0005 M AICl3
pH = 3.5


Sewage
effluents


Initial
volume
(1 )


Final
volume
(ml)


Final
percent
recovery


'Na te r
type


Reference


19-190


76-96


20
effluent



30
effluent



30


76-96


Gerba
et al.
(1978)

Landry
et al.
(1978)


Landry
et al.
(1978)





-8-



MATERIALS AND METHODS 'A

Viruses Used and Their Assay

Plaque-purified poliovirus type 1 (Sabin), poliovirus type 2 (natu-
ral isolate), poliovirus type 3 (natural isolate), ECHO 1 (Farouk),
ECHO 4 (Pesacek), and Coxsackie B3 (Nancy) were used in these studies.
Poliovirus types 2 and 3 were isolated from wastewater sludge.

Virus stocks were produced by infecting a monolayer of human am-
nion cells (AV3) or Rhesus monkey embryonic kidney cells (MA104) growing
in 32 ounce bottles. Viruses were allowed to adsorb to the host cells
for one hour at room temperature after which 40 ml of Eagle's Minimal
Essential Medium (MEM).with 10% Fetal Calf Serum (FCS) was added. The
monolayers were then incubated for 48 hours at 370C. The virus was
harvested by decanting the fluid, centrifuging at 1500 RPM for 15 minutes,
and freezing the resultant supernatant at -70OC in small aliquots for
later use.

Viruses used in seeded experiments were assayed on AV3, MA104, or
BGM (Buffalo Green Monkey) host cells using a routine plaque procedure.
For this assay monolayers were prepared by incubating host cells at
370C in 32 ounce bottles containing 40 ml of Eagle's MEM with 10% FCS
until confluent monolayers were obtained (approximately 60 hours). At
confluence, the growth media was removed and the cells washed with 30 ml
of a pre-trypsin solution. The monolayers were then treated with 10 ml
of a standard trypsin-versene solution to separate the cells from the
glass surface. After approximately 1 minute the latter solution was
decanted and the monolayer maintained at room temperature until the cells
were unbound (approximately 5 minutes). Then, 200 ml of Eagle's MEM with
10% FCS was added to suspend the cells and subsequently distributed to
forty 2-ounce glass or plastic tissue culture bottles in 5 ml aliquots.
The bottles were incubated at 370C for 48 hours or until monolayer con-
fluence was evident. At this stage the tissue cultures were ready for
use in the standard plaque assay.

Mature tissue cultures were infected by decanting the growth medium
and adding 0.2 ml of sample adjusted to isotonicity and pH=7.2-7.4.
Samples were diluted using either Eagle's MEM with 10% FCS and antibiotics
or phosphate-buffered saline (PBS) with 5% FCS and antibiotics. The tissue
cultures were maintained at room temperature for one hour to allow for ,
virus adsorption to the host cells. After the adsorption period the mono-
layers were overlayed with five ml of methyl cellulose and incubated at
371C until the plaques were approximately 1-5 mm in diameter. At this
stage the monolayers were stained using crystal violet or neutral red and
the number of plaques determined.


Water Samples

Seawater was collected from Crescent Beach, Florida, and stored at
40C prior to use. The seawater had a pH of 8.2 and a specific conductance


.*^J^-aKaiVKa^ar-a..^-,^,^^^^,^. ,_-_. --.--_ ________________,__









of 41,000 jmhos/cm (29.6 ppt salinity). Gainesville tap water used
had a pH of 8.4 and a specific conductance of 250 pmhos/cm and was
dechlorinated using sodium thiosulfate prior to viral seeding. Ortho-
tolidine titrations (Standard Methods, 1976) were performed to deter-
mine the concentration of sodium thiosulfate necessary for complete
dechlorination.

Concentration Step: Viral Adsorption and Elution

All experiments were performed in batch processes. The viruses
used were suspended in three liters of seawater and adsorbed to a series
(3.0 im > 0.45 pjim 0.45 pim or 3.0 jim -* 0.45 pm -> 0.25 pm) of epoxy-
fiberglass filters (Filterite Corp., Timonium, Md.) in the presence of
0.0005 M AlCl at pH = 3.3-3.5. Viruses were eluted from the membrane
filters using 0.5% (w/v) technical, purified, or isoelectric casein
(Difco, Detroit, Mich.) in 0.05 M glycine buffer at pH = 9.0 or 10.0,
or 1% (w/v) non-fat dry milk (NFDM) (Atlantic and Pacific Tea Co.,
Montvale, N.J.) in 0.05 M glycine buffer at pH = 9.0. Additional eluents
used were 3% beef extract at pH = 9.0 (Katzenelson et al., (1976) and
0.05 M glycine, pH = 11.5 (Farrah et al., 1977). Both the filtrates
and eluates were assayed to determine percent adsorption and elution,
respectively.

In experiments using large volumes of water (20 to 100 gallons),
suspended viruses were adsorbed to a 0.25 pm or 0.45 pm 10-inch epoxy-
fiberglass cartridge filter (Filterite Corp., Timonium, Md.) in the
presence of 0.0005 M AlC13 at pH = 3.3-3.5. This was done by pumping
the prepared sample through the filter, using a gasoline (Sears Roebuck
and Co., Chicago, Ill.) or electric (Sturdy Puppy, ITT Jabsco Prod.,
Costa Mesa, Calif.) powered centrifugal pump. Flow rates as high as
10 GPM were achieved using the gasoline powered pump. When 50 gallons
of secondary effluent was processed a 3.0 jim 10-inch Filterite cartridge
filter was used as a prefilter. Viruses were eluted by pumping 1 liter
of 1% NFDM in 0.05 M glycine at pH 9.0 through the filters after a 1
minute contact period was allowed. In seeded experiments the filtrate
and eluate were assayed to determine percent adsorption and elution,
respectively.

For the concentration step the concentration factor ranged from
100 in the small volume trials to 400 in the-large vol ume- trials.


Reconcentration of filter eluates

Glycine eluates were reconcentrated according to Farrah et al. (1977).

Beef extract eluates were concentrated by organic flocculation as
described by Katzenelson et al. (1976).





-10-


Solutions of casein and non-fat dry milk flocculate at pH 4.5-4.6
which is the isoelectric point of casein (Jenness and Patton, 1959).
Eluates obtained by the above concentration step were flocculated at
pH = 4.5-4.6 by the addition of 1 M glycine (pH = 2.0) and then centri-
fuged at 4500 RPM for 4 minutes. Eluates may also be allowed to settle
and only the lower portion centrifuged. This was ascertained by seeding
30 ml of a 1% NFDM solution in 0.05 M glycine, adjusting the pH to 4.5,
and allowing the flocs to settle undisturbed. The supernatant was with-
drawn and the loose sediment adjusted to pH = 7 with 1 M glycine (pH = 11.0). .
Upon assay, an average of 117% of the added poliovirus was recovered
in a final volume of 7 ml. This data suggests that the centrifugation
step may also be carried out at a lower speed with no effect on virus re-
covery. Pellets obtained from centrifugation were redissolved in 0.15
M Na HPO (pH = 9.0). The concentration factor for this step ranged from,
10 (smalt volume) to 40 (large volume). The final sample was sonicated
at 35 watt power output for 10 to 15 seconds with a Sonifier Cell Disrupter
(model WI85D, Heat Systems Ultrasonics, Inc., Plainview, N.Y.).

When using casein or NFDM it was found to be advantageous to add a
small quantity (one squirt) of an antifoam agent (Dow Corning Corp.,
Midland, Mich.) to inhibit foam production during elution and pellet re-
suspension. The use of an antifoam agent did not reduce polio-
virus numbers or cause cytotoxicity to AV3 or MA104 host cells (Table 2
and 3). -

When large volumes of water were processed using NFDM the final
concentrates were dialyzed against PBS for 18 to 24 hours at 4C prior to
virus assay. This step was necessary to prevent cytotoxity to host cells
caused by factors present in the undialyzed fluid.


Virus Recovery From Sediments

Marine sediments used in this study were a sandy sediment collected
at Crescent Beach, Fla. and an organic muck sediment sampled at the Inter-
coastal Waterway near St. Augustine; Fla. The sediments were retrieved
with the aid of a Ponar dredge and were stored at 40C until use.

lOg-sediment samples were mixed with 20 ml of seawater which has
been seeded with poliovirus type 1. The mixtures were mechanically shaken
for 30 minutes and then centrifuged at 4000 RPM for 4 minutes. The Te
supernatants were poured off and assayed for viruses to determine the per-
cent virus adsorption to the sediment. A blank, which consisted of seawater
without sediment, was run simultaneously with the sediment samples.

Ten eluents were investigated for their ability to desorb viruses
from marine sediments. These eluents were 4M urea + 0.05M lysine, 0.6M
Na trichloroacetate + 0.02M glycine, 1.0% isoelectric casein, 1% non-fat
dry milk, humic substances .(color units = 5,700),0.3% Na pyrophosphate,
10% fetal calf serum (FCS) in phosphate buffered saline (PBS), distilled
water, 3% beef extract and 0.25 M glycine + 0.05M EDTA. All the eluents






-'.. .... ... ..-. iM .-l... -- -l. "llll





-11 -



were buffered to pH = 9.0 except for distilled water (pH = 7.0) and
glycine-EDTA (pH = 11.5). Thirty ml of a given eluent were added
to lOg of wet sediment. The mixtures were vortexed for 30s and then
shaken for 30 min. However the shaking time was only 4.5 min and 1 min
for glycine-EDTA and urea-lysine, respectively. The samples were
centrifuged at 4000 RPM for 4 min and the supernatants were assayed
for viruses to determine the % of virus eluted from the sediment.





-12-


Table 2: The effect of Antifoam A Spray
of poliovirus type 1 using AV3


Total PFU before
treatment (x 10,3)


With Antifoam


on the recovery
host cells.


Total PFU after
treatment (x 103)


6.6

6.8


Mean: 6.0


Mean: 6.6


Without Antifoam


6.0


Mean: 6.6


Mean: 6.6


-Note: 50 ml of a 0.05 M glycine buffer solution (pH = 8.5) were seeded
with poliovirus type 1 (Sabin), stirred with a magnetic bar
for 1 minute and then assayed for virus. Two squirts of
Antifoam A Spray (Dow Corning Corp., Midland, Mich.) were
added to two test tubes while two other tubes received no
treatment. All tubes were then strongly mixed for 5 minutes
with a magnetic stirring bar and then assayed for virus on
AV3 host cells.


Treatment












Table 3: The effect of Antifoam A Spray on the recovery
of poliovirus type 1 using MA104 host cells.


PFU/ml


Treatment (a)


Run 1(b)


Run 2


Run 3


Mean


MEM
+ Antifoam 440 440 420 430



MEM
(control) 450 390 370 400



(a) 14.9 ml of Eagle's MEM were added to each of 6 test tubes and
0.1 ml of poliovirus was added to give a final virus titer of
approximately 400 PFU/ml. Three tubes were treated (one squirt)
with Antifoam A Spray (Dow Corning Corp., Midland, Mich.) while
the three other tubes did not receive Antifoam. All 6 tubes
were vortexed for 10 sec and the fluids assayed on MAI04 host
cells.


(b) Each run represents
bottles.


the mean number of 4 to 5 tissue culture


I ^^^BMM--____


-13-






-14-


Concentration of Indigenous
Virus from Field Samples


City of Tampa Sewage Treatment Plant Effluents and Tampa Bay Water

Table 4 shows conductivity, salinity, and pH values of sewage treat-
ment plant effluents and Tampa Bay water which were examined for the presence
of naturally occurring enteroviruses. Unchlorinated secondary effluent was
collected immediately prior to tertiary treatment. Tertiary effluent was
collected after approximately 10 minutes of chlorination (=3 ppm available
chlorine). This effluent was dechlorinated prior to processing as described
for Gainesville tap water. Tertiary effluent from the City of Tampa sewage
treatment plant is discharged.to Tampa Bay by an underwater outfall. Sampling
of the outfall was accomplished by anchoring a boat directly over the "boil"
created by the discharge and pumping water from a five (5) foot depth into
a holding tank on board the boat. Tampa Bay water was collected from a site
30 yards seawater of the outfall from a five foot depth. It is evident from
conductivity and salinity values for effluents and bay water (Table 4) that
the sample collected from the outfall site was quite dilute with effluent.
The virus concentration method, using 1% non-fat dry milk, is described
in details in the "Results and Discussion" section.

Recovery of indigenous viruses from estuarine water and sediment in St. Augustine,
Florida

The presence of indigenous viruses was monitored in two stations in
St. Augustine, Fla (Figure 1). These are shellfish beds and shellfishing is
prohibited. The first sampling point is on the Matanzas River just south
of the Bridge of Lions in St. Augustine. The second sampling station is
in the Salt Run at a location north of the public boat ramp. The station on
the Matanzas River is in an area which is subject to fecal pollution from
wastewater treatment plants (see Figure 1).
Forty to fifty gallons of estuarine water were sampled and processed
following the non-fat dry milk method described previously.
Fifty to one hundred grams of sediment were sampled at both stations.
They were immediately stored in ice and brought back to the laboratory where
they were processed. The time between sampling and processing did not exceed
5 hours. The sediment samples were eluted with urea-lysine as described
previously. The urea-lysine eluent must be prepared a few hours before
use since older solutions may lead to lower recovery from sediment. The sedi-
ment eluates were concentrated according to the following procedure: A1C13
was added to give a final concentration of 0.005M. The eluate was then
adjusted to pH 7 by the addition of IM sodium carbonate and mixed for 5-10
minutes. The flocs formed were collected by centrifugation at 5000 RPM
for 5 min. The supernatant was discarded and the floc was mixed with 5 volumes
.lIM riIHA..i'. hberf ,xtract. pll-Q.0. Most of the floc could be dissolved in
tii *.olii lionl. ic *..iamp I wa.. the vn .cri ui lu ed at. 5)000 RPM for 5 mrin. and
the supernatant was adjusted to pH7 and dialyzed against. phosphate buffered
saline at pH=7.0 overnight at 40C. After dialysis the sample was reconcen-
trated by the organic flocculation method of Katzenelson et al. (1976).
Our method resulted in a 3 ml concentrate from 50 to lQOg sediment sample.





-15-


Concentrates from water and sediment were assayed on BGM cells and
examined for cytopathic effects (CPE) for up to three weeks. The 50% tissue
culture infective dose (TCID5 ) was determined according to Reed and Muench
(1938). Virus isolates were Tdentified by using pools of neutralizing
antiserum (Lim and Benyesh-Melnick, 1960).






-16-


Water quality parameters of City of Tampa
sewage treatment plant effluents and Tampa
Bay water.


Water sample


Conductivity
(minhos/cm)


Salinity a)
(ppt)


Secondary effluent
(unchlorinated)


Tertiary effluent
(chlorinated)


Tertiary effluent
outfall, Tampa Bay


Tampa Bay, 30 yards
from outfall


(a) Salinity values were computed from


Table 4:


0.7


6.5


960



884



14,820



15,150


0.6


6.8


10.7


10.9


7.7


conductivity measurements.






-17-


Figure 1: Location of wastewater treatment plants and virus sampling stations
in St. Augustine, Fla.

^ Wastewater treatment plants

0 Virus sampling (Water and sediments)


















1












I






-18-




-19-


RESULTS AND DISCUSSION

Comparison of Existing Methods for the Recovery of Poliovirus from Seawater

Two existing methods of concentrating viruses were examined with respect
to recovery efficiency, using samples of seawater. Table 5 compares polio-
virus recoveries using methods involving: 1) Elution with glycine (pH = 11.5)
followed by inorganic flocculation using AIC13 (Farrah et al., 1977), and
2) Elution with beef extract (pH = 9.0) followed by organic flocculation (Kat-
zenelson et al., 1976).
All methods examined shared a common first concentration step, that is the
adsorption of viruses to epoxy-fiberglass (Filterite) filters in the presence
of 0.0005 M AlC13 at pH = 3.3-3.5. The modified method of Farrah et al.
(1977) used in our study involved elution with 0.05 M glycine buffer (pH = 11.5)
and reconcentration using Al (OH)3 flocs. Use of this method yielded mean re-
coveries of 47% elution., 25% from the reconcentration step, and 12% over-
all recovery. Farrah et al. (1977) reported that 85% to 95% of poliovirus ad-
sorbed to Al (OH)3 flocs were recovered by elution with the glycine-FCS mixture.
The particular problem in the recovery of poliovirus in these trials (Table 5)
was the elution of viruses adsorbed to the flocs. Greater than 90% of viruses
present in the filter eluates were adsorbed to the inorganic flocs.
The second existing method of concentrating viruses is based upon elution
of viruses adsorbed to membrane filters with 3% beef extract (pH = 9.0) followed
by organic flocculation (Katzenelson et al., 1976). Use of beef extract method
yielded higher poliovirus recovery than the glycine method and resulted in an
overall recovery of 56% (Table 5). .Using this method, Katzenelson 1977 was
able to recover 63% of added poiluvirus from 35 liters of seawater.


The Use of Technical, Purified, and Isoelectric Casein in the Concentration of
Poliovirus from Seawater

A set of preliminary experiments were performed to learn whether dilute
casein solutions could be used to elute poliovirus adsorbed to membrane filters
and further concentrate the viruses by organic flocculation. Table 6 shows
percent viral recovery using 0.2% technical casein in 0.05 M glycine'tpH = 11.0-
11.5), 0.5% technical casein in 0.05 M glycine (pH = 11.5). All trials yielded
greater than 75% recovery from elution and greater than 65% recovery from or-
ganic flocculation (reconcentration). Overall recoveries for the three tials
were greater than 50%.
The main advantage of the beef extract method is that elution is carried
out at pH = 9.0, thus not affecting the survival of pH-sensitive viruses (reo-
viruses, rotaviruses, and adenoviruses) which are inactivated by exposure to
pH = 11.5 for even short times (Fields and Metcalf, 1975, Sobsey et al., 1980).
Furthermore, even the exposure of poliovirus type 1 to pH = 11.5 gTycTne buffer
for more then 10 minutes may be virucidal (Gerba et al., 1977b). For this
reason, one must work with great speed when using pH = 11.5 glycine buffer as
an eluent for enteric viruses.






-20-





Table 5: Comparison of two existing methods for the concentration
of poliovirus type 1 from seawater.


Method(a) Percent Percent Percent
elution reconcentration overall
recovery

Elution with 0.05 M glycine
(pH = 11.5) followed by
inorganic f pculation
using AICI3 47 25. 12


Elution using 3% beef
extract (pH = 9.0) followed
by organic flocculationkc) 86 66 56


(a) Poliovirus type 1 (Sabin) suspended in 3 liters of seawater, was
adsorbed to a series (3.0 pm -> 0.45 im -> 0.25 pm) of Filterite
filters in the presence of 0.0005 M A1CI at pH = 3.3-3.5. Viruses
were eluted with 30 ml of a designated eluent (see references below).
Final concentrates obtained were 2 to 4 milliliters. Numbers given
are mean values for 2 to 4 trials.

(b) Trials performed according to Farrah et al. (1977).


(c) Trials performed according to Katzenelson et al. (1976).






-21-


Table 6: Use of technical and purified casein in the concentration of polio-
virus type 1 from seawater.


Treatment(a)


Total PFU
adsorbed
to filters


Percent
elution


Organic flocculation
percent recovery


Percent overall
recovery


0.2% technical
casein in
0.05 M glycine
(pH = 11-11.5)


0.5% technical
casein in
0.05 M glycine
(pH = 11.5)


0.5% purified --
casein in
0.05 M glycine
(pH = 11.5)


4.06 X 106




5.47 X 106




4.64 X 106


(a) Poliovirus type 1 (Sabin), suspended in 3 liters of seawater, was adsorbed to a
series (3.0 um > 0.45 m -- 0.25 pim) of Filterite filters in the presence of 0.0005 M
AIC,l pH = 3.5. Viruses were eluted using 30 ml of a designated eluent. Organic
floc ulation was induced by adding 0.5 M glycine (pH = 2) to the casein eluates
until a pH of 4.5 was reached. The floc formed was pelleted by centrifugation at
4500 RPM for 4 minutes and re-dissolved in 3 ml 0.15M Na2HPO4,'pH = 9.0. Values
given are mean values from duplicate or triplicate trials.





-22-


It then became of interest to examine the effect of pH upon the elution of
poliovirus type 1 from membrane filters. Table 7 shows that elution was most
efficient at pH = 10.0 (100%) and least efficient at pH = 11.5 (64%). It is
worth noting that viral elution was relatively high (76%) even at pH = 8.0.
Use of 0.5% isoelectric casein in 0.05 M glycine (pH = 10.0) permitted
the highest recovery efficiency for elution (>100%) of the three types of casein
examined so far (Table 3). Overall viral recovery from 3 liters of seawater
using isoelectric casein was 83%.


Use of Non-Fat Dry Milk for the Concentration of Poliovirus from Seawater

While contemplating the potential use of casein for viral detection and
concentration from seawater, the thought arose that since caseins and lipids
(fats) are the major components of milk, then perhaps non-fat dry milk (NFDM)
could be used as an inexpensive alternative for casein. Preliminary unseeded
experiments showed that a 1% (w/v) solution of NFDM in 0.05 M glycine behaved
like purified or isoelectric caseins in that flocculation occurred at pH = 4.5
and centrifugation produced a pellet and a clear supernatant.
Using 1% NFDM in 0.05 M glycine, a study was undertaken to investigate
the influence of pH on the efficiency of elution of poliovirus type 1 from mem-
brane filters. As shown in Table 9 the elution was maximum (116%) at pH =
9.0 and decreased to 35% and 43% at pH = 8.0 and pH = 7.0, respectively.
Since the ultimate goal of a concentration technique is to achieve a high
concentration-factor, it was decided to examine the effect of NFDM concentra-
tion on the efficiency of poliovirus recovery from seawater, the rationale beinq
that the more dilute the NFDM solution used, the smaller the centrifuged pellet
and hence the smaller- the volume of final concentrate. As shown in Table 10
efficiency decreased with decreasing NFDM concentration (from 71% with 1% NIDM
to 35% with 0.25% NFDM). However, recovery from organic flocculation was always
high and ranged from 81% to 100%. Use of NFDM concentrations of 1%, 0.75%,
0.50% and 0.25% yielded overall poliovirus recoveries of 71%, 48%, 33%, and
28%, respectively.


Concentration of Poliovirus from Large Volumes of Seawater and Tap Water Using
the NFDM Technique

As the NFDM technique proved efficient for the concentration of poliovirus
from small volumes of seawater (3 liters), its application to the processing of
large volumes of seawater and other types of water became of interest.
Twenty gallons ('*76 liter) samples of seawater were adjusted to pH =- 3.3-
3.5 and 0.0005 M AlC13, seeded with poliovirus, and processed by the NFDM techni-
que (Table 11). Recoveries obtained for two trials were 54% and 87% elution
recovery, 87% and 115% reconcentration recovery, and 74% and 100% overall reco-
very; the mean overall recovery for the two trials being 74%.
Next, fifty gallon (189 liters)-samples of dechlorinated tap water were seeded
with high (=7 x 10' PFU) and low (=600 PFU) inputs of poliovirus type 1 and
processed by the NFDM technique. As shown in Table 12 high virus input trials
resulted in a mean overall recovery of 80% while low input trials recovered an
average of 75% of the initial virus titer. Concentration factors ranging from
2363 to 6750 were obtained in these trials.






- f,= Ma.. ..a ..-- :.E .. =. ,,- -,z :. -' A-, .f -. -- ^ -: ,- ; ^- B .tS-B^- ,, i .m __'-,.- wk











Effect of pH on elution of poliovirus type 1 from
membrane filters using 0.5% purified casein.


pH of casein


Percent elution(a)


11.5


(a) Poliovirus 1 (Sabin), suspended in 1 liter of seawater,
was adsorbed to a series (3.0 lm 0.45 um 0.25 um)
of Filterite filters in the presence of 0.0005 M AIC13
at pH = 3.5. The adsorbed viruses were eluted with
0.5% purified casein in 0.5 M glycine (30 ml) adjusted
to various pHs with 0.5 M glycine. Each number repre-
sents the mean for 2 to 4 trials.


Table 7:






-24-


Use of isoelectric casein in the concentration of polio-
virus type 1 from seawater.


Total number of
viruses adsorbed
to membrane filters
PFU


Percent
elution


Organic
flocculation
percent
recovery


Percent
overall
recovery
efficiency


2.06 x 106 136 80 1I(


2.51 x 106


Mean:


120


Note: Poliovirus type 1 (Sabin), suspended in 3 liters of seawater, was adsorbed to a
series (3.0 pm -- 0.45 vm 0.45 pm) of Filterite filters in the presence of
0.0005 M AlC13 at pH = 3.5. Viruses were eluted from the filters with 30 ml
of a 0.5% isoelectric casein solution in 0.05 M glycine (pH = 10.0).
Organic flocculation was undertaken by adding 0.5 M glycine (pH = 2) to the
eluate until a pH of 4.5 was reached. The floc formed was pelleted by centri-
fugation and redissolved in 2.5 ml of 0.15 M Na2HPO4, pH = 9.0.


Table 8:






-25-


Table 9: Effect of pH on elution
Filterite filters using
(NFDM).


of poliovirus type 1 from
1% (w/v) non-fat dry milk


pH of NFDM eluent Percent elution(a)


7.0 43

8.0 35

9.0 116

10.0 86

11.5 69



(a) Poliovirus 1 (Sabin), suspended in 1 liter of sea-
water, was adsorbed to a series (3.0 pm 0.45 -
0.45 tim) of Filterite filters in the presence of
0.0005 M AiC13 at pH = 3.5. Viruses were eluted with
10 ml 1% NFDM in 0.05 M glycine. Each number re-
presents the mean of 2 to 4 trials.


-.~-= ~W ~ ~





-26-


Table 10:


Concentration of poliovirus type 1
concentrations of non-fat dry milk


from seawater using various
(NFDM).


Treatment(a)


1 .00% (,,
NFDM(b)


0.75%
NFDM


0.50%
NFDM


0.25%
NFDM


Total PFU
adsor)ed
(x 10)


Percent
elution


Organic
flocculation
percent
recovery


51



1.86



1 .36



1 .22


(a) Poliovirus type 1, suspended in seawater, was adsorbed to a series (3.0 im >
0.45 um-+ 0.45 pm) of Filterite filters in the presence of 0.0005 M AICI3
at pH = 3.5. Viruses were eluted with the various concentrations of NFDM
in 0.05 M glycine, pH = 9.0. Organic flocculation was undertaken by adding
0.5 M glycine to the filter eluate until a pH of 4.5-4.6 was reached. The
floc formed was pelleted by centrifugation at 4500 RPM for 4 minutes and
the pellet redissolved in 0.15 M NapHPO pH = 9.0 Each number represents
the mean value obtained from duplicate trials.


(b) Trials using 1% NFDM used
through 0.4E5 im Filterite
seawater samples and were


seawater samples of 20 gallons (76 liters) processed
cartridge filters. Other trials used 1-3 liters
processed as described above.


............*.r~ ~
........................M.. At -~


Percent
overall
recovery





-27-


Table 11:


Concentration of poliovirus from 20 gallons (76 liters)
of seawater using the NFDM technique.


PFU adsorbed to
membrane filters


Percent
elution


Percent
reconcentration


Percent
overall recovery


6.98 x 107 54 87 47


3.16 x 107 87 115 100


Note: Poliovirus type 1 (Sabin), suspended in 20 gallons (76 liters) of seawater was
adsorbed to a 0.45 vm Filterite cartridge (10") in the presence of 0.0005 M AlCl3
pH = 3.3-3.5. Viruses were eluted with 1 liter of 1% NFDM in 0.05 M glycine (pH
= 9.0). Filter eluates were brought to pH 4.5 with 1 M glycine (pH = 2.0)
and the resultant flocs centrifuged at 4500 RPM for 4 minutes. The pellets
were then resuspended in 0.15 M Na2HPO4, pH = 9.0.





-28-


Table 12:


Concentration of poliovirus type 1 from 50 gallons
(189 liters) of tapwater by the non-fat dry milk
(NFDM) technique.


Total virus input
PFU


High virus input(a)

7.12 x 107

7.17 x 107



Low virus input(ab)


Percent
elution


Organic
flocculation
percent
recovery


118


569


(a) Poliovirus type 1, suspended in 50 gallons (189 liters) of dechlorinated
tapwater, was adsorbed to a 0.25 pm or 0.45pm Filterite cartridge filter
in the presence of 0.0005 M AlCI at pH = 3.5. Viruses were eluted from
the filters using 1% NFDM in 0.05 M glycine, pH = 9.0. Filter eluates
were brought to pH = 4.5-4.6 with 1 M glycine (pH = 2.0) and the resultant
floc centrifuged at 4500 RPM for 4 minutes. The pellets were then resus-
pended in 10-63 ml Na2HPO4, pH = 9.0.

(b) Concentrates from low virus input trials were dialyzed for 18-24 hours
against PBS at 40C prior to direct assay on host cells.


Final
vol ume
(ml)


Percent
overall
recovery




-29-


These results agree with those reported by other researchers using large
volumes of water but different methods. Wallis et al. (1972a, 1972b, 1972c)
and Sobsey et al. (1973) reported poliovirus recoveries ranging from 61% to
92% from large volumes of tap water using two step filter-adsorption elution.
Using filter-adsorption elution and aluminum hydroxide flocculation reconcen-
tration, Farrah et al. (1977) were able to recover 40% to 50% of poliovirus
added to 500 gallons of tap water. Katzenelson et al. (1976) recovered 74%
of added poliovirus from 500 liters of tap water using beef extract elution
and organic flocculation. Using low numbers (16 to 50 PFU) of added polio-
virus per 100 gallons of tap water, Hill et al. (1974) were able to recover
25% to 50% of the initial virus titers using two step filter-adsorption
elution. However, Sobsey (1979) reported that the above method recovered
only <1% to 25% of low numbers of nine enteroviruses, each added to 100
gallons of tap water.


Concentration of Other Enteroviruses from Seawater Using the NFDM Technique

The concentration of six enterovirus (poliovirus type 1, 2, and 3,
coxsackie B3, ECHO 1, and ECHO 4) by the NFDM technique is shown in Table
13. It is evident that this method is efficient for the recovery of the
three types of poliovirus as well as coxsackie B3; overall recoveries for
the above four viruses ranged from 59% to 75%. Using NFDM, 31% overall
recovery of ECHO 4 was obtained, but only 8% overall recovery was obtained
using ECHO 1. The poor performance of the NFDM technique with regard to
ECHO 1 recovery efficiency is mainly due to the inability to concentrate
this virus on membrane filters and casein flocs. Other researchers working
in this area have also encountered recovery problems regarding ECHO 1.
Charles (1979) reported that ECHO 1 was poorly adsorbed to a sandy soil and
.to isoelectric casein and NFDM flocs as compared to poliovirus type 1,
coxsackie B3, and ECHO 4. ECHO 1 was found to adsorb poorly to Filterite
filters and lagoon sludge solids as compared to poliovirus type 1 (Pancorbo
et al., unpublished observation). Goyal and Melnick (1978) observed that
ECHO 1 was adsorbed significantly less to soils than poliovirus types 1, 2,
and 3, coxsackie B3, and other ECHO virus types and suggest the presence
of intratypic differences in adsorptive potential between ECHO virus isolated
strains.











1



.-i. j A *; a ^ .< i S j -^ ,. .- .' '. ..v ,. -- '* .- .






-30-


Table 13:


Concentration of six enteroviruses from seawater
by the non-fat dry milk (NFDM) technique.


Virus(a) Virus Percent Organic Percent
input PFU elution flocculation overall
(x 106) percent recovery
recovery


Polio 1(b)

Polio 2

Polio 3

Coxsackie B3

ECHO 1

ECHO 4


31.6-69.8

29.9-30.9

61.1-69.6

0.46

17.0

0.8-1.6


100


(a) The various viruses, suspended in 1-3 liters of seawater, were adsorbed
to a series (3.0 pm 0.45 Pm 0.45 urnm) of Filterite filters in the
presence of 0.0005 M AlC13 at pH = 3.5. The filters were eluted with
1% NFDM in 0.05 M glycine, pH = 9.0. Filter eluates were brought to
pH = 4.5-4.6 with 1 M glycine (pH = 2.0) and the resultant floc centri-
fuged at 4500 RPM for 4 minutes. The pellets were then resuspended in
0.15 M Na2HPO4, pH = 9.0. Each number represents the mean of duplicate
trials.

(b) The polio 1 trials used seawater samples of 20 gallons (76 liters) pro-
cessed through 0.45 um Filterite cartridge filters. Other trials used
1-3 liters seawater samples and were processed as described above.






-31-


VIRUS RECOVERY FROM MARINE SEDIMENTS

Prior to evaluating the ability of various eluents to desorb virus
from marine sediments, experiments were undertaken to study the sorption
ability of the sediments toward viruses. Table 14 shows that the sandy
sediment sampled at Crescent Beach, Fla. retained 99% of added viruses.
The Intercoastal Waterway sediment adsorbed 100% of added virus (only two
samples were run for this sediment). Gerba et al., (1977) found similar
results with estuarine sediments from Galveston Bay, Texas.
Ten eluents were investigated for their ability to desorb viruses from
the Crescent Beach sediment. Three to six replicates were run for each
eluent.
It was observed that virus elution from the sediment surface was
generally relatively low and ranged from <1% to 43.9% (Table 15). Urea-
lysine and TCA-glycine were found to be the most efficient among all the
eluents tested. A surprising finding was the low recovery (<1%) obtained
with 0.25 M glycine + 0.05 M EDTA pH = 11.0. Similar results were obtained
with the Intercoastal Waterway sediment (Table 16). These findings do
not agree with those of Gerba et al. (1977) who reported that glycine-EDTA
eluted more than 50% of adsorbed viruses. In our study we made sure that
the viruses were exposed to the high pH for no more than 15 minutes. Urea-
lysine, which eluted 43.9% of poliovirus, has been successfully used for
virus elution from sludge (Farrah et al., submitted for publication), and
from positively charged microporous filters (Chang et al., unpublished).
We have (Chang et al., unpublished; Farrah et al., submitted for publication).
a two-step concentration scheme for urea-lysine eluates. This method led
to a 50% virus recovery from these eluates.
Addition of distilled water (Table 15) to the marine sediment resulted
in a poor virus recovery (1.6%) and this agrees with the results reported
by Gerba et al. (1977). The use of 2% isoelectric casein resulted in a
35% recovery while less than 10% recovery was achieved with 3% beef extract
(Table 15).
It was therefore decided to use 4M urea + 0.05M lysine (pH 9.0) for
the detection of indigenous viruses in estuarine sediments.





-32-


Table 14: Adsorption of Poliovirus type 1 to a marine sandy sedimenta




Run Total Virus Imput (PFU) Total PFU % Virus
retained by adsorbed to
sediment sediment


8.8 x 106


2.0 x 106


1.9 x 106


2.6 x 106


2.0 x1106


8.74 x 106


1 .98 x 106


1 .87 x 106


2.56 x 106


1.98 x 106


99.3


99.4


98.9


98.7


99.2


(a) 20 ml of seawater seeded with poliovirus type 1 were added to
O1g of sediment. (From Crescent Beach, Fla.). The mixtures
were shaken for 30 min and centrifuged at 4000 RPM for 10 min.
The supernatants were assayed to determine the % virus adsorbed
to the sediment.


- .----'~Td~J&~.~L





-33-


Table 15: Elution of Poliovirus Type 1 from a Marine Sandy Sedimenta


Eluent


4M Urea + 0.05M
Lysine (pH = 9.0)

0.6M TCA + 0.02M
Glycine (pH= 9.0)

2% Isoelectric Casein
(pH1 = 9.0)

1% Isoelectric Casein
(pH = 9.0)

1% Non-Fat Dry Milk (pH = 9.0)


Humic Substances
(color units = 5,700;


pH = 9.0)


0.3% Na Pyrophosphate
(pH = 9.0)

3% Beef Extract (pH = 9.0)

10% FCS in Phosphate
Buffered Saline (pH = 9.0)

Distilled Water

0.25M Glycine + 0.05M
EDTA (pH=9.0)


43.9 + 14.3

41.3 1.3

35.0 + 6.5

23.4 + 6.0


16.0 +


3.8


11.3 + 0.6

11.2 + 2.7

9.3 + 2.9

6.9 + 1.1

1.6 + 0.2

< 1%


(a) Thirty ml of eluent were added to lOg of sediment. The samples
were vortexed for 30s and then shaken for 30 min (the shaking time
was only 4.5 min and 1 min for glycine-EDTA and urea-lysine, re-
spectively). The samples were centrifuged at 4000 RPM for 4 min
and the supernatants were assayed for viruses.

(b) Mean of 3 to 6 replicates.


K


% Plution
Mean + S.D.





-34-


Table, 16:


Recovery of Poliovirus Type 1 from Intercoastal Water-
way Sediment.


Eluent % Reconcentration %
Elution step Overall
% Recovery Recovery


3% Beef Extract
(pH = 9.0) 11 87



0.25M Glycine +
0.05M EDTA
(pH = 11.5) <1 <1


9.6





-35-


RECOVERY OF INDIGENOUS ENTEROVIRUSES FROM SEAWATER AND MARINE
SEDIMENTS

Virus Recovery from Tampa Bay.

In February, 1979, a trip was made to the City of Tampa Sewage Treatment
Plant for the purpose of testing the efficacy of the NFDM technique in the
recovery of naturally occurring enteroviruses (Table 17).
Fifty gallons (189 liters) of unchlorinated secondary treated effluent
were processed by membrane filtration using a series (3.0 pm 0.45 pm) of
Filterite cartridge filters followed by NFDM elution and organic flocculation
to yield a final concentrate of 22.7 ml and 27.8 ml for the two filters,
respectively, as each filter was eluted separately. The final concentrates
were assayed following chloroform treatment to remove bacterial and fungal
contamination. Concentrates from both the prefilter and filter were positive
with regard to enteroviruses (Table 17). No attempts were made to quantify
viruses since the method was designed specifically for marine waters.
Chlorinated tertiary effluent was collected immediately prior to discharge
into Tampa Bay after approximately 10 minutes contact time with about 3 ppm
available chlorine. According to the sewage treatment plant operator the
concentration of available chlorine should have only been 1 ppm (personal
communication). This latter fact implies that the excess chlorine addition
was due to either human error or a desire to produce high quality water with
respect to the presence of viruses. No virus was detected in a sample of
chlorinated tertiary effluent. Similarly no virus was detected in Tampa Bay
water (Table 17).

Detection of indigenous enteroviruses in estuarine water and sediment in
St. Augustine, Fla.

Indigenous enteroviruses were monitored at two stations (Salt Run and
Matanzas River) in St. Augustine, Fla. (See Figure 1). Estuarine water was
processed according to the non-fat dry milk method which was discussed pre-
viously. Virus detection in sediments was performed according to the urea-
lysine method. The results are shown in Table 18. In June 1980 no virus
was detected in Salt Run in both water and sediment. In Matanzas River
(near the Bridge of Lions in St. Augustine, Fla.) viruses were detected
in both estuarine water and sediments.
The virus concentrations found were 207 TCID50/50 gallons of water and -
41.4 TCID /1OOg of wet sediment. Poliovirus type I and type 3 were identi-
fied in cocentrates from water and sediment, respectively. Salt Run sediment
was sampled again on September 1980 and enteroviruses were detected at a
concentration of 18 TCID0/10OOg of sediments.
Virus concentration in Matanzas River is within the range found by
other investigators. Gerba et al. (1977) reported virus concentrations
of 2-16 PFU/10 liters in coastal canals in Texas (2-16 PFU/10 liters is
equivalent to 38-304 PFU/50 gallons). However, Edmond et al. (1978)
found lower virus concentrations (21-42/100 gallons) in Miami Beach marine
outfall. Virus concentration in Matanzas River sediment is similar to that
found by DeFlora et al. (1975) in Italy.









Table 17:


Use of the non-fat dry milk (NFDM) technique for the recovery of naturally occurring entero-
virus from sewage treatment plant effluents and Tampa Bay water.


Water sample Volume processed Final volume Volume assayed Virus Concentration
(gallons) (ml) (ml) presence factor


Secondary effluent
(unchlorinated) 50 27,8(a) 13.10 + 3743
(b) "3,743
22.7(b) 15.20 +

Tertiary effluent
(chlorinated) 100 23.4 5.60. N.Dc 16,154


Tertiary effluent
outfall, Tampa Bay 80 30.2 14.65 N.D 10,000


Tampa Bay water,
30 yards from
outfall 75 18.2 2.80 N..D 15,604


(a) Volume of final concentrate from the elution of the 0.45 Pm filter.

(b) Volume of final concentrate obtained from the elution of the 3.0 pm pre-filter.

(c) Not detected.


P




-37-


Table 18: Detection of indigenous enteroviruses in estuarine water and sedi-
ment in St. Augustine, -Fla. (June 1980).


Sample Salt Run Matanzas River
type (Near Bridge of Lions)

Water N.D(a) 207 (P1)(b)
(TCID50/50 gallons)

Sediment N.D 41.4 (P3)(c)
(TCID50/I100g)


Not detected
Poliovirus type 1
Poliovirus type 3





-38-


CONCLUSIONS

The method of beef extract elution and organic flocculation has recently
been used for the concentration of poliovirus in tap water, seawater, waste
water effluent, and anaerobic sludge. This study has essentially shown that
enteroviruses (poliovirus types 1, 2, and 3, coxsackie B3, and ECHO 1 and
4) can be recovered from seawater or tap water by elution and organic
flocculation using 1% NFDM in 0.05 M glycine.
Elution with NFDM is most efficient at pH=9.0 and this is an advantage
over methods which use glycine buffer at pH=11.5 due to the virucidal effect
of such a pH.
Flution and organic flocculation with technical casein, purified casein,
isoelectric casein, NFDM, or beef extract resulted in high overall recoveries
(51% to 83%) of poliovirus type 1 from seawater. Results obtained here are
in agreement with those reported by Katzenelson (1977), pertaining to the
recovery of poliovirus from seawater using the beef extract method.
High overall recoveries (72% to 81%) oF both high and low quantities
of poliovirus type 1 were recovered from tap water using the NFDM technique.
The use of the NFDM technique resulted in the efficient elution of
(71% to >100%) of six enterovirus from membrane filters. Problems were
encountered during the organic flocculation step where low recoveries of
ECHO 1 and 4 (10% and 43%, respectively) were observed.
Of the ten eluents investigated for desorption of viruses from marine
sediments, 4M urea-O.05M lysine was found to be the most efficient.
The methods develop in the course of this study were successful in
the recovery of indigenous enteroviruses in estuarine water and sediment.





-39-


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Full Text

PAGE 1

CES researcli center Publication No. 53 TECHNIQUES FOR VIRUS DETECTION IN THE MARINE ENVIRONMENT By Gabriel Bitton, Samuel R. Farrah, Edward M. Hoffmann, Bruce N. Feldberg and Y. J. Chou Department of Environmental Engineering Sciences Department of Microbiology and Cell Sciences University of Florida Ga i nes vi 11 e UNIVERSITY OF FLORIDA fW(

PAGE 2

'j TECHNIQUES FOR VIRUS DETECTION IN THE MARINE ENVIRONMENT By Gabriel Bitton, Samuel R. Farrah, Edward M. Hoffmann, Bruce N. Feldberg andY. J. Chou PUBLICATION NO. 53 FLORIDA WATER RESOURCES RESEARCH CENTER RESEARCH PROJECT TECHNICAL COMPLETION REPORT OWRT Project Number A-036-FLA Annual Allotment Agreement Numbers 14-34-0001-9010 14-34-0001-0110 .._ .. ___ '.' ....... .... Report Submi tted January .: The work upon which this report is based was supported in part by funds provided by the United States Department of the Interior, Office of Water Research and Technology as Authorized under the Water Resources Research Act of 1964 as amended.

PAGE 3

TABLE OF CONTENTS Page LIST OF TJ\BLES ..................................... ;... iii LIST or FIGURES ..... v ABSTRACT .. ; ............................................ '. vi PUBL I CATIONS ...... ...... '.' .................... '. vi i THESIS .. .......................................... vii INTRODUCTION ...... ... '.' .... . . . . 1 LITERATURE REVIHi .... .............................. ." 2 Occurrence of Viruses in Marine Waters....... ....... 2 -Virus Survival in Marine Waters....... .... .......... 2 Potential Uealth Hazards Due to the Presence of Enteric Virus-es 'j n Ma ri ne Wa ter.s . . . . . . . . 3 Melhodolo9Y For Virus Detection in the Marine Environment... 3 For Virus Detection in Sediment................. 4 M/rIEH[J\LS J\ND METHODS......... ........ ........ .......... ..... 8 Virus Used and their Assay.................................. 8 ltJa ter Samples............................................... 8 Concentration Step: Viral Adsorption and Elution............ 9 Reconcentration of Filter Eluates........................... 9 Vi rus Recovery from Sedi ments. . .. 10 Concentration of Indigenous Viruses from Field Samples...... 14 RESULTS AND fHSCUSSION .......................... . 19 oi_f;)5i.st}l1g f9.r_.the_ Poliovirus. -." .' ""--From Seawa fer :-:-::-: '. : .-:-. -19--' -The Use of Technical, Purified and Isoelectric Casein in the Concentration of Poliovirus from Seawater................... 19 Use of Non-Fat Dry Milk for the Concentration of Poliovirus from Seawater............................................... 21 Concentration of Poliovirus from Large Volumes of Seawater and Tapwater Using the NFDM Technique....................... 21 Concentration of Other Enteroviruses From Seawater Using the NFDMTechnique .................................... ......... 29 i

PAGE 4

'\ .j j I I TABLE OF CONTENTS -Continued Virus Recovery From Marine Sediments .. Recovery of Indi genous Enterovi ruses From Seawater and Marine Sediments .. 0 0 0 0 0" 0 0 0 CONCLUSIONS 0 0000. 0 o. 0 000. 000000000 0 0 0 REFERENCES 0. 000 0 .0 0.; 0 0 0 0 ii Page 31 35. 38 39 l ;1 i Tab I i i $ i i 1 f I I 1 ( 11 li 1 = 14 15 16

PAGE 5

Taole 1 2 3 4 5 6 7 9 10 11 12 LIST OF TABLES Virus concentration from large volumes of seawater and sewage effl uents .................................. e' The effect of Antifoam A Spray on the recovery of pol iovi rus type lus-ing AV3-ho.st cells ................................ .. The effect of Antifoam A Spray on the recovery of poliovirus type 1 us i ng MAl 04 has t-tell s ................................ quali ty parameters of Ci ty of Tampa sewage treatment plant effl uents and Tampa 'Bay water ........................ .. Comparison of two existing methods for the concentration of poliovirus type 1 from seawater ................. Use of technical and purified casein in the concentration of po 1 i ovi rus type 1 from seawa ter .............. .............. .. LffN:t of pUon elution of poliovirus type 1 from membrane fTIlC'rs using 0.5% purified casein ........................ .. Use-of isoelectric the concentration of poliovirus 6,.7 12 13 16 20 21 23 type 1 ft'om seawa ter ................ o. 24 Effect of pH on elutionof poliovirus type 1 from Filterite filters using 1% (wiv) non-fat dry milk (NFDM) ............. -. Concentration of poliovirus type 1 from seawater using var-ious concentrations of non-fat dry milk (NFDM) ............. Concentration of poliovirus from 20 gallons (76 liters) of seawater using the NFDM technique ...................... Concentration of poliovirus type 1 from 50 gallons (189 liters) of tapwater by the non-fat dry milk (NFDM) technique 25 26 27 28 13 Concentrat-ion of c,ix froHl.',piJVIiJtf'r by non-fat dry mi 1 k .. : .. Adsorption of Poliovirus type 1 to a marine sandy sediment.. 32 Elution of Poliovirus type 1 from a marine sandy sediment ... 33 Recovery of Poliovirus type 1 from intercoastal waterway sediment ......................................... '............. 34

PAGE 6

List of Tables (continued): Table 17 Use of non-fat dry mil k (NFDM) techni que for the recovery of naturally occurring enterovirus from sewage treatment plant effluents and Tampa Bay water......................... 36 18 Detection of indigenous enteroviruses in estuarine waterand sediment in St. Augustine, Fla. (June 1980) ............ 37

PAGE 7

LIST OF FIGURES 1 Location of wastewater treatment plants and virus samp-1 i ng s ta ti ons inS t. Augus ti ne, Fl a ..................... 18 37 & ;;4

PAGE 8

ABSTRACT This study dealt with the developement of methodology for virus recovery from the mari ne envi ronment. We have explored a vari ety of methods for the re.covery of enteroviruses from seawater which has been inoculated with high liters of poliovirus 1, 2, and 3, coxsackie B3and echovirus 1 and 4. We have developed a technique based on the adsorption of viruses to Filterite filters, elution with 1% non-fat dry milk (NFDM), and organic flocculation of the eluates. This method resulted in the efficient recovery of most enteroviruses tested except for echovirus type 1. The method was also suitable for vi rus recovery from tap water Various eluents were tested fOt' their ability to desorb viruses from marine sediments. In 1 aboratory experiments 4M urea-O .05M lysine was the most efficient in virus recovery from sediment .. Although the method is time consuming, it results in a small volume of concentrate. The methods developed in the course of this study were used-to re..: cover indigenous viruses from estuarine water and sediments. Enteroviruses 'tJere found in both water and sediment in a section of Matanzas River whi ch was closed with regard to shellfish ..

PAGE 9

PUBLICATIONS G. Bitton, B.N. Feldberg andS.R. Farrah. 1979. Concentration of enteroviruses from seawater and tapwater by organic flocculation using non-fat dry milk and casein. Water Air and Soil Pollution. 12: G. Bitton, P. Chou and S.R. Farrah. Virus recovery from marine sediments. (In Preparation). THESIS feldherg. B.N. 1979. The use of non-fat dry milk for the concentration of cnteroviruscs from seawater, tapwater, and sewage treatment plant thesis, 75 pp. "Department of Environmental Lngirwerinq Sciences. University of Florida, Gainesville, Florida. 32611

PAGE 10

-1INTRODUCTION Wastes resulting from man's activities have been traditionally (and are still) discharged into the ocean. These include liquid wastewater, sludge, oil, industrial and thennal wastes. With ever increasing populations along the coastlines around the world, it is by now realized that the ocean can no more-be considered as the lIinfinite sink" for the disposal of-fecal and other wastes. The coastal waters of the USA receive more than 8 billion gallons of municipal sewage in a single day. Approxi mately half of this domestic sewage receives secondary treatment (McNulty, 1977). Ocean outfal1s are commonly used for the disposal of non treated or partially treated sewage. Their main advantages are low cost as compared to tertiary of wastewater Their design is based on location, depth_, distance from shore, and mixing regime .. Over the'past 25 years much information has beerl gaiheredconcerning the fate of enteric bacteria in marine waters These pathogens are generally eliminated by physical dilution and by die-off mechanisms,which .include temperature, .predation, nutrient deficiency, algal and bacterial antibiotics or tOXins, solar radiation, heavy metals and salinity .. (MitChell, 1968; Mitchell and Chamberlin, 1975). Other factors, such as sedimentation, lead to the accumulation of the bacterial pathogens in marine sediments. (Gerbaet.al., 1978; Rittenberg et al., 1958). With regard to viruses more infonnat-ion is being gathered about their occurrence and fate in marine and estuarine waters (Edmond et al., 1978; Goyal et al., 1978; 1979; LaBelle et al. 1980). However, more efforts must be spent on the improvement or development of methodology for vi rus detection in marine and estuarine waters as well as sediments. This report explores the methodological aspects of virus occurrence in the marine environment.

PAGE 11

-2-LITERATURE REVIEW We will briefly review the occurrence of viruses in the marine/estuarine en vi ronment, the potenti a 1 publ i c health haza rds resul ti ng from thei r entry in ocean waters, and the existing methodology for their detection in that particular environment. Occurrence of Viruses in Marine Waters The' contamination of seawater by viruses mainly occurs through the disposal of sewage or sewage effluents into estuarine waters or offshore dis, posal via sewage outfalls. A survey carried out in the Mediterranean sea by the Environmental Health Laboratory of Hebrew University led to the detection of several types of enteric viruses (Poliovirus, Echovirus, Coxsackievirus) as far as 1500 m from the discharge point. Coliform monitoring showed that these bacteria were less stable than viruses in seawater. (Shuval et al., 1971). Another virus survey was carried out along the Houston ship channel and in Galveston Bay, Texas. (Metcalf et al ., 1974). This study showed that sewage treatment plants may discharge from 70 x 106 to 1746 x 106 viruses perday .. Examination of the Houston ship channel for viruses led to the detection of enterovir-uses as far as 8 miles' (=:13 kilometers) from the contamination source. Ruiter and Fujioka (1978) have shown that up to 8xl010 viruses are discharged daily into Mamala Bay via a sewage outfall. More recently human viruses were detected in coastal waters and sediments (Edmond et al. 1978; Goyal et al., 1978; 1979; LaBelle et al., 1980). These studies have essentially shown that marine/estuarine waters are frequently contaminated with viruses and that classical indicator bacteria are not suitable for demonstrating the presence or absence of viruses in the marine environment. These findings prompted world-wide research on the factors which control the fate of viruses in rnarine waters. Virus Survival in Marine Waters Laboratory studies have shown that virus survival is p'rimarily controlled by 'physical, chemical, and biological factors (Bitton, 1978). Some of these laboratory experiments were confirmed by "in situ" studies. However, lithe vi rucl da 1 agent" remains yet to be identifi demonstrated that temperature is the most decisive factor controlling virus survival in seawater and other coastal waters as well. Viruses displa'y an affinity for suspended solids in the aquatic environment (Bitton,1980a). These solids may be silts, clay minerals, cell debris and particulate organic matter. The solid-associated viruses tend to settle into bottom sediments where they reach higher concentrations than in the overlying waters (Gerba et al., 1978; Goyal et al., 1977). Therefore, sediments may serve as reservoirs for viruses as It/ell as for enteric bacteria. This has a public health significance since resuspension of the upper layers of sediments may increase the virus concentration in the water column. Sediment resuspension is particularly significant in shallow waters and is.due to motor boat activity, currents, swimming and changes in water qual1ty.

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I -3-potential Health Hazards Due to the Presence of Enteric Viruses in Marine Waters In the last decade, water has been recognized as a possible route for the transmission of enteric viral diseases. (Berg, 1967). We have now evidence that the infectious hepatitis virus may be transmitted via the wa ter route. However there is alack of clem; 0109; ca 1 data ng the association between swimming in water and viral diseases. Recently, tt-hasbeen demonstrated that there was a statistically significant association between swimming and shigellosis. Shigella sonnei was isolated from the Mississippi River near Dubuque, Iowa and was shown to be responsible for disease in individuals who had water in their mouth while swimming (Rosenberget al., 1976). These are interesting data since Shigella has a low infectious dose (10 to 100 bacterial cells) as compared to other patho gens. This infectious dose is not very far from the infectious dose of enteric vi ruses .': Viral diseases may also be contracted following the consumption of shellfish which have been harvested from beds contaminated with sewage effluents (Metcalf, 1976). Shellfish (oysters, clams, mussels) have the ability to concentrate viruses within their gastrointestinal tract. Viruses may later diffuse from the alimentary tract to other tissues. Although infectious hepatitis and gastroenteritis are the only diseases shown to be associated with shellfish consumption, it is possible that other viral diseases are transmitted via this route. for virus detection in the marine environment Microporous filters have been mostly used for virus concentration from marine and estuarine waters (Goyal and Gerba, 1981). The virus concentra-tor developed by Wallis et al., (1972) was used by Metcalf et al., (1974) to concentrate viruses from estuarine and surface waters. Clarifying filters (1-pm orlon fil ters) were used to remove 1 arge parti cl es that tended to clog the Millipore filters that were used as virus adsorbents. It was noted that the clarifying filters tended to adsorb viruses as larger volumes of water were processed. Metcalf and co-workers (1974) used a modified version of the previous method to detect viruses in Houston ship channel and Galveston Bay. A procedure for concentration of enteroviruses from 15 to 100 gallons of turbid estuarine water using a filter aid (Celite) and Millipore membranes'tlas described by Hill, et al., (1974). However, these procedures permitted recoVery were used to test the procedures. _. Sobsey et al., (1977) used asbestos-fiberglass (Cox) filters in place of the nitrocellulose (Millipore) filters previously used. Since these filters were more resistant to clogging than Millipore filters, the clarifying filters were eliminated. The virus concentrator used by Sobsey et al., (1977) employed a single prefilter along with two flat sheets of membrane filters as the virus adsorbents. Virus in estuarine water at pH 3.5 and 0.0005 M aluminum chloride adsorbed to the filters and could be recovered by treating the filters with glycine buffer at pH=ll .5. However, virus in this eluate could not be reconcentrated in a smaller volume using a second membrane I, ___ 4-*;& B"MIMfl* ," d,i;a

PAGE 13

;, T -.;. -4-adsorption-elution step as was used when tapwater was sampled for virus (Sobsey et al., 1973). Adjusting eluates to pH 3.S resulted in the formation of large amounts of floc that rapidly clogged filters used for reconcentration. However, the authors (Sobsey et a1 q 1977) observed that flocculation could be increased by addition of ferric chloride. The f10cs that formed wereeffi ci ent virus adsorbents and cou1 d be used to reconcentrate the virus to a final volume of 20 m1. The flat filters used by Sobsey et al., (1977) permitted relatively low initial flow rates and also clogged rapidly Freq.uent1y, 1-2 hours were requi red to process 50 gallons. of estuarine water. Since p1 eated fi ber-g1ass fil ters were shown to permit processing of large volumes of tapwater at high flow rates (Farrah, et a1., 1976), these filters were tested for their ability to concentrate vi rus from estuari ne water by Payment et a1 ., (1977). The use of pleated fiberglass filters permitted the concentration of virus from liters of estuarine water. Virus was recovered by treati,ng the filters with high pH glycine. A two-step flocculation procedure using aluminum chloride was used to concentrate virus to a final volume of 20-40 ml. Thi s reconcentration procedure was found to g1v.e inconsistent recoveries. when estuarine water with different amounts of organic compounds u.:1d from different sites were tested. Therefore, this reconcentration procedure was replaced by a procedure using aluminum hydroxide flocculation and hydroextraction. (Farrah et al., 1977). The completed procedure using pleated membrane filters as the virus adsQrbent, glycine buffer to elute adsorbed virus, and aluminum hydroxide flocculation along with hydroextraction to reconcentrate eluted virus, was used in virus monitoring in Galveston-Bay (Goyal et al., 1978) and in seawater near a deep sea sewage putfa 11 off Mi ami, Flori da (Edmond et a 1 ., 1978). It thus appears that the pleated Filterite microporous filters offer large surface areas for virus adsorption and are relatively resistant to clogging. The optimal virus adsorption to these filters requires an AIC13 concentration of 0.0005M to O.0015M and a pH of 3.5. However, elutlOn of adsorbed viruses with glycine buffer at high pH (pH=ll .S) may be detrimental to some pHsensitive viruses such as adenoviruses or rotaviruses. Therefore one needs to look fOI" e1uents which work efficiently at pH=9.0-9.S. In 1976, Katzenelson and his collaborators described a method which responded to this need. These investigators showed that beef extract could be used as an eluent in virus concentration from tapwater. After elution at pH=9.0, viruses could be concentrated-via org-anic flocculation of the beef extract at pH=3.5. The sediment obtained from flocculation could be resolubilized with O.lSM Na2HP04 at alkaline pH. It is therefore useful to explore virus -methodology which uses eluents that work well at pH
PAGE 14

-5-currents, dredging or changes in water may lead to an increase of virus concentration in the water column. Few studies have been undertaken on the development of methods for virus detection in sediments. De Flora et al. (1975) showed anaccumula t'lon of viruses in marine sediments in the vicinity of sewage outfalls in Italy. They used sterilized seawater for eluting viruses from sediments. Glycine buffer (0.05M; pH=8.5) and nutrient broth (pH=8.5) have also been used for virus rerovery from mar-ine sediments (Metcal f et ala 1974; Vaughn and Metcalf, 1975). none of the above investigators de termined the efficiency of the eluents used. Gerba et al. (1977b) described a method for virus recovery from marine sediments. The procedure consists. of eluting the sediment with 0.25M glycine buffer, pH=1l.5, containing 0.05M EOTA. Following a lO-minute contact time, the sediment was spun down and the supernatant was concentrated via membrane fil tration.. Virus. was recovered from sediment with an efficiency of 50%. This method has two major drawbacks: The pH of the el uent is too high and may 1 ead to inactivation of pH-sensitive viruses as was discussed previously. The second drawback is the clogging of microporous filters used in the recon centration step.' It is therefore necessary to evaluate a wide range of eluents for virus recovery from marine and estuarine sediments. These eluents should be used at a pH lower than 11.5. Moreover; some other means of eluate reconcentration (other thilln membrane filtration) should be explored. Since no study has dealt with methodology for virus recovery frolll' freshwater sediments, some of the findings may be helpful in the development of methodology-for virus recovery from these types of sediments.

PAGE 15

Tabl e 1 Virus concentration from large volumes of seawater and sewage effluents. -------.-------.---.--. Concentration Adsorption I niti aJ Final Final t'ia ter Reference system conditions volume volume percent type (1) (m 1 ) recovery --------------_.Seawater 2 step adsorption 0.D005 M A1C13 95 10 63 Coastal r,letca 1 f elution pH = 3.5 et a 1 (1974) Adsorption, elu0.0015 A1C13 378 20 53 Seawater Gerba ti on, i nO,rgani c pH = 3.5 et .a 1 flocculation (1978 ) I C.;) I Adsorption, elu0.0015 M A1C13 400 10-40 60-80 Estuarine Fa rrah tion, inorganic pH = 3.5 et al fl occul ati on (1977) v, elu0.0015 M A1C13 378 20-100 50 Seawater Payment tion, pH = 3.5 et a 1 fl occu1 at) on (1976) il ;I( ,'-I, Adsorption, e1u0.0005 M. A1C13 189 15-20 41 Seawater Sobsey tion, inorganic pH =3.5 et a 1 flocculation (1977) ,;'i 1'-1 4-'! ;i' i -'. u, !It ,n aM

PAGE 16

r Sewage effl uents Concentrati on system p, Adsorption, elution, inorganic flocculation Adsorption, beef extract elution, organic ;': flocculation Adsorpti oni e 1 uti on ,I,i i norgani c:"'! fl occul a ti:9n _________ ________________ .. __ ........ ...... .. __ _. ______ __ Table ------continuedi Adso rpt ion conditions Initial volume (1 ) 0.0005 M A1C13 19-190 -pH = 3.5 0.0005 M A1C13 76-96 pH = 3.5 0.0005 M A1C13 76-96 pH = 3.5 Fi na 1 volume (ml) 20 ? ? Final percent recovery 50 \':a ter type 20 effl uent 85 30 effluent 36 3 Reference Gerba et al (1978) Landry et al (1978) Landry et al (1978) if I., .... __ -,-.:.... __ .;..... ________________ .. -!!'! ';'ft ,f;!1; ',Li it I, <'1\1' !,g V I --.J I

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-8-MATERIALS AND METHODS Viruses Used and Their Assay Plaque-purified poliovirus type 1 (Sabin), poliovirus type 2 (natu ral isolate), poliovirus type 3 (natural isolate), ECHO 1 (Farouk), ECHO 4 (Pesacek), and Coxsackie B3 (Nancy) were used in these studies. Poliovirus types 2 and-3 were isolated from wastewater sludge. Virus stocks were produced by infecting a monolayer of human am-nion cells (AV3) or Rhesus monkey embryonic kidney cells (MA104) growing in 32 ounce bottles. Viruses were allowed to adsorb to the host cells for one hour at room temperature after which 40 ml of Eagle's Minimal Essential Medium (MEM) with 10% Fetal Calf Serum bFCS) was added. monolayers were then incubated for 48 hours at 37 C. The virus was harvested by decanting the fluid, centrifuging at 1500 RPM for 15 minutes, and freezing the resultant supernatant at -700C in small aliquots for 1 ater use. Viruses used in seeded experiments were assayed on AV3, MA104, or BGM (Buffalo Green Monkey) host cells using a routine plaque procedure. For this assay monolayers were prepared by-incubating host cells at 370C in 32 ounce bottles containing 40 ml of Eagle's MEM with 10% Fes until confluent monolayers were obtained (approximately 60 hours). At confluence, the growth media was removed and the cells washed with 30 1111 of a pre-trypsin solution. The rnono1ayers were then treated with 10 III 1 of a standard trypsin-versene solution to separate the cells frolll the glass surface. After approximately 1 minute the latter solution was decanted and the monolayer maintained at room temperature until the cells unbound (approximately 5 minutes). Then, 200 ml of Eagle's MEM with 10% FCS was added to suspend the cells and subsequently distributed to forty 2-ounce glass or plastic tissue culture bottles in 5 ml aliquots. The bottles were incubated at 370C for 48 hours or until monolayer con fluence was evident. At this stage the tissue cul tures were ready for use in the standard plaque assay. -,Mature tissue cultures were infected by decanting the growth medium and adding 0.2 ml of sample adjusted to isotonicity and pH=7.2-7.4. Samples were diluted either Eagle's MEM with 10% FCS and antibiotics or phosphate-buffered saline (PBS) with 5% FeS and antibiotics. The tissue cultures were maintained at room temperature for one hour to,aljow for, virus adsorption to theChost-cells.Aftertheadscirption-period tne monolayers were overlayed with five ml of methyl cellulose and incubated i'lt 3i1C until the plaques were approximately 1-5 mm in diameter. At this stage the monolayers were stained using crystal violet or neutral red and the number of plaques determined. vJater Sampl es Seawater was collected from Crescent Beach, Florida, and stored at 40e prior to use. The seawater had a pH of 8.2 and a specific conductance

PAGE 18

-9-of 41,000 ppt salinity). Gainesville tap water used had a pH of 8.4 and a specific conductance of 250 and was dechlorinated using sodium thiosulfate prior to viral seeding. Ortho to1idine titrations (Standard Methods, 1976) were performed to determine the concentration of sodium thiosulfate necessary for complete dechlorination. Concentration Step: Viral Adsorption and Elution All experiments were performed in batch processes. The viruses used were suspended in three 1 iters of seawater and adsorbed to a series (3.0 pill > 0.45 111111>0.45 11m or 3.0 11m 0.45 pm -)0 0.25 11m) of epoxyfiberglass fnters (Filterite Corp., Timonium, Md.) in the presence of 0.0005 M Ale'3 at pH = 3.3-3.5. Viruses were eluted from the membrane filters using (w/v) technical, purified, or isoelectric casein (Difco, Detroit, Mich.) in 0.05 M glycine buffer at pH = 9.0 or 10.0, or 1% (w/v) non-fat dry milk (NFDM) (Atlantic and Pacific Tea Co., N.J.) in 0.05 M glycine buffer at pH = 9.0. Additional eluents used were 3% beef extract at pH = 9.0 (Katzenelson et al., (1976) and 0.05 N glycine, pH = 11.5 (Farrah et al., 1977). Both the filtrates and eluates were assayed to determine percent adsorption and elution, respecti ve1y. In experiments using large volumes of water (20 to 100 gallons), suspended viruses were adsorbed to a 0.25 jlm or 0.45jlm 10-inch epoxyfiberglass cartridge filter (Filterite Corp., Timonium, Hd.) in the presence of 0.0005 M A1C13 at pH = 3.3-3.5. This was done by pumping the prepared sample through the filter, using a gasoline (Sears Roebuck and Co Chicago, Ill.) or electric (Sturdy Puppy, ITT Jabsco Prod., Costa Mesa, Calif.) powered centrifugal pump. Flow rates as high as 10 GPM were achieved using the gasoline powered pump. When 50 gallons of sccondaryeffl uent was processed a 3.0 pm 10-inch Fil terite cartridge filter was used as a prefilter. Viruses were eluted by pumping 1 liter of1% NFDM in 0.05 M glycine at pH 9.0 through the filters after a 1 minute contact period was allowed. In seeded experiments the filtrate and eluate were assayed to determine percent adsorption and elution, respectively. '_ For the concentration step the concentration factor ranged fDQm 100 i Reconcentration of filter eluates Glycine eluates were reconcentrated according to Farrah et a1. (1977). Beef extract eluates were concentrated by organic flocculation as described by Katzenelson et al. (1976).

PAGE 19

. .: -10Solutions of casein and dry milk flocculate at pH 4.5-4.6' which is the isoelectric point of casein (Jenness and Patton, 1959). Eluates obtained by the above concentration step were flocculated at pH = 4.5-4.6 by the addition of 1 M glycine (pH = 2.0) and then centri-fuged at 4500 RPM for 4 minutes. Eluates may also be allowed to settle and only the lower portion centrifuged., This was ascertained by seeding 30 ml of a 1% NFOM solution in 0.05 M glycine, adjusting the pH to 4.5, andall owi-ngthe flocs to settl e undisturbed. The supernatant was wi thdrawn and the loose sediment adjusted to pH = 7 with 1 M glycine (pH=11.0). Upon assay, an average of 117% of the added pol iovi rus was recovered in a final volume of 7 ml. This data suggests that the centrifugation step may also be carried out at a lower speed with no effect on virus recovery. Pellets obtained from centrifugation were redissolved in 0.15 H Na;;>HPOa (pH = 9.0). The co_ncentration factor for this step ranged from' 10 lr val ume)-, ,to 40 (l a rge volume). The fi na 1 samp 1 e was soni ca ted, at 35 watt power output for 10 to 15 seconds with a Soni fier Cell Oi srupter (model WI850, Heat Systems Ultrasonics, Inc., Plainview, N.Y.). -_ ... ,When usi ng casei n or NFOM it was found to be advantageous to add a small quantity (one squirt) of an antifoam agent (Dow Corning Corp., rvlidland, Mich.) to inhibit foam production during elution and pellet re-' suspension. The use of an antifoam agent did not reduce polio-' _virus number$ or cause cytotoxicity to AV3 or MA104 host cells (Table 2 and 3)., __ When large volumes of water were processed using NFOM the final concentrates were di a lyzed against PBS for 18 to 24 hours at 4C prior to virus assay. This step was necessary to prevent cytotoxity to host cells caused by factors present in the undialyzed fluid. I Vi rus Recovery From Sedimehts Marine sediments used in this study were a sandy sediment collected at Crescent Beach, Fl a. and an organi c muck sediment sampled at the Intercoastal Waterway near St. Augustine; Fla. The sediments were retrieved\'lith the aid of a Ponar dredge and were stored at 40C until use. 109-sediment samples were. mixed with 20 ml of seawater which has been seeded with poliovirus type 1. The mixtures were. mechanically.,.., shaken fo r30 mi nu tes:..and thencenctrifuged 4000 ,---supernatants were poured off and assayed for vi ruses to determine the percent virus adsorption to the sediment. A blank, which consisted of seawater wi thout sediment, was run simul taneously wi th the sediment sampl es. Ten eluents were for their ability to desorb viruses from marine sediments. These eluents were 4M urea + lysine, 0.6M Na trichloroacetate + O.02M glycine 1.0% isoelectric casein, 1% non-fat dry milk, humic substances _{COlor units =. 5,700) ,0.3% Na pyrophosphate, 10% fetal calf serum (FCS) in phospbate buffered saline (PBS), distilled water, 3% beef extract and 0.25 M glycine + 0.05M EOTA. All the eluents :1 .. -I':' ; '1-.

PAGE 20

-11were buffered to pH = 9.0 except for distilled water (pH = 7.0) and glycine-EDTA (pH = 11.5). Thirty ml of a given eluent were added to 109 of wet sediment. The mixtures were vortexedfor 30s and then shaken for 30 min. However the shaking time was only 4.5 min and 1 min for glycine-EDTA and urea-lysine, respectively. The samples.were centrifuged at 4000 RPM for 4 min and the supernatants were assayed for viruses to determine the % of virus eluted from the sediment.

PAGE 21

-12-Table 2: The effect of Antifoam A Spray on the recovery of poliovirus type 1 using AV3 host cells. Treatment vl"ith I\ntifoarn Without Antifoam Total PFU befor3 treatment (x 10 ) 6.4 5.5 Mean: 6.0 6.6 6.5 Mean: 6.6 Total PFU after treatment (x 103 ) 6.6 6.8 Mean: 6.6 7.1 6.0 Mean: 6.6 -Note: 50 ml of a 0.05 M glycine buffer solution (pH = 8.5) were seeded. with poliovirus type 1 (Sabin). stirred with a magnetic bar for 1 minute and then assayed for virus. Two squirts of Antifoam A Spray (Dow Corning Corp., Midland. Mich.) were added to two tes t tubes whil e two other tubes recei ved no treatment. All tubes were then strongly mixed for 5 lIlinutes with a magnetic stirring bar and then assayed for virus on AV3 host cells. :;& 1 m SMIII ""

PAGE 22

I -13-Table 3: The effect of Anti foam A Spray on the recovery of poliovirus type 1 using MA104 host cells. pFU/ml Treatment(a) Run l(b) Run 2 Run 3 Mean MEM + Antifoam 440 440 420 430 MEM {control} 450 390 370 400 (a) 14.9 ml of Eagle's MHl were added to each of 6 test tubes and 0.1 ml of poliovirus was added to give a final virus titer of approximately 400 PFUjml. Three tubes were treated (one squirt) with Antifoam A Spray (Dow Corning Corp., Midland, Mich.) while the three other tubes did not receive Antifoam. All 6 tubes were vortexed for 10 sec and the fl ui ds assayed on MAl 04 host cell s. (IJ.) Each run represents the mean number of 4 to 5 ti ssue cul ture bottl es

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-14Concentration of Indigenous Virus from Field Samples City of Tampa Sewage Treatment Plant Effluents and Tampa Bay Table 4 shows conductivity, salinity,and pH values of sewage treat--ment plant effluents and Tampa Bay water which were examined for the presence of naturally occurring enteroviruses. Unchlorinated secondary effluent was collected immediately prior to tertiary treatment. Tertiary effluent was collected after approximately 10 minutes of chlorination ppm available chlorine). This effluent was dechlorinated prior to processing as described for Gainesville tap water. Tertiary effluent from the City of Tampa sewage treatment plant is discharged. to Tampa Bay by an underwater outfall. Sampling of the outfall was accompl i shed by anchoring a boat di rect1y over the IIboi 1" created by the discharge and pumping water from a five (5) foot depth into a holding tank on board the boat. Tampa Bay water was collected from a site 30 yards seawater of the outfall from a fi ve foot depth. It is evi dent from conductivity and salinity values for effluents and bay water (Table 4) that the sample collected from the outfall site was quite dilute with effluent. The virus concentration method, using 1% non-fat dry milk, is described in details in the IIResults and Discussionll section. Recovery 'Of i ndi genous viruses from estuari ne water and sedime!l.! __ Flori da The presence of indigenous viruses was monitored in two stations in St. Augustine, Fla (Figure 1). These are shellfish beds and shellfishing i> prohibited. The first sampling point is on the Matanzas River just south of the Bridge of Lions in St. Augustine. The second sampling station is in the Salt Run at a location north of the public boat ramp. The station on the Matanzas River is in an area which is subject to fecal pollution from wastewater treatment plants (see Figure 1). Forty to fifty gallons of estuarine water were sampled and processed' following the non-fat dry milk method described previously. Fifty toone hundred grams of sediment were sampled at both stations. They were immedi ately stored in ice and brought back to the 1 aboratory where they were processed. The time between sampling and processing did not exceed 5 hours. The sediment samples were eluted with urea-lysine as described previously. The urea-lysine eluent must be prepared a few houts before usesinceolder solutions'may'1ead to lower recovery from sediment. The sediment eluates to the following procedure: nlC1 3 was added to glve a flnal concentratlon of 0.005M. The eluate was then adjusted to pH 7 by the addition of 1M sodium carbonate and mixed for 5-10 minutes. The flocs formed were collected by centrifugation at 5000 RPM for 5 min. The superniltant was discarded and the floc was mixed with!) volumes I'llTl\!t IH'pr C'Xj)"Mt. pll"C),a, of thp floc coulct lw dissolved in ltd', '.lIll/tioll. Ih(' ',.11111'1(' w.-t', tllt'l\ Ct'II!.I'irll
PAGE 24

-15-Concentrates from wa ter and sedi ment were as sayed on cell sand examined for cytopathic effects (CPE) for up to three weeks. The 50% tissue cul ture infective dose (TCID50 ) was determined according to Reed and Muench. (1938). Virus isolates were fdentified by using pools of neutralizing .. antiserum (Lim and 1960). -.

PAGE 25

. -16e 4: Water qual i ty parameters of Ci ty of Tampa sewage treatment plant effluents and Tampa Bay water. ... Hater sample Secondary effluent (unchloriDated) Tertiary effluent (ch 1 ori na ted) Tertiary effluent outfall, Tampa Bay Tampa Bay, 30 yards from outfall Conductivity (pmhos/cm) 960 884 14,820 15,150 Salinity(a) (ppt) 0.7 0.6 10.7 10.9 (a) Salinity values were computed from conductivity measurements. pH 6 f' ,l 6.8 7.7 7.7

PAGE 26

'!tz -17-Figure 1: Location of wastewater treatment plants and virus sampling stations. in St. Fla. '* Wastewater treatment plants o Virus sampl ing (Hater and sediments)

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ST. AUGUSTINE AIRPORT IS ... KING ST. -18-= 112 O. = SCALE MilES AnANTIC OctAH

PAGE 28

-19RESULTS AND DISCUSSION Comparison of Existing Methods for the Recovery of Poliovirus from Seawater Two existing methods of concentrating viruses were examined with respect to recovery efficiency, using samples of seawater. Table 5 compares polio virus recoveries using methods involving: 1) Elution with glycine (pH = 11.5) fol-lowed-by inorganic flocculation using A1C13 (Farrah et al., 1977), and 2) Elution with beef extract (pH = 9.0) followed by organic flocculation (Kat zenelson etal., 1976). All methods examined shared a common first concentration step. that is the adsorption of vi ruses to epoxy-fi bergl ass (Fil teri tel fi 1 ters in the presence of 0.0005 M Alel 3 at pH = 3.3-3.5. The modified method of Farrah et al. (1g.77) used in our study involved elution with 0.05 M glycine buffer (pH = 11.5) and reconcentration using Al (OH)3 flocs. Use of this method yielded mean re coveries of 47% el ution., 25% from the reconcentration step. and 12% overall recovery. Farrah et al. (1977) reported that 85% to 95% of poliovirus ad sorbed to Al (OH)3 flocs were recovered by elution with the glycine-FCS mixture. The particular problem in the recovery of poliovirus in these trials (Tahle 5) was the elution of viruses adsorbed to the floes. Greater than 90% of viruses present in the filter eluates were adsorbed to the inorganic flocs. The second existing method of concentrating viruses is based upon elution of viruses adsorbed to membrane filters with 3% beef extract (pH = 9.0) followed by flocculation (Katzenelson et al., 1976). Use of beef extract method yielded higher poliovirus recovery than glycine method and resulted in an overall recovery of 56% (Table 5) .. Usinq this method, Katzenelson 1977 was ab 1 e to recover 63% of added po.-Ii uvi rus from 35 1 i ters of seawater. Use of Technical, Purified, and Isoelectric Casein in the Concentration of Poliovirus from Seawater A set of preliminary experiments were performed tol earn whether di 1 u'Ce casein could be used to elute poliovirus adsorbed to membr:ane filters and further concentrate the viruses by organic flocculation. Table 6 shows percent-viral recovery using 0.2% technical casein in 0.05 M glycini tpH = 11.011.5),0.5% technical casein in 0.05 M glycine (pH = 11.5). All trials yielded greater than 75% recovery from elution and greater than 65% recovery from or ganic flocculation (reconcentration). Overall recoveries for the three tials were greater than 50%. The main advantage of the beef extract method is that elution is carried out at pH = 9.0, thus not affecting the survival of viruses (reo viruses, rotaviruses, and adenoviruses) which are inactivated by exposure to pH = 11.5 for even short times (Fields and 1975, Sobsey et al., 1980). Furthermore, even the exposure of pol i ovi rus type 1 to pH = 11.5 gTyCfne buffer for more then 10 minutes may be virucidal (Gerba et al., 1977b). For this reason, one must work with great speed when using pH = 11.5 glycine buffer as an eluent for enteric viruses.

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-20Table 5: Comparison of two existing methods for the concentration of pol i ovi rus type 1 from seawater. f1ethod(a) Percent el ution Percent reconcentration ------.---_._------------_._-----------------_. Elution with 0.05 M glycine (pH = 11.5) followed by inorganic using A1C13 { ) Elution using 3% beef extract (pH = 9.0) follQwed by organic flocculationtc) 47 86 25 66 Percent overall recovery 12 56 (a) Poliovirus type 1 (Sabin) suspended in 3 liters of seawater, was adsorbed to a series (3.0 l1m -)-0.45 11m -}-0.25 11m) of Filterite filters in the presence of 0.0005 M A1Cl at pH = 3.3-3.5. Viruses were eluted with 30 ml of a designated efuent (see references beloW). Final concentrates obtained were 2 to 4 milliliters. Numbers given are mean values for 2 to 4 trials. (b) Trials performed according to Farrah et al. (1977). (c) Trials performed according to Katzenelson et al. (1976).

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-21I Table 6: Use of technical and purified casein in the concentration of polio virus type 1 from seawater. Trea tment (a) 0.2% technical casein in n.05 M glycine (pH = 11:-11.5) 0.5% technf'cal casein in 0.05 M glycine {pH = 11.5) 0.5% puriffed .. casein in 0.05 M glycine (pH = 11.5) Total PFU adsorbed to filters 4.06 X 106 5.47 X ;06 4.64 X 106 Percent elution 77 76 81 Organic flocculation percent recovery 79 67 72 Percent overall -recovery 56 51 58 (a) Poliovirus type 1 (Sabin), suspended in 3 liters of seawater, was adsorbed to a series (3.0 11111 >-0.45 11m .>-0.25 11m) of Filterite f.ilters in the presence of 0.0005 J\lClv pH = 3.5. Viruses were eluted using 30 m1 of a designated eluent. Orga.niC flocculation was induced by adding 0.5 M glycine (pH = 2) to the casein eluates until 8 pH of 4.5 was reached. The floc formed was pe11eted by centrifugation at 4500 RPM for 4 minutes and re-disso1ved in 3 m1 O.15M Na2tlP04,'pH = 9.0. Values given are mean values from duplicate or triplicate trials. .....

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-22-It then became of interest to examine the effect of pH upon the elution of poliovirus type 1 from membrane filters. Table 7 shows that elution was most efficient at pH = 10.0 (100%) and least efficient at pH = 11.5 (64%). It is worth noting that viral elution was relatively high (76%) even at pH = 8.0. Use of 0.5%isoelectric casein in 0.05 M glycine (pH = 10.0) permitted the recovery efficieny for elution (>100%) of the three types of casein exarnlned so far (Table 2). Overall viral recovery from 3 liters of seawater using isoelectric casein was 83%. Use of Non-Fat Dry Milk for the Concentration of Poliovirus from Seawater While contemplating the potential use of casein for viral detection and concentration from seawater, the thought arose that since caseins and lipids (fats) are the major components of milk, then perhaps non-fat dry milk (NFDM) could be used as an inexpensive alternative for casein. Preliminary unseeded experiments showed that a 1% (w/v) solution of NFDM in 0.05 M glycine behaved like purified or isoelectric caseins in that flocculation occurred at pH = 4;5 and centrifugation produced a pellet and a clear supernatant. Using 1% NFDM in 0.05 M glycine, a study \lIas undertaken to investigate the influence of pH on the efficiency of elution of poliovirus type 1 from membrane filters. As shown in Table 9 the elution was maximum (1l6%) at pH = 9.0 and decreased to 35% and 43% at pH = 8.0 pH = 7.0, respectively. Since the ultimate goal of a concentration technique is to achieve a high concentration-factor, it was decided to examine the effect of NFDM concentra tion on the efficiency of poliovirus recovery from seawater, the rationale beinq that the more dilute the NFDM solution used, the smaller the centrifuged pellet and hence the smaller' the volume of final concentrate. As shown in TablelO efficiency decreased with dect'easing NFDM concentration (from 7lX with 1% NI-l)M to 35% with 0.25% NFDM). However, recovery from organic flocculation was always high and ranged from 81% to 100%. Use of NFDM concentrations of 1%, 0.75%, 0.50% and 0.25% yielded overall poliovirus recoveries of 71%, 48%, 33%, and 28%, respectively. Concentration of Poliovirus from Large Volumes of Seawater and Tap Water Using the NFDM Technique As the NFDM technique proved efficient for the concentration of poliovirus from small volumes of seawater (3 liters), its application to the processing of large volumes of seawater and other types of water became of interest. Twenty gallons (,,76 liter) samples of seawater were adjusted to pll ::.. 3.33.5 and 0.0005 M A1C13, seeded with poliovirus, and processed hy the NrDM techni que (Table 11). Recoveries obtained for two trials were 54% and nn', elution recovery, 87% and 115% reconcentration recovery, and 74% and 100% overall reco very; the mean overall recovery for the two trials being 74%. Next, fifty gallon (189 liters)-samples of dechlorinated tap water were seeded with high x 10/ PFU) and low PFU) inputs of poliovirus type 1 and processed by the NFDM technique. As shown in Table 12 high virus input trials resulted in a mean overall recovery of 80% while low input trials recovered an average of 75% of the initial virus titer. Concentration factors ranging from 2363 to 6750 were obtained in these trials.

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Tabl e 7: Effect of pH on el ution of pol iovirus type 1 from membrane filters using 0.5% purified casein. pH of casein Percent elution(a) 8 76 9 74 10 100 64 (a) Poliovirus 1 (Sabin), suspended in 1 liter of seawater, was adsorbed to a series (3.0 + 0.45 + 0.25 of Filterite filters in the presence of 0.0005 M Ale13 at pH = 3.5. The adsorbed viruses were eluted with 0.5% purified casein in 0.5 M glycine (30 ml) adjusted to various pHs with 0.5 M glycine. Each number represents the mean for 2 to 4 trials.

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-24Table 8: Use of isoelectric casein in the concentration of poliovirus type 1 from seawater. Tota 1 number of viruses adsorbed to membrane filters PFU 6 2.06 x 10 6 2.51 x 10 Mean: Percent elution 136 104 120 Organic fl occul ation percent recovery 80 54 67 Percent overall recovery efficiency 110 56 83 Note: Poliovirus type 1 (Sabin), suspended in 3 liters of seawater, was adsorbed to a series (3.0 pm + 0.45 pm + 0.45 pm) of Filterite filters in the presence of 0.0005 M Ale13 at pH = 3.5. Viruses were eluted from the filters with 30 m1. of a 0.5% isoelectric casein solution in 0.05 M glycine (pH = 10.0). Organic flocculation was undertaken by adding 0.5 M glycine (pH = 2) to the eluate until a pH of 4.5 \lIas reached. The floc formed was pelleted by centrifugation and redissolved in 2.5 ml df 0.15 M Na2HP04 pH = 9.0. ll!\l!! _1iIIII!!-------r -----....... -------.-------

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-25Table 9: Effect of pH on elution of poliovirus type 1 from Filterite filters using 1% (w/v) non-fat dry milk (NFDM) pH of NFDM eluent 7.0 8.0 9.0 10.0 11.5 Percent e1ution(a) 43 35 116 86 69 (a) Poliovirus 1 (Sabin), suspended in 1 liter of water, was adsorbed to a series (3.0 11m + 0.45 +-0.45 pm) of Filterite filters in the presence of 0.0005 M A1e13 at pH = 3.5. Viruses were eluted with jQ ml 1 % NFDM in 0.05 1'1 glyci ne. Each number represents the mean of2 to 4 trials.

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-26Table 10: Concentration of poliovirus type 1 from seawater using various concentrations of non-fat dry milk (NFDM). Treatment (a) 1 .00% (b) NFDM 0.75% NFDM 0.50% NFDM 0.25% NFDM Total PFU adsorged (x 10 ) 51 1.86 1.36 1.22 Percent elution 71 58 35 35 Organic flocculation percent recovery 101 84 94 82 Percent overall recovery 71 48 33 2B (a) Poliovirus type 1, suspended in seawater, was adsorbed to a series (3.0 0.45 11m+ 0.45 11m) of Filterite filters in the presence of 0.0005 M A1C13 at pH = 3.5. Viruses were eluted with the various concentrations of NFDM in 0.05 M glycine, pH = 9.0. Organic flocculation was undertaken by adding 0.5 M glycine to the filter eluate until a pH of 4.5-:-4.6 was reached. The floc formed was pelleted by centrifugation at 4500 RPM for 4 minutes and the pellet redissolved in 0.15 M Na?HP04 pH = 9.0 Each number represents the mean value obtained from trials. (b) Trials using 1% NFDM used seawater samples of 20 gallons (76 liters) processed through 0.45 pm Filterite cartridge filters. Other trials used 1-3 liters seawater samples and were processed as described above. ,,C ,-J.,..l.' ".1 !! l q .-; i .. f

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-27Table 11: Concentration of poliovirus from 20 gallons (76 liters)" of seawater using the NFDM technique. PFU adsorbed to membrane filters 7 6.98 x 10 3".16 x 107 I Percent el ution 54 87 Percent reconcentration 87 115 Percent overall recovery 47. 100 Note: Poliovirus type 1 (Sabin), suspended in 20 gallons (76 liters) of seawater was adsorbed to a J-lm Filterite in the pres:nce of 0.0005.M A1C13 pH = 3.3-3.5. V1ruses were eluted.w1th lllter of 1% NFDM 1n 0.05 M glyc1ne (pH = 9.0). Filter eluates were brought to pH 4.5 with 1 M glycine (pH = 2.0) and the resul tant flocs centrifuged at 4500 RPM for 4 minutes. The pellets were then resuspended in 0.15 M Na2HP04 pH = 9.0.

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-28-Table 12: Concentration of poliovirus type 1 from 50 gallons (189 liters) of tapwater by the non-fat dry milk (NFDM) technique. Total virus input PFU High vi rus input{a) 7.12 x 10 7 7.17 xl 0 7 Low virus input(a,b) 641 569 Percent elution 72 118 Organi c flocculation percent recovery 110 68 Final volume {ml} 80 55 28 30 Percent overall recovery ._---.... --79 81 72 78 --"--,-,-,,--(a) Poliovirus type 1, suspended in 50 gallons (189 liters) of dechlorinated tapwater, was adsorbed to a 0.25 ]1mor 0.45]1m Fi1terite cartridge filter in the presence of 0.0005 M A1C13 at pH = 3.5. Viruses were eluted from the filters usin'g 1% NFDM in 0.05 M glycine, pH = 9.0. Filter eluates were brought to pH = 4.5-4.6 with 1 M glycine (pH = 2.0) and the resultant floc centrifuged at 4500 RPM for 4 minutes. The pellets were then resus-pended in 10-63 ml Na2HP04 pH 9.0. ". (ti) Concentratesfrom-low -virus, input trials were dialyzedfor.1B-:24 hours against PBS at 40C prior to direct assay on host cells. ________________________________________________________ .......... __________ .. t ..

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-29These resul ts agree wi th those reported by other researchers using large volumes of water but different methods. Hallis et al. 1972b, 1972c) and Sobsey et al. (1973) reported poliovirus .recoveries ranging from 61% to 92% from large volumes of tap water using two step filter-adsorption elution. Using elution and aluminum hydroxide flocculation reconcentration, Farrah et al (1977) were able to recover 40% to 50% of poliovirus added to 500 gallons of Katzene-lson et al. (1976) recovered 74% of added pol iovi rus from 500 1 iters of tap water using beef extract el ution ilnd organic flocculation. Using low numbers (16 to 50 PFU) of added poliovirus per 100 gallons of tap water, Hill et cil. (1974) were able to recover 25% to 50% of the ini tial vi rus titers using two step filter-adsorption elution. However, Sobsey (1979) reported that the above method recovered only <1% to 25% of low numbers of nine enteroviruses, each added to 100 gallons of tap water. Concentration of Other Enteroviruses from Seawater Using theNFDM Technique The concentration of six enterovirus (poliovirus type 1,2, and 3, coxsackie B3, ECHO 1, and ECHO 4) by the NFDM technique is shown in Table 13. It is evi dent that thi s method is effi cient for the recovery of the' three types of pol iovirus as well as coxsackie B3; overall recoveries for the above four vi ruses ranged from 59% to 75%. Us i ng NFDM, 31 % avera n recovery of ECHO 4 was obtained, but only 8% overall recovery was obtained using ECHO 1. The poor performance of the NFDM technique with regard to ECUO 1 recovery effi ciency is mainly due to the inabi 1 ity to concentrate this virus on membrane filters and casein flocs. Other researchers working in this area have also encountered recovery problems regarding ECHO 1. Charles (1979) reported that ECHO 1 was poorly adsorbed to a sandy soil and to i soe 1 ectri c case; nand NFDM flocs as compa red to. pol i ovi rus type 1, coxsackie B3, and ECHO 4. ECHO 1 was found' to adsorb poorly to Filterite filters and lagoon sludge solids as compared to poliovirus type 1 (Pancor!=o et al., unpublished observation). Goyal and Melnick (1978) observed that ECHO 1 was adsorbed significantly less to soils than poliovirus types 1, and 3, .c()xsackie B3, and other ECHO virus types and suggest the presence of intratypic differences in adsorptive potential between ECHO virus isolated strai ns.

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-30Table 13: Concentration of six enteroviruses from seawater by the non-fat dry milk (NFDM) technique. Virus(a) Polio 1 (b) Polio 2 Polio 3 Coxsackie ECHO 1 ECHO 4 83 Virus input PFU (x 106 ) 31.6-69.8 29.9-30.9 61.1-69.6 0.46 17.0 0.8-1.6 Percent elution 71 76 111 87 78 72 Organic floccul ation percent recovery 100 78 72 75 10 43 Percent avera 11 recovery 71 59 75 65 8 31 (a) The various viruses, suspended in 1-3 liters of seawater, were adsorbed to a seri es (3.0 lIm -+ 0.45 lIm -+ 0.45 lIm) of Fi lted te fi 1 ters in the presence of 0.0005 M Ale13 at pH = 3.5. The filters were eluted with 1% NFDM in 0.05 M glycine, pH = 9.0. Filter eluates were brought to pH = 4.5-4.6 with 1 M glycine (pH = 2.0) and the resultant floc centrifuged at 4500 RPM for 4 minutes. The pellets were then resuspended in 0.15 M Na2HP04' pH = 9.0. Each number represents the mean of duplicate trials. (b) The polio 1 trials used seawater samples of 20 gallons (76 liters} pro cessed through 0.45 lIm Filterite cartridge ather trials used 1-3 liters seawater samples and were processed as described above. WE' wz=t e' =eo

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I l -31VIRUS RECOVERY FROM MARINE SEDIMENTS Prior to evaluating the ability of various eluents to desorb virus from marine sediments, experiments were undertaken to study the sorption ability of the sediments toward viruses. Table 14 shows that the sandy sediment sampled at Crescent Beach, Fla. retained 99% of added viruses. The Intercoastal Waterway sediment adsorbed 100% of added virus (only two samples were run for this sediment). Gerba et a1., (1977) found similar results with estuarine sediments from Galveston Bay, Texas. Ten eluents were investigated for their ability to desorb viruses from' the Crescent Beach sediment. Three to six replicates were run for each eluent. It was observed that virus elution from the sediment surface was generally relatively low and ranged from <1% to 43.9% (Table 15). Urea lysine and TCA-glycine were found to be the most efficient among all the eluents tested. A surprising was the low recovery %) obtained with 0.25 1'-1 glycine + 0.05 M EDTApH = 11.0. Similar results were obtained with the Intercoastal Waterway sediment (Table 16). These findings do not agree with those of Gerba et al. (1977) who reported that glycine-EDTA eluted more than 50% of adsorbed viruses. In our study we made sure that the viruses were exposed to the high pH for no more than 15 minutes. Urea-' ,lysine, which eluted 43.9% of poliovirus, has been successfully used for virus elution from sludge (Farrah et a1., submitted for publication), and from positively charged microporous filters (Chang et al., unpublished). We have (Chang et al., unpubl ished; Farrah et al., submitted for publication). a two-step concentration scheme for urea-lysine eluates. This method led to a 50% virus recovery from these eluates. Addition of distilled water (Table 15) to the marine sediment resulted in a poor virus recovery (1.6%) and this agrees with the results reported by Gerba et al. (1977). The use of 2% isoelectric casein resulted in a 35% recovery while less than 10% recovery was achieved with 3% beef extract (Table 15). It was therefore decided to use 4M urea + 0.05M lysine (pH 9.0) for the detection of indigenous viruses in estuarine sediments. i I I i

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-32-Table 14: Adsorption of Poliovirus type 1 to a marine sandy sedimenta Run Total Virus Imput (PFU) Total PFU % Virus retained by adsorbed to sediment sediment 1 8.8 x 106 8.74 x 10 6 99.3 2 6 2.0 x 10 1 .98 x 10 6 99.4 3 1.9 x 106 1 .87 x 10 6 98.9 4 2.6 x 10 6 2.56 x 106 98.7 5 2.0 xll06 1 .98 x 10 6 99.2 ------.--(a) 20 ml of seawater seeded with poliovirus type 1 were added to 109 of sediment. (From Crescent Beach, Fla.). The mixtures were shaken for 30 mi nand centri fuged at 4000 RPM for 10 mi n. The supernatants were assayed to determine the % virus adsorbed to the sediment. ) -: ;,-:-.-=::, ..

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-33Table 15: Elution of Poliovirus Type 1 from a Marine Sandy Scdiment a Eluent 4t<1 Urea + 0.05M Lysine (pH = 9.0) 0.6M TCA + 0.02M Glycine (pH= 9.0) 2% Isoelectric Casein (pH = 9.0) 1% Isoelectric Casein (pH = 9.0) .1% Non-Fat Dry Milk (pH = 9.0) Humic Substances (color units = 5,700; pH = 9.0) 0.3% Na Pyrophosphate (pH = 9.0) 3% Beef Extract (pH = 9.0) 10% in Phosphate Buffered Saline (pH = 9.0) Distilled Water 0.25M Glycine + 0.05M EOTA (pH=9.0) % 61ution Mean + S.D 43.9 + 14.3 41.3 + 1.3 35.0 + 23.4 + 6.0 16.0 + 3.8 11.3 + 0.6 11 .2 + 2.7 9.3 + 2.9 6.9+"1.1 1 .6 + 0.2 < 1% (a) Thirty ml of eluent were added to 109 of sedimenf.The samples were vortexed for 30s and then shaken for 30 min (the shaking time was only 4.5 min and 1 min for glycine-EDTA and urea-lysine, re. specttvely). The samples were centrifuged at 4000 RPM for 4 min and the supernatants were assayed for viruses. (b) Mean of 3 to 6 repl icates. 27T'"FM

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-34-Table. 16: Recovery of Poliovirus Type 1 from Intercoastal Water. way Sediment. Eluent % Reconcentration % E1 ution step Overall % Recovery Recovery 3% Beef Extract (pH = 9.0) 11 87 9.6 0.25M Glyci ne + O.05M EOTA (pH = 11.5) <1 <1 <1 ., ---.------. -.. --

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1 -35RECOVERY OF INDIGENOUS ENTEROVIRUSES FRm1 SEAHATER AND i1ARINE Virus Recovery from Tampa Bay. In February, 1979, a trip was made to the City of Tampa Sewage Treatment Plant for the purpose of testing the efficacy of the NFDM technique in the recovery of na tura 11y occurri ng enterovi ruses (Table 17) Fifty gallons (189 liters) of unchlorinated secondary treated effluent were processed by membrane filtration using a series (3.0 + 0.45 of Filterite cartridge filters followed by NFDM elution and organic flocculation to yield a final concentrate of 22.7 ml and 27.8 ml for the two filters, respectively, as each filter was eluted separately. The final concentrates were assayed following chlorofonn treatment to remove bacterial and fungal contami nat'i on. Concentrates from both the prefil ter and fil ter were positi ve with regard to enteroviruses (Table 17). No attempts were made to quantify viruses since the method was designed specifically for marine waters. Chlorinated tertiary effluent was collected immediately prior to discharge into Tampa Bay after approximately 10 minutes contact time with about 3 ppm available chlorine. According to the sewage treatment plant operator the concentration of available chlorine should have only been 1 ppm (personal cOfl1lllun.ication). This latter fact implies that the excess chlorine addition was due to either human error or a desire to produce high quality water with respect to the presence of viruses. No virus was detected in a sample of chlorinated tertiary effluent. Similarly no virus was detected in Tampa Bay water (Table 17). Detection of indiqenous enteroviruses in estuarine water and sediment in St. Augustine, Fla. Indigenous enteroviruses were monitored at two stations (Salt Run and Matanzas River) in St. Augustine, Fla. (See Figure 1). Estuarine water was processed according to the non-fat dry milk method which was discussed pre viously. Virus detection in sediments was performed according to the urealysine method. The results are shown in Table 18. In June 1980 no virus WilS detected in Salt Run in both water and sediment. In Matanzas (near the Bridge of Lions in St. Augustine, Fla.) viruses were detected in both estuarine water and sediments. The virus concentrations found were 207 TCID50/50gaJlonsof wafer 41.4 TCID /100g of wet sediment. Poliovirus type 1 and type 3 were identified in caRcentrates from water and sediment, respectively. Salt Run sediment was sampled again on September 1980 and enteroviruses were detected at a concentra ti on of 18 TCI D50/1 OOg of sedi ments Virus concentration 1n fvlatanzas River is within the range found by other investigators. Gerba et al. (1977) reported virus concentrations of 2-16 PFU/10 liters ;n coastal canals in Texas (2-16 PFU/10 liters is equivalent to 38-304 PFU/50 gallons). However, Edmond et al. (1978) found lower virus (21-42/100 gallons) in Hiami Beach marine outfall. Virus concentration in Matanzas River sediment is similar to that found by DeFlora et al. (1975) in Italy.

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I,' :'\ Table 17: Use of'the dry milk (NFDt) technique for the recovery of natut'al1y occurring entero virus from sewage treatment plant effluents and Tampa Bay water. Ha ter sample Secondary effluent (unchlorinated) Tertiary effluent (chlori nated) Tertiary effluent outfall, Tampa Bay Tampa Bay water, 30 yards from outfa 11 Volume processed (gallons) :,L 50 ;"i 100 <' SO 75 I;'j !l Final volume Volume assayed (ml) (ml) 27.S(a) 13.10 22.7(b) 15.20 23.4 5.60, 30.2 14.65 18.2 2.S0 (a) Volume of finaloncentrate from the elution of the 0.45 llm filter. ( Virus presence + + N.Dc N.D N .. D (b) Volume of final (c) Not detected. ncentrate obtained from the elution of the 3.0 llm pre-filter. Concentra ti on factor 3,743 16,154 10,000 15,604 ")."'" ..... ;'10 I ",*r' .. ,': 1,'-, 'ij', .. '.' ,:/,', I w 0'\ I

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! .1 -37Table 18: Detection of indigenous enteroviruses in estuarine water and sediment in St. Augustine, -Fla. (June 1980). Sampl e type Water (TCID50/50 gallons) Sediment (TCIOSO/1 OOg) (a) Not detected (b) Poliovirus type 1 (c) Poliovirus type 3 Salt Run Matanzas River (Near Bridge of lions) N.O(a) 207 (Pl)(b) N.D 41.4 (P3)(c) ,'<-.-

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-38CONCLUSIONS The method of beef extract elution and organic flocculation has recently been used for the concentration of poliovirus in tap water, seawater, waste water effluent, and anaerobic sludge. This study has essentially shown that enleroviruses (poliovirus types 1, 2, and 3, coxsackie B3, and ECHO 1 and 4) can be recovered from seawater or tap water by elution and organic flocculation using 1% NFDM in 0.05 M glycine. Elution with NFDM is most efficient at pH=9.0 and this is an advantage methods '.',h"j ch use 91 yci ne buffer at pH=ll. 5 due to the vi ruc; da 1 effect (Jf such a pH, Flution and organic flocculation with technical casein, purified casein, isoelectric casein, NFDM, or beef extract resulted in high overall recoveries (Sl% to 83%) of poliovirus type 1 from seawater. Results obtained here are in agreement with those reported by Katzenelson (1977), pertaining to the recovery of poliovirus from seawater using the beef extract method. High overall recoveries (72% to 81%) of both high and low quantities of poliovirus type 1 were recovered from tap water using the NFDM technique. The use of the NFDM technique resulted in the efficient elution of (71% to >100%) of six enterovirus from membrane filters. Problems were encountered during the organic flocculation step where low recoveries of ECHO 1 and 4 (10% and 43%, respectively) were observed. Of the ten eluents investigated for desorption of viruses ft'om marine sediments, 4M urea-0.05M lysine was found to be the most efficient. The methods develop in the course of this study were successful in the recovery of indigenous enteroviruses in estuarine water and sediment.

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1 1 '. -39-REFERENCES Berg, G. 1967. Transmission of Viruses by the Water Route. Wiley Interscience, N.Y. Bitton, G. 1978. Survival of enteric viruses. IN: Water Pollution Microbiology, Vol. 2, R. Mitchell, ed., Wiley Interscience, N.Y. nitton, G. 1980a. Adsorption of viruses onto surfaces: Technological and ecological implications. IN: Adsorption of Microorganisms to Surfaces, G. Bitton and K.C. Marshall, eds., Wiley Interscience, N.Y. Bitton, G. 1980b. Introduction to Environmental Virology. Wiley, N.Y. 326 p. Charles, M.J. 1979. Development of methods for virus recovery from soils. Master'.s Thesis, University of Florida. Gainesville, Fla. De Flora, S., De Renzi, G.P., and Badolati, G. (1975).' Detection of animal viruses in coastal seawater and sediments. App1. Microbial. 30: 472-475. Edmond, T.D., Schaiberger, G.E., and Gerba, C.P. (1978). Detection of enteroviruses near deep marine sewage outfalls. Marine Pollute null. 9: 246-249. Farrah, S.R., C.P. Gerba, C. Wallis, and J.L. Melnick. 1976. Concentration of viruses from large volumes of tap water using pleated membrane filters. Appl. Environ. Microbiol. R: 221-226. Farrah, S.R., S.M. Goyal, C.P. Gerba, C. Wallis, and J.L. Melnick. 1977. Concentration of enteroviruses from estuarine water. Appl. Environ. 33: 1192-1196. Fields, H.A. and T.G. Metcalf. 1975. Concentration of adenovirus from seawater. Water Res. 9: 357-364. Gerba, C.P., S.R. Farrah, S.M. Goyal, C. Wallis and J.L. Melnick. 1978. Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater. Appl.Environ. Microbiol Gerba, C.P., Goyal, S.M., Smith, E.M., and J.L. (1978). Distribution of viral and bacterial pathogens in al coastal canal community. Marine Pollute Bull. 8: 279-282. Gerba, C.P., and McLeod, J.S. (1976). Effect of sediments on the survival Escherichia coli in marine waters. Appl. Environ. Microbiol. 32: 114-120. j 1 I 1 I I

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