IDENTIFICATION OF ENTERIC VIRUSES AND
SALMONELLAE FROM SEWAGE

B7

Henry Harold Bloom

A THESIS
Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1958

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ABSTRACT

The utilization of improved tissue culture techniques has led
to the finding of many viral agents in the excreta of man and animal.
A new group of viruses, enteric cytopathogenic human orphan (Echo),
has been isolated in tissue culture, along with the polio and some
of the Coxsackie viruses.

Some of the Echo viruses have been in­

criminated as the etiological agents for a number of diseases, in­
cluding aseptic meningitis, summer diarrhea of infants and mild para­
lysis.

The ease of detecting these enteric viruses in sewage would

greatly aid in epidemiological investigations.
Along with the viruses, salmonellae are also present in sewage.
New techniques, such as the gauze pad sample and new media, offer the
prospect of better evaluation of these organisms in sewage.
When it was determined that tetrathionate enrichment broth was
not ideally suited for the isolation of salmonellae from sewage (32
of 1U2 samples positive), selenite brilliant green enrichment medium
was used.

The isolations of salmonellae increased dramatically (62

of 92 samples positive) with the employment of SBG medium.
A total of 1018 sewage samples were tested in monkey kidney
tissue culture and/or suckling mice to detect enteric viruses.
agents were isolated on 186 occasions.

Viral

Thirty-five of the isolates

were identified as Echo or polioviruses, 76 were identified as
Coxsackie viruses, 36 samples were positive both in tissue culture and
in suckling mice, an additional 17 viruses were not identified, and 22
tissue culture cytopathogenic agents were lost in passage or storage.
Echo 1 and/or 13 was identified on 10 occasions and was the Echo

virus most often isolated.
Samples were collected from 7 plant locations to determine the
effect of sewage treatment on the viability of viruses and salmonellae.
Enteric viruses and salmonellae were found in all stages of sewage
treatment tested, including final effluent.
Sewage samples provide a source of material from which the
enteric viral flora of a population may be determined.

In addition,

the prevalence of salmonellae in a community may be estimated by the
testing of appropriate sewage samples.

TABLE OF CONTENTS
PAGE
INTRODUCTION .........................................

1

REVIEW OF THE LITERATURE.............................

2

MATERIALS AND M E T H O D S ................................. 17
Collection of S a m p l e s ............................... 17
Virus I s o l a t i o n ..................................... 18
Virus S t r a i n s ....................................
Mice

19

......................... • ................... 21

Tissue Culture...............
Preparation of Antisera

21
.............

23

Neutralization T e s t s ..............................

25>

Salmonella Isolations

27

RESULTS

.....................

....

............................................. 29

DISCUSSION............................................. E>2
S U M M A R Y ...............................................61
BIBLIOGRAPHY........................................... 65

LIST OF TABLES

TABLE
I#

PAGE
Echo Virus Strains Used in the Preparation of
Antisera..................... . • . . • ........

II.

Serum Neutralization Titers Against Echo Prototype
Strains Prepared in Rabbits

III.

•

2h

Viruses Isolated from Sewage by Tissue Culture and
Mouse Inoculations

IV.

20

31

Virus Isolations in Suckling Mice from Sewage
Specimens

.............. . . .

32

V.

Virus Isolations in Tissue Culture from Sewage . . .

3h

VI.

Identity of Viruses Isolated in Tissue Culture . . .

36

VII.

• • • . . • • • •

Antigenic Types and Some Biologic Characteristics
of Viruses Isolated in Tissue Culture from Sewage
Samples

VIII.

36

Viruses Isolated in Tissue Culture not Identifiable
as Echo Viruses 1-lii, Poliovirus Types I, II, or
III or Coxsackie Viruses

IX.

.........

UO

Duplicate Isolations of Cytopathogenic Agents in
Tissue Culture and Coxsackie Viruses in Suckling
Mice

X.

.........

Tissue Culture Cytopathogenic Agents Recovered from
Sewage but Lost on Subsequent Passage or Storage .

XI.

1|2

U3

Isolation of Enteric Viruses from Various Locations
in a Sewage P l a n t ............ • • • • .........

bS

LIST OF TABLES (continued)

TABLE
XII*

PAGE
Comparison of Tetrathionate Broth and Selenite
Brilliant Green Media in the Isolation of
Salmonella from Sewage •

XIII.

............. • • • . •

Isolation of Salmonella from Gauze Pads and Raw
Sludge According to Location • • • • • • . . » • •

XIV.

U6

Isolation of Salmonella from Gauze Pads Located at
Various Points in a Treatment Plant

XV.

It7

• • • • • • •

50

Virus Isolations from Sewage Specimens as Compared
to Isolations Obtained from Stool Specimens

...

53

ACKNOWLEDGEMENTS

Appreciation is expressed to the Surgeon General of the United
States Navy and to all other members of the Medical Department who
made possible this tour of duty at Michigan State University.

My

special thanks to Captain S. A. Britten, M.C., USN for his efforts
in my behalf.
I shall always think highly of Dr. ¥. N. Mack for his guidance
and his friendship during the course of this study and will try to
duplicate his high standards of personal and professional principles.
This work could not have been accomplished without the able
assistance of Mrs. Russell Krueger and Miss Mary E. Fields.
Dr. W. L. Mallmann has been most generous with his assistance
in the Salmonella work and will also be remembered for his personal
kindness.

INTRODUCTION

The isolation of viral agents has become more commonplace since
the refinement of tissue culture methods, within the past ten years.
Epidemiological studies of the viral flora of selected groups have
been made possible through these new techniques.
The purpose of this study was to identify a number of viruses
that were isolated from samples collected at two sewage plants.

It

was necessary to limit the study to a particular group of viruses
since sewage represents such a heterogeneous mixture of human and
animal waste products, that may contain a variety of viral entities.
The enteric virus group was selected as the focal point of the study.
This group consists of the polioviruses, Coxsackie viruses and the
Echo viruses (enteric cytopathogenic human orphan).

In addition to

the virus identification, attempts were made to isolate salmonellae
from similar types of sewage specimens*

Sewage can serve as a source

for epidemiological information in attempting to determine the
presence of disease-producing bacteria and viruses in a community
at any given time, since mass rectal swabbing is impractical.

Infor­

mation was also sought on the effect sewage disposal treatment had
in removing the viruses and salmonellae present in the sewage prior
to discharge from the plant.

2
REVIEW OF THE LITERATURE

Poliovirus was first isolated from sewage by Paul et al., (1939),
although the virus had been isolated from stool samples by Sawyer
(1915) and Harmon (1937).

A series of three papers followed in which

the recovery of poliovirus from stools and from sewage was discussed.
Trask et al., (19U0) reported that the ease of detection of the
virus was related directly to the non-paralytic type of poliomyelitis
and inversely to age.

An important contribution made in this paper

was that at least one infective monkey dose per gram could be
extracted from one stool, indicating that relatively large quantities
of virus were present in stool specimens from patients.

The method

of extracting the virus from the stools was relatively simple.

The

stools were diluted 5 to 10 times by weight in cold distilled water
and after mixing were allowed to settle 2 to 10 hours in the cold.
A portion was shaken with 10 per cent ether by volume and was again
held overnight in a refrigerator.

The supernatant fluid was inocu­

lated into monkeys after centrifugation at 1000 rpm for 25 minutes.
The second paper by Paul et al., (19U0) was an extension of the
work presented in the first paper.

Since they had shown that polio­

virus was present in stools of patients, they were interested in
detecting the virus in the sewage from the hospital.

During two of

three large urban epidemics of poliomyelitis the virus was isolated
from samples of sewage.

To avoid the dilution effect of the sewage

system, the samples were collected in the irranediate vicinity of the
isolation hospitals.

3
The third paper in the series dealt with methods of detecting
the virus in sewage and stools,

Gard (19U0) described the precipi­

tation of the virus from the specimens with £0 per cent ammonium
sulphate followed by dialysing the precipitate.

Basically, this

method was employed by Melnick (19U7) with the addition of an ultra­
centrifugation procedure in an effort to concentrate further the
virus.

After this basic knowledge of poliovirus was presented,

several new thoughts on the pathogenesis of poliomyelitis were
brought forth in the literature.

Paul and Trask (19h2) placed polio­

virus among the intestinal diseases since the virus could be re­
covered from the feces of a convalescent patient for as long as two
or three weeks following a severe or mild attack of the disease.

To

demonstrate the foresight of these workers a quotation is taken from
the paper: "From the epidemiological standpoint, from the engineering
standpoint, and from the public health standpoint, the intestinal
tract seems like a dangerous place for this virus to be."

Further

recoveries of the poliovirus from sewage followed, including one
from New York City reported by Trask and Paul (19U2).

The largest

single obstacle in the path of these experimental procedures was
the absolute dependence upon the monkey as a test animal.

The very

high cost of using this animal was instrumental in the appearance of
reports based on a single positive isolation from relatively few
samples.

The earlier work of Paul and Trask was later confirmed by

Kling et al«> (19U2) and still later by Melnick (19U7).

Horstmann

and Melnick (19U6) also reinforced the findings that poliovirus
persisted in the stools after acute infections.

h
The Coxsackie virus group was first described by lalldorf (19U8)
and since the isolation of the virus depended on the utilization of
suckling mice rather than the more expensive monkey, attempts were
made immediately to isolate this group of viruses from sewage•
Melnick et al., (19lt9) recovered the virus from sewage in six differ­
ent cities during the summer and fall of 19U8.
reported during the winter months.

No isolations were

Clark et al., (19^1) recovered

the virus from sewage in Canadian cities during a summer season when
there was a low incidence of reported poliomyelitis cases.

In an

attempt to improve the methods of isolation Kelly (19^3) described
a method which consisted of virus adsorption on, and elution from,
ion-exchange resins.
Aside from the difficulty involved in isolating the virus from
the sample per se, an additional problem further complicates the
procedure.
ation.

The actual sampling method must be taken into consider­

A dip sample, of approximately 100 ml, normally taken for

routine physical, chemical and standard biological tests, is at best
a random sample of the contents of the flow through a sewage system
and there is a good chance that particular viruses or bacteria are
not present in a given sample.

To increase the probability of

collecting samples containing virus, larger quantities of sewage were
collected and processed.
gallons.

Melnick (19U7) used samples of two to three

The use of a gauze pad sampling procedure was described by

Moore (19^8) for the detection of the typhoid bacillus in sewage.

A

detailed description of this method is presented in the materials and
methods section of this thesis.

Kelly (1953) employed this sampling

5
method for monitoring sewage over a 2h to U8 hour period and reported
that the number of Coxsackie isolations was increased consistently
over the isolations obtained using the dip samples.

Melnick et al.,

(195U) also reported that the gauze pad method of obtaining sewage
samples increased the number of Coxsackie isolations considerably
over the dip sample method of collection.
Since the gauze pad was suspended in the sewage flow for long
periods of time (up to seven days), the viability of the viruses in
this environment was of considerable importance.

The work of Rhodes

et al., (1950) revealed that poliovirus survived, in experimentally
contaminated river water, at least 188 days when the stored water was
kept at U C and the stool diluted 1 to 200.

In a second study,

(Kelly et al., 1955) the seasonal distribution of Coxsackie virus in
the sewage of Albany, Mew York, area was determined.

Coxsackie

viruses were present continuously between June and November and only
sporadically during the remainder of the year.

The effect of sewage

plant treatment on the viability of Coxsackie virus was also investi­
gated.

Coxsackie viruses were recovered regularly from the effluent

as well as from the influent of the plant during the summer and fall,
and therefore, sewage does not appear to affect enteric viruses to
any great extent.
With the advent of the wide-spread use of roller-tube tissue
culture technique as applied to the isolation and identification of
poiioviruses by Robbins et al., (1952), it became practicable, from
an economic standpoint, to conduct extensive clinical and epidemio­
logical investigations of poliomyelitis.

Tissue culture techniques

were refined to such an extent that other viruses besides polioviruses were isolated and recognized from fecal material.

A number

of types of Coxsackie viruses were isolated and identified in the
HeLa strain of human cancer cells by Sickels et al., (1955) and
Crowell and Syverton (1955) as well as other viruses which are
occasionally found in the feces, such as herpes simplex (Weller,
1953), mumps (Henle et al., 195U) and the adenoviruses (Rowe et al.,
1957).

At times, more than one virus was found in a stool specimen

(Kibrick, 1955).

With the development of a procedure by Dulbecco

and Vogt (195U) for the preparation of cell suspensions of monkey
kidney by the use of a trypsinization procedure, an important step
in tissue culture methodology was achieved.

This technique, further

improved by Younger (1951*), provided a method for the preparation of
a monolayer of cells grown on any glass surface in such containers
as Petri dishes, test tubes or bottles held in a stationary position
Comparative studies by Melnick et al., (195U) demonstrated that
epithelial monolayer cultures from monkey kidneys are more sensitive
for primary isolation of poliovirus than suspended fragments of
kidney tissue or of fibroblast cultures from monkey testis.

Wenner

and Miller (195U) also reported that for primary isolation of polio­
virus, monkey kidney epithelial cells proved superior to testicular
cultures and also to HeLa cultures with which they were compared.
Melnick (1955a) has shown that poliovirus can be isolated at
about twice the frequency from stools as from rectal swabs.

While

many individuals yielded positive stools but negative swabs, the
reverse situation was a rare occurrence. During tissue culture

7
investigations using stools from suspected poliomyelitis patients
viruses were isolated that were not identifiable as poliovirus or
Coxsackie virus by serological or suckling mice virulence tests.
Melnick (1955b) tentatively placed them in a group which he called
"orphan viruses," since there was so little known about their re­
lationship to disease.

These agents have been isolated in the United

States and in other countries.

In this country, Robbins et al.,

(1951), Riordan et al., (1952), Kibrick et al., (1953), Melnick et
al., (1953) and Steigman et al., (195U) have all isolated a number of
these viruses, chiefly from the stools of patients with aseptic
meningitis or non-paralytic poliomyelitis.

In Egypt they were iso­

lated from the blood, throat and rectal swabs of young children
brought to a public clinic with various non-differentiated clinical
symptoms (Horstmarm, 1955)*

Melnick and Agren (1952) also isolated

an "orphan" virus from the stool of a normal Egyptian child.
A study was undertaken by Ramos-Alvarez and Sabin (195U) to
determine if avirulent poliomyelitis viruses occurred in nature.
Rectal swabs were taken of 1566 healthy children in Ohio.

In

addition to five strains of poliomyelitis virus and one strain of
Coxsackie virus that were isolated, 25 other enteric viruses were
found.

Twenty-two of these viruses were unrelated to three serologic

types previously isolated by other workers.
It can readily be seen that a problem existed in classifying
these "orphan viruses."

In an attempt to solve this dilemma, the

National Foundation for Infantile Paralysis formed a committee to
help resolve this matter of nomenclature.

The committeefs title was

8
the "Committee on the ECHO Viruses” (Enteric Cytopathogenic Human
Orphan Viruses).

The first report of the committee (1955) contained

a list of the 13 strains that were designated prototype strains of
the Echo group of viruses.

These strains did not reveal any high

degree of cross-reactions among one another and were considered to
be separate and distinct viral entities.
An additional 1771 stool specimens were obtained from healthy
children in Mexico by Ramos-Alvarez and Sabin (1956) and 26l of the
samples were positive for viruses other than poliovirus and Coxsackie
virus.

Cross-neutralization tests between the strains isolated from

healthy children in Ohio and healthy children in Mexico revealed
that Echo 7 was not present in any of the Mexican isolates, while it
was present in 32 per cent of the strains isolated in Ohio.

Echo 2,

with an incidence of hh per cent in Ohio, constituted only eight per
cent of the Mexican strains.

Approximately 90 per cent of the

Mexican strains were not neutralized by Echo strains 1 to 13.
Gelfand et al., (1957) studied a group of 150 young children
in Louisiana during a two-year sampling period in an attempt to
determine their normal enteric viral flora over a fairly long period
of time.

Stool specimens were obtained monthly and viral isolation

was accomplished in monkey kidney cell cultures.

A marked seasonal

effect, with a predominance of positive specimens in the summer and
fall months was observed, with parallelism between the polio and non­
polio viruses.
Ormsbee and Melnick (1957) classified 30 Echo viruses from 1558
fecal samples taken during a 29-month period, from 136 children of

9
preschool age in Charleston, West Virginia.

Twenty of the 30 were

typed Echo 3, 6, 7> 9* 11, lU, or a new type, designated Echo 15.
A previous paper by Honig and Melnick (1956) on this same group of
children reported that there was a repeatable seasonal incidence
noted in the Echo virus isolations, with over 90 per cent isolated
from June to October.
The Committee on the Echo viruses, in its second report (1957),
stated in part: '‘recognizing that the poliomyelitis, Coxsackie and
ECHO viruses all inhabit the alimentary tract as well as share other
properties, has changed the name of its Committee on the ECHO Viruses
to the Committee on the Enteroviruses.”

Six new antigenically

distinct members of the Echo group were listed; Echo li; to 19.

Since

the publication of the Committe's first report (1955), it was dis­
covered that the Echo 13 prototype strain (Hamphill) actually was a
mixture of Echo 1 and Echo 13 strains, or was serologically related
to Echo 1.
With the refinement in tissue culture methods Kelly et al.,
(1957) were able to approach the problem of enteric viruses in sewage
on an expanded scale.

In addition to the Coxsackie viruses which she

and her group searched for in 1955, an attempt was made, using
suckling mice and tissue culture, to detect other enteric viruses
which might be present in sewage.

Three hundred and eight sewage

samples were tested for pathogenic agents in newborn mice.

Two

hundred and eight of them were tested for cytopathogenic agents in
HeLa cell cultures which were described by Scherer et al., (1953)
and the remaining 100 in monkey kidney epithelium cultures.

10
Coxsackie viruses were isolated on 119 occasions, polioviruses were
isolated 97 times and unknown agents were encountered on five occasions.
The authors reported that they did not observe great differences in
sensitivity between HeLa cell and monkey kidney cell cultures.
To isolate and classify these Echo viruses was in itself a sig­
nificant advancement, a task that is continuing.

However, until it

can be ascertained to what extent these viruses function as disease
producing agents, much work remains.

The Committee on Enteroviruses

(1957) published a summary of available information showing the associ­
ation of enteroviruses with human disease and it is presented here:

Enteroviruses

Associated Disease

Poliovirus

Paralysis (complete to slight muscle weakness)
Aseptic meningitis
Undifferentiated febrile illness particularly
during the summer

Coxsackie viruses,
Group A

Herpangina
Undifferentiated febrile illness particularly
during the summer
Aseptic meningitis (Types A7, A9)

Coxsackie viruses,
Group B

Aseptic meningitis
Pleurodynia
Undifferentiated febrile illness with
pharyngitis
Myocarditis or encephalomyocarditis during
neonatal period and early childhood
Mild paralysis (?)

Echo viruses

Aseptic meningitis (Types 2, 3, U, 5, 6, lh,
16)
Summer rash (Types h, 9, 16)
Summer febrile illness
Mild paralysis (?) (Type 6)
Summer diarrhea of infants and children (Type
18 and others)

11
According to Rivers (19U6), the clinical picture of polio­
myelitis is one of a biphasic nature, with both phases not seen in
all cases.

Phase one may simulate a mild upper respiratory infection,

with headache or a nonexudative pharyngitis, or it can be ushered in
by what appears to be a simple gastro-intestinal disturbance with
nausea and vomiting.

The second phase involves a disturbance of the

central nervous system (CNS) and may appear immediately following
phase one or several days after the remission of phase one symptoms.
The symptoms seen at the onset

of CNS disturbances are: muscle

tenderness and stiffness, rigidity of the neck and back with pain
when an attempt is made to place the chin on the chest.

Lumbar

puncture reveals an abnormal number of granulocytes and lymphocytes
in the cerebrospinal fluid (CSF)• Later when paralysis is apparent,
a mononuclear pleocytosis is nearly always present, ranging from 15>
to 200 cells per cu. mm.

Increased protein is usually demonstrable

in the CSF and persists for some weeks after the cell count has
returned to normal.

Paralysis usually reaches its maximum extent

before the fifth day following its apoearance.
The aseptic meningitis syndrome has been described by Kibrick
(1957)*

The most prominent clinical features of this disease

complex are an abrupt onset in most of the cases characterized by
headache, stiff neck, stiff back and vomiting.

Sore throat, abdomi­

nal pain and myalgia were reported from a smaller number of patients.
The majority of the patients were febrile after admission and the
temperature remained elevated from one to four days, ranging from
100 to 102 F.

In the CSF the number of cells rarely exceeded 500

12
per cu. ram. and the levels of protein were normal or slightly ele­
vated.

Muscle weakness was consistently of a mild or moderate degree

and in most instances tended to disappear with the passage of time.
Karzon (1957) reported a clinical syndrome, resembling aseptic
meningitis, occurring in epidemic form in western New York State
during the summer of 1955.

The features of this disease, from which

Echo 6 was isolated from 75 of the 8U hospitalized patients, included
an acute onset with severe frontal headache, nausea and vomiting,
pains in the abdomen, limbs or chest, and signs of meningeal irrita­
tion.

Transient muscle weakness, especially of the anterior neck

muscles, occurred in some of the cases, but there was no residual
paralysis.

It was of interest to note that Echo 6 virus was iso­

lated from the CSF from seven of the patients.
Svedmyr (1957) claimed that the etiological role of Echo 6
virus in aseptic meningitis appeared to be quite evident in the
material studied during an epidemic in Sweden.

In a review on the

etiologic role of Coxsackie B and Echo viruses in meningitis, Rhodes
and Beale (1957) cite numerous references on the isolation of these
viruses from feces and CSF of patients clinically classified as
showing the aseptic meningitis syndrome.
Lehan et al., (1957) isolated Echo

h

virus

from stools of

aseptic meningitis patients during an epidemic in Iowa.

A signifi­

cant rise in titer against Echo U virus was reported in the majority
of the paired serum specimens collected.
A report, by Nihoul, (195?), incriminated Echo 9 virus as the
agent responsible for an outbreak of aseptic meningitis in Belgium.

13
Echo 9 virus was isolated from 28 CSF samples and 122 stool specimens.
From the previous information it may be concluded that the
aseptic meningitis syndrome may be caused by various viral agents
that produce a similar clinical appearance.
Group A Coxsackie viruses have been associated with herpangina.
Herpangina, is mainly a disease of children occurring in the summer
months.

It is characterized by fever, pharyngitis and small ulcer­

ative lesions on the base of the tongue and the tonsils (Parrott,
1957).
Group B Coxsackie viruses are recognized as the important
etiological agents of pleurodynia or Bornholm disease, which was
first clearly described by Sylvest (1932).

This condition is

characterized by fever, severe attacks of muscle oafn, usually
affecting the lower thorax and abdomen, and pharyngitis.

Lazarus et

al., (1952) described an outbreak of pleurodynia in Washington in
1950 from which Coxsackie B virus was isolated and incriminated as
the etiological agent.
Enders (1957) has reviewed the findings of a number of workers
concerning acute aseptic myocarditis of infants.

This disease has

been found in young infants and clinical symptoms are those of
anorexia, vomiting, cyanosis and dyspnea, together with a tachy­
cardia.

In fatal cases, the heart was dilatated and sometimes

hypertrophic.

No association with pathogenic bacteria could be

demonstrated.

Coxsackie B virus has been isolated on at least two

occasions from the myocardial tissue of infants, diagnosed as acute
aseptic myocarditis cases.

1U
A study by Ramos-Alvarez (1957) included stool samples from 100
infants and children, up to the age of four years, coming to a
clinic for the treatment of diarrhea.

His findings indicated that

various types of Echo viruses were associated significantly with the
disease.

Since these fecal specimens were collected only during the

summer months this syndrome has been termed "summer diarrhea."
Since many of the "orphan" viruses have been suspected as the
etiological agents for various diseases and doubtless many others
will be thought to be the causes of other maladies, a set of guide­
lines has been offered by Huebner (1957).

"In order that a virus

be regarded as an established cause of a specific human illness, the
following conditions seem to be necessary:
1.
2.
3.
U.
5.
6.
7.
8.
9.

Virus as "real" entity.
Origin of virus.
Antibody response.
Characterization and comparison with known agents.
Constant association with known illness.
Studies with human volunteers.
Epidemiologic studies.
Prevention by specific vaccination.
Financial support."

A review of the literature on the occurrence and survival of
salmonellae in sewage was made by Rudolfs et al., (1950).

One of the

highlights of this review article was Gray's (1929) seven isolations
of paratyphoid organisms from 20 samples of Edinburgh, Scotland,
sewage.

All isolations were from a district which had suffered an

outbreak of the disease two years previously.

More than a year

later, Begbie (1930) reported seven isolations of the organisms from
58 samples of Edinburgh sewage.

Bruns and Sierp (1927) noted that

Salmonella paratyphoid B was still found in experimentally inoculated

15
sludge samples even after prolonged aeration.

In a search for

typhi, Hajna (1935) found six samples positive out of 22 tested from
five different plants.

In the raw sludge, three out of seven samples

from two plants showed positive results.
positive results from nine samples tested.

Plant effluents showed no
The presence of salmonellae

in irrigation waters was reported by Dunlop et al., (1951).

Using

tetrathionate enrichment broth followed by streaking on bismuth
sulfite agar plates, 23 of 113 samples were positive for salmonellae
comprising 13 species.

Four of 21 one-ml samples, five of 12 ten-ml

samples and lh of 66 one-hundred-ml samples were also positive for
salmonellae.

In a later paper Dunlop et al., (1952) compared re­

coveries of Salmonella from naturally-contaminated irrigation water
with tetrathionate broth and with Galton's modification (1950).
Galton's modification consisted of the addition of sodium-sulfathiazole to Kauffmann's brilliant-green tetrathionate broth.

This

modified medium resulted in higher recovery rates than did the
regular tetrathionate broth.

In this study, the volume of irrigation

water sampled ranged from 333 ml to 3000 ml and the material was
concentrated ten-fold, using a continuous centrifuge, before inocu­
lation into the enrichment media.
Stokes and Osborne (1955) described a selenite brilliant green
(SBG) enrichment medium for the isolation of Salmonella. This liquid
primary enrichment medium was reported to be more effective than the
more commonly used selenite F broth.

In addition to preventing

excess growth of non-salmonella organisms, SBG medium supported
abundant growth of salmonellae from very small inocula.

Thirteen

16
smooth stock strains of Salmonella were tested by Kenner et al.,
(1937) utilizing SBG medium in conjunction with the membrane filter.
Recovery rates of kO to 60 per cent were obtained with the SBG medium
as compared to 10 per cent or less using various formulations of SS
medium (Salmonella-Shigella) with the membrane filter procedures.
Moore (1930) described a method of sampling sewage for salmonellae
over a long period of time, by using a gauze swab suspended in the
sewage.

This method was used by Moore (1931) for the detection of

typhoid carriers.

By suspending these gauze pads at various points

along a sewerage system, it was possible to locate the dwellings,
from which S. typhi had been excreted into the system.

Follow-up

rectal swab studies of the occupants revealed the carrier.

This

method has been used with success by Moore et al., (1932), Kelly et
al., (1933) and Greenberg et al., (1937).

It was observed by Kelly

(1933) that S. montevideo was isolated five times from the final
effluent of a sewage plant in New York State.

17
MATERIALS AND METHODS
Collection of Samples
From September 1955 through August 1957 biweekly samples of
sewage were collected from the East Lansing and Lansing sewage
plants.

During the latter part of August and the months of September

and October 1957 daily samples were obtained from seven sampling
points located in the East Lansing plant.
A pumping station, serving the northside and eastside sections
of Lansing, was the location from which samples representing the
Lansing area were obtained.

Lansing is a highly industrialized city

with a population of 92,129 (1950).

The flow of sewage through the

pumping station contained both residential and industrial wastes.
The sewage plant in East Lansing serves a population of 1*0,000
when the university is in session.

The final effluent was not

chlorinated when it was passed into the Red Cedar River.
Three types of sewage samoles were obtained from the East
Lansing plant.

The samples collected were 100 ml dip samples, gauze

pad samples and raw sludge samples.

The gauze pads were similar to

those described by Moore (1950) and consisted of adsorbent cotton
placed between two layers of cheese cloth, 1* inches by 1* inches
square, which were sewed together around the periphery.

The pads

were placed directly into the flow and remained in contact, at the
various sampling stations, from 2l* to 72 hours.

Approximately 150 ml

of raw sludge were collected from an outlet on a sludge pump and
represented a 2l* hour sample, as the raw sludge was kept in a holding
tank and pumped to the digestors once daily.

At the Lansing pumoing

13
station only dip samples and gauze pad samples were collected.
Specimens were processed in the laboratory within one hour of
collection in the case of salmonellae examination or were frozen and
held at -20 C until they could be processed for subsequent virus
isolation techniques.
Processing of the Samples:
Virus Isolation
For the isolation of viruses, the fluid was extracted from the
gauze pad samples by placing the pad in the barrel of a 100 cc
syringe and forcing the liquid through by exerting Dressure with the
plunger.

This was followed by washing the pad with approximately

1±0 ml of distilled water.

The liquid was then centrifuged at 2h00

rpm for 5 minutes to remove the larger particles.

The supernatant

fluid was placed in two 30 ml lusteroid ultracentrifuge tubes and
placed in a Rotor B for ultracentrifugation.

The samples were ultra-

centrifuged at U2,0U0 rpm (llU,6l0 g) for one hour in a Spinco Model
E Ultracentrifuge.

The sediment, in the

form of a pellet, was re­

suspended in 3 ml of supernatant fluid and the remainder was dis­
carded.

To the 3 ml, 1^00 units of penicillin per ml and h mg of

streptomycin per ml were added to eliminate bacteria.

The specimens

were then held for 2h to i±8 hours at 5 C before they were tested for
sterility by inoculating a loopful of the specimen into a tube of
brain heart infusion broth containing one per cent agar.

The

specimen was considered to be free of contaminating bacteria if the
appearance of the brain heart infusion broth indicated no bacterial
activity at the end of a 72 hour incubation period at 37 C.

If,

19
however, the specimen proved to be contaminated, an additional step
was included in an effort to prepare the specimen for tissue culture
inoculation.

The contaminated specimen was transferred to a 7 ml

Pyrex glass tube which was placed in an angle-head rotor and cen­
trifuged in an International Refrigerated Centrifuge, Model PR-1.
The specimen was centrifuged at 9,000 rpm for 30 minutes while the
temperature was kept at li C.

The supernatant fluid was removed and

3000 units of penicillin per ml and 12 mg of streptomycin per ml were
added.

The sediment was discarded.

A sterility test was made again

and the specimen was discarded if still found to be contaminated.
Virus was recovered from specimens receiving the second high speed
centrifugation, indicating that the method used in eliminating con­
taminating organisms was effective and did not destroy or remove the
virus.
The outlined procedures for the processing of the gauze pad
samples also apply to the dip and sludge samples, with the exception
that there was no need of the extraction procedure.

Virus Strains
The strains of Echo viruses used in the study are listed in
Table I.

The titers obtained in monkey kidney cultures are presented

along with those reported by Wenner et al., (19^5), Ormsbee and
Melnick (19^7), Microbiological Associates Inc. (195>7) and Stulberg
et al., (19^8) for comparison.

The titers, from the Michigan State

University laboratory, represent the average of three titrations.
With Echo viruses 3, U, 5, and lU, there appears to be a wide vari­
ation in titer among the three laboratories.

This range in titers

20
was not unexpected, since each shipment of tissue culture represented
the cells of a different monkey.

Slight variations in viral sus­

ceptibility within the same species can be expected.
Dr. C. S. Stulberg generously provided Echo types 7 through 13
and Echo types 1 - 6 and ll± were purchased from the American type
Culture Collection.

Polio Type I (Mahoney) and Polio Type III

(Saukett) strains were kindly supplied by the School of Public Health,
University of Michigan.

Polio Type II (Lansing) has been maintained

in this laboratory for a number of years.

The titers of these polio

strains, in monkey kidney cells, representing an average of three
titrations, were 10®*° TCI'50 per ml for Type I, 10^*^ TCB50 per ml for
Type II, and Type III was 1 0 * ^ TCD5>0 per ml.
TABLE I
ECHO VIRUS STRAINS USEL IN THE PREPARATION OF ANTISERA

Echo
virus
type

Prototype
strain

1
2
3
h
5
6
7
8
9
10
11
12
13
114

Farouk
Cornells
Morrisey
Pesascek
Noyce
D *Amori
Wallace
Bryson
Hill
Lang
Gregory
Travis
Hamphill
Tow

Source of
strain
ATCC**
ATCC
ATCC
ATCC
ATCC
ATCC
Stulberg
Stulberg
Stulberg
Stulberg
Stulberg
Stulberg
Stulberg
ATCC

______________ TDC50 per ml*_______ _ _
Micro.
MSB Stulberg Assoc. Archetti Ormsbee
(1958)
(1957)
(1955)
(1957)
6.8
6.3
5.8
6.5
6.5
8,b
8.U
6.b
6.8
5.6
7.1
8.2
7.1
6.7

9.2
8.8
8.2
9.5
9.5
8.8
8.6
7.6
6.5
6.5
7.7
8.5
8.U
9.U

7.0
6.1
6.2
5.8
7.7
8.3
8.2
6.5
7.U
6.2
6.9
8.3
6.7
5.7

7.7
5.8
7.5
li.5
8.8
8.U
8.5
7.8
7.2
7»k
7.0
8.8
8.1

^Expressed as the log of the TCD50.
^American Type Culture Collection, Washington, D. C.

7.3
6.5
6.0
5.8
6.5
6.8
6.5
6.5
5.8
6.3
7.3
7.3
7.3

21
Mice
A colony of Swiss strain mice was maintained to supply the
suckling mice necessary for the detection of Coxsackie viruses
present in the sewage specimens.
One to three-day old mice were used.

Six-hundredths ml of the

specimen was inoculated intraperi tone ally into all but two suckling
mice of a litter*

The uninoculated two were held as controls and

were marked by cutting off the tips of their tails.

All of the mice

were held for a 21 day period and observed for signs of Coxsackie
infection, manifested by paralysis of the hind-limbs and occasional
deformity in the fore-limbs, with death occurring within a few hours
after onset of symptoms.

Mice dying within 2U hours of inoculation

were not considered 11specific” deaths.
The brains from dead and paralyzed mice were harvested.

A 20

per cent suspension was prepared in nutrient broth, for two addition­
al passages in suckling mice.

The harvest from the third passage

was injected intracerebrally in 21-day old mice, 0.03 cc per mouse,
to rule out the presence of a virus pathogenic for older mice.

Tissue Culture
Monkey kidney cells were purchased from Microbiological Associ­
ates, Bethesda, Maryland.

Round, screw cap tubes, 16 x 125 mm,

containing a confluent monolayer of monkey kidney epithelial cells
on one side of the tube were shipped weekly by air express.

The

growth medium used by the company contained calf serum two per cent,
lactalbumin hydrolysate 0.5 per cent and Hank's Balanced Salt So­
lution 97.5 per cent.

Upon arrival the 0.5 ml of growth medium was

replaced with 0.5 ml of Mixture 199, developed by Morgan £t al.,
(1950) containing two per cent inactivated horse serum buffered to
pH 7*2 with 2.8 per cent NaHCO^.

The monkey kidney cells were held

2h hours at 37 C after arrival before they were inoculated.

After

inoculation the medium was changed every third day in original iso­
lation techniques and on the fifth day following inoculation in the
neutralization tests.
Each of three tubes of monkey kidney cells was inoculated with
0.03 ml of the processed sewage specimen.

The cells were observed

every other day for signs of cytopathogenic effect (CPE).
was progressive in character.

The CPE

The first visible sign of viral

activity was the degeneration of the epithelial cells shown by small
clusters of cells becoming rounded and transparent.

In this early

stage, a partial release of some of the cells from the glass surface
of the tubes could be observed.

As viral multiplication continued

some of the cells became spindle shaped and more of the cell popu­
lation showed the characteristic rounding of the epithelial cells.
As the infection progressed there was a sloughing of the cells from
the glass surface which greatly interrupted the continuity of the
monolayer.

The tissue culture was harvested when the majority of

the cells were released from the glass surface.

On primary isolation,

the cell cultures were observed over a 20 day period.

CPE was usual­

ly observed anytime from the second to the twelfth day after inocu­
lation with sewage specimens.

In many cases, only one or two of the

three inoculated tubes were suspected as containing a virus infection.
Only tubes demonstrating CPE were harvested and passed.

The first

23
passage, after the original isolation, consisted of inoculating each
of three tubes of monkey kidney cells with 0,1 ml of undiluted tissue
culture fluid.

The CPE observed usually occurred much sooner and

affected all three tubes.

During the early part of the study all

suspected viral isolates were passed three times after original iso­
lation before a virus was considered ready for identification proce­
dures.

In the latter part of the study it was found that the titer

of the virus was, in most cases, directly related to the time of
onset of complete CPE.

This differentiation was also used by Melnick

(1957) in studying unknown viruses, thought to belong to the entero­
viruses grouo, isolated in West Virginia.

An arbitraty method was

chosen to determine whether the virus was passed more than once; on
first passage if marked CPE was observed in less than four days
following inoculation no further passages were made.
The tissue culture fluid from all positive tubes was pooled,
harvested and stored at -20 C.
Preparation of Antisera
The antisera were prepared by inoculating rabbits with the
strains listed in Table I.
as antigens.

Undiluted tissue culture fluids served

Rabbits received three injections each of 0.25 ml,

0.5 ml and 1.0 ml, given intravenouslsr, on successive weeks.

This

was followed by booster injections of 1.0 ml given at various inter­
vals.

In Table II are presented the serum neutralization titers

obtained and the total volume of antigen each rabbit received.
The method of producing specific antiserum of high titer
differed from Sabin and Ramos-Alvarez (195U), Melnick (1957), and

2h

TABLE II
SERUM NEUTRALIZATION TITERS AGAINST ECHO
PROTOTYPE STRAINS PREPARED IN RABBITS
Volume of TC
Antigen
Injected**

Virus

Echo

1
2
3
h

9,0 ml
11.0

5

12.0
16.0
19.0
20.0
9.5
7.0
11.5
12.5
12.0
8.0

6
7

8
9
10
11
12
13
Hi

8.5
8.5

MSU
hOO
600
1800
100
2800
1700
5700
1600
5300
690
2200
2600
1600
800

Serum Neutralization Inde:**
Melnick and
Microbiological
Associates (1957)
Kanda (1957)
3li0
1900
270
11

n5o
3100
900
ii90
2800
3li00
600
1600
650
335

1000
2000
800
25
1000
1600
2000
800
3200
800
650

Uooo
3200
1*00

*Titers are listed as 50 per cent end point per O.l ml serum
against 100 TCD50 of virus.
^Divided among a basic series consisting of 13 injections,
totaling 6.5 ml, and booster injections given at various intervals
thereafter.

25
Microbiological Associates Inc, (1957) because the initial volumes
injected in this laboratory were smaller and the period of time in
which the animals were inoculated was longer.

However, the total

volume injected into each rabbit was the same or greater than that
used by the other investigators and it was felt that the longer
period of injection would help to produce high titers in the anti­
sera.
Antisera against poliovirus types I, II, and III, prepared in
monkeys, was kindly supplied by Dr. H. A. Wenner.
Neutralization Tests
The lit Echo prototype specific rabbit antisera were divided into
four pools designated A, B, C and L according to the work of Melnick
(1955).

Pool A contained antisera 2, 3, 5, and 6.

Pool B consisted

of Echo antisera 1, 12 and 13, and Pool C was composed of Echo anti­
sera 7, 8, 9, 10, and 11.
and lU.

Pool D was comprised of Echo antisera 1*

The composition of these pools was based on the geographical

distribution of the original prototype strain isolations, with the
exception of Pool D which was made up of the two poorest antiserumproducing members of the group.
The pools were changed when it appeared that only seven members
of the Echo virus group were being identified from the unknown iso­
lates.

A pool containing Echo virus antisera 1, 3, 13 and ll* was

designated E and a pool containing 6, 7, and 9 was labeled F.
Viruses to be identified, which were non-infectious for suckling
mice, were titrated in monkey kidney cell cultures using 10-fold
dilutions made in Medium-199*

The TCD50, for each unknown virus, was

26
calculated according to the method of Reed and Muench (1938)*

These

titers are shown in Table III and represent the highest dilution in
which 5>0 per cent of all inoculated tissue culture tubes showed
demonstrable CPE.

The unknown viruses were mixed, at a dilution

containing 200 TCD^O, with equal volumes of the pooled antisera.
Each antiserum in the pool contained from 10 to iiO units of antiviral
activity.

One unit was equivalent to the highest dilution of anti-

serum neitralizing 100 TCD50 of homologous virus.

The serum-virus

mixtures were allowed to incubate at room temperature for one hour.
One tenth ml of the mixture was placed in each of three monkey
kidney tissue culture tubes.
each test.

Two control series were included in

One set of tubes contained 100 TCD5>0?s of virus in

normal rabbit serum and one group of tubes was uninoculated.

The

tubes were incubated at 37 C and were observed daily over a 10 day
period for evidence of viral activity.
In most of the identification procedures either Pool E or Pool
F antisera neutralized the unknown virus.

The next step was to

perform the neutralization test against each of the component members
of the pool.

If Pool E or Pool F did not neutralize the unknown

virus, a series of neutralization tests, using rabbit antiserum
prepared against each of the poliovirus types was performed.

In the

event that the unknown virus specimen contained more than one type
of poliovirus, a mixture composed of equal volumes of all three types
of poliovirus antiserum was included.

The same number of units of

virus and antiserum as described above was used in this screening
procedure.

Failure of the unknown virus to be neutralized in the

27
poliovirus screen resulted in the screening of the unknown virus
against the remaining members of the Echo group.

Pool G contained

Echo antiserum 2, ii, and 5 while Pool H contained Echo antiserum 8,
10, 11, and 12.
When the unknown virus was not neutralized by pools G and H, it
was designated as an "Unknown11 and subjected to further tests in
suckling and 21-day old mice to detect the presence of Coxsackie B
virus.

Salmonella Isolations
For the isolation of species of Salmonella, only gauze pad and
sludge samples were utilized.

The extraction of the liquid from the

pads was similar to the procedure outlined in the section on virus
isolation.

In many instances, the same pad or sludge sample served

as a source both for virus and salmonellae isolation techniques.
Approximately one ml of material served as an inoculum for 150 ml of
enrichment medium*.

The enrichment media used in the study were

Difco tetrathionate broth and Selenite Brilliant Green (SBG) develop­
ed by Stokes and Osborne (1955).

The formulation of Stokes' and

Osborne's medium is as follows:
Per cent
Peptone ....... ..... ..... ..............
Yeast extract
.......................
Mannitol ................................
Sodium selenite
.................... .
Sodium taurocholate
................
Brilliant green .........................
Phosphate buffer ,pH 7*0 •••••...........

0.5
0.5
0.5
0.U
0.1
0.0005
0.025 molar

*Later work has shown that 25 ml of SBG medium is sufficient
for one ml of inoculation.

28
”In the preparation of this medium, the first five
ingredients were dissolved in somewhat less than the re­
quired amount of water and adjusted to pH 7.0 by the ad­
dition of a few drops of £N HCL. The phosphate buffer
and brilliant green were then added and the volume of the
medium was adjusted to the required level with water*
The phosphate buffer was prepared by- mixing appropri­
ate quantities of 0.2^M solutions of K^POi^ to give a
solution of pH 7.0 and this was added to the medium in a
ration of one part of buffer to ten parts of medium.
The brilliant green had a dye content of 93 per cent.
One ml of a 0,1 per cent aqueous solution of the dye was
added to each 200 ml of medium. Since the dye solutions
lost considerable color on standing overnight in the labo­
ratory, a fresh solution was prepared on each day that a
new batch of medium was to be made.’1
The inoculated enrichment media were incubated for 18 to 2h
hours at 37 C.

One large loopful of material was then streaked onto

a Difco bismuth sulfite agar plate which was incubated at 37 C for
2k to U8 hours.

Typical salmonellae-like colonies were picked and

inoculated into triple sugar iron agar tubes.

At least four sus­

picious colonies were picked from each plate when possible.

Transfers

into tubes of Stuartfs urea broth, Simon's citrate agar, nutrient
agar and one per cent solutions of lactose, sucrose, salicin and
mannite, made up in a purple broth base, were made from tubes of
triple sugar iron agar showing salmonella-like reactions.

Final

serological typing was generously performed by the Salmonella Typing
Station of the Michigan Department of Health Laboratories.

29
RESULTS

During 27 consecutive months of sampling, a total of 951 sewage
specimens were tested in monkey kidney tissue culture.
are presented the composite data.

In Table III

Sewage specimens from East Lansing

accounted for 585 of the samples tested and the remaining 366 were
collected from Lansing.

The per cent positive isolations of enteric

viruses accomplished in East Lansing was lii.2 per cent (83 of 585),
which was exactly twice the 7.1 per cent recovery rate from Lansing
(26 of 366).

An analysis of the differences in the recovery rates

from the two locations revealed that the number of positive dip
samples was approximately equal; 10 of 189 samples were positive from
Lansing and 7 of 179 samples were positive from East Lansing.

The

gauze pad samples in East Lansing yielded a much higher number of
viruses, U5 of 285, than did similar types of samples from Lansing,
16 of 177.

The fact that raw sludge samples were only collected from

the East Lansing sewage plant must also be taken into consideration.
However, even though the sludge samples accounted for 20.7 per cent
of the total number of samples and 11.0 per cent of the positive
samples, the differences between the two locations was still signifi­
cant: 11 per cent of the specimens tested were positive from East
Lansing as opposed to 7*1 per cent from Lansing, after excluding the
sludge samples from the calculations.
Table III also includes a summary of the mouse inoculation tests
carried out in parallel with the tissue culture studies.

A total of

859 sewage specimens from East Lansing and Lansing were tested in
infant mice.

Lansing contributed 303 specimens of which six were

30
positive (2.0 per cent).

Seventy of the 556 specimens from East

Lansing were positive (12,6 per cent).

The dip samples from Lansing

were comparable to those of East Lansing in that 5 of 155 (3*2 per
cent) were positive in mice as opposed to seven of lUl samples (5.0
per cent that were positive in the East Lansing series.

There was a

dramatic difference in the number of positive isolations obtained from
the gauze pad samples.

Only one of 1U8 samoles was positive from

Lansing, while there were 38 of 310 (12.3 per cent) positive samples
from East Lansing.

The raw sludge specimens yielded positive results

in 25 of 105 attempts (23*8 per cent).
The information presented in Table III does not take into con­
sideration the number of duplicate isolations obtained from the same
specimen in tissue culture and mouse inoculation tests.

From the 951

tissue culture isolation procedures, 110 tissue culture cytopathogenic
agents were found (11.6 per cent).

An infectious agent was demon­

strated in 76 of 859 specimens tested in infant mice (8.8 per cent),
and since there is no evidence in the literature to indicate that all
paralyzed suckling mice were not infected with either Coxsackie A or
Coxsackie B virus, these were recorded as Coxsackie viruses.

The

number of sewage specimens tested in mice on a monthly basis during
the course of the study is presented in Table IV.

During the last

four months of 1955, l6l specimens were processed and three samples
contained Coxsackie viruses.

During the 12 months of 1956, 26 of 3U5

specimens tested were positive in mice (7.5 per cent).

During the

10 months of 1957 in which 353 samples were collected, the number of
isolations obtained in mice was 26 (11.0 per cent).

31

TABLE III
VIRUSES ISOLATED FROM SEWAGE BY TISSUE CULTURE
AND MOUSE INOCULATIONS
Tissue Culture
Number
Number Per cent
tested positive positive

Mouse Inoculations
Number
Number Per cent
tested positive positive

Sample

Location

Gauze
pad

Lansing
East
Lansing

177

16

9.0

11*8

1

0.7

285

1*5

15.8

310

38

12.3

Lansing
East
Lansing

189

10

5.3

155

5

3.2

179

7

3.9

11*1

7

5.0

121

32

26.1*

105

25

23.8

366

26

7.1

303

6

2.0

585

83

ll*.2

556

70

12.6

951

110

11.6

859

76

8.8

Dip

Sludge

Totals

East
Lansing

Lansing
East
Lansing

Grand Total

32
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33
By combining the number of samples tested in each month of each
year (Table IV, 1955-1957) of the study, a pattern of the seasonal
occurrence of Coxsackie virus was indicated*

The months of the lowest

incidence of recoveries were December through June.

In July there was

a noticeable increase in the recovery of Coxsackie viruses (8.U per
cent) . The peak occurrence was in August when 21 of 100 samples were
positive in infant mice.

A decrease from this peak of 21 per cent

positive specimens was seen during the month of September when the
incidence fell to 10.7 per cent.

A slight rise was seen in October

over the previous month, but was still considerably below the peak
incidence in the month of August, when 19 of the 129 samples (l5.2
per cent) were positive in mice.
The number of specimens tested in monkey kidney cell cultures
during the study is shown in Table V.

During the last four months of

1955, 172 specimens were processed and yielded 11 tissue culture
cytopathogenic agents (6 .I4 per cent) . For the year 1956 in which 399
samples were tested, Ii3 positive isolations were accomplished (13*3
per cent) and in 1957, 56 of the U20 specimens were positive (13.3
per cent)•
By combining the number of samples tested in each month of each
year (Table V, 1955-1957) of the study, a pattern of the seasonal
occurrence of tissue culture cytopathogenic agents from sewage was
obtained.

These agents were isolated during each month, with the

peak incidence occurring during the month of October, when 18.9 per
cent of the 111 samples tested were positive.

Since the differences

in per cent recovery between the months of August, September, and

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35
November did not vary more than three per cent (16.0, 17ih, and 16.h
per cent respectively) lower than the October peak, the results
indicated that there was a long period of high incidence.

Marked

reduction in incidence was noted during December (h*8 per cent),
January (6.1 per cent), February (6.5 per cent), March (7-8 per cent),
and April (5*2 per cent).

The small number of isolations obtained

during the month of May in both years of the study (2.8 per cent) may
be attributed to the smaller number of samples tested (36) or in part
due to the heavy rainfall in the area.

The recovery rate of cyto­

pathogenic agents was increased to 7.6 per cent during the month of
June and was further elevated during the month of July, in which 11
per cent of the specimens tested in tissue culture yielded cyto­
pathogenic agents.
Serum neutralization tests in monkey kidney culture tubes were
used to identify 35 of the viruses isolated from sewage in tissue
culture.

Nine of the lh Echo types included in the identification

schema were present (Table VI). Echo 1 or 13 virus was identified
most frequently and occurred on 10 occasions.

Since the prototype

Echo 13 virus has been reported by the Corrcnittee on the Enteroviruses
(1957) to contain an Echo 1 contaminant, these two strains were
grouped together, since cross-reactions were seen in most cases.
7 virus was identified from six of the samples.

Echo

Next, listed in the

order of their most frequent appearance were Echo 6 (four isolations),
Echo 8 (four isolations), Echo 3 (three isolations), Echo lh (two
isolations), and Echo 9 (one isolation).

In addition to the Echo

viruses that were identified, four samples yielded poliovirus Type

36

TABLE VI
IDENTITY OF VIRUSES ISOLATED
IN TISSUE CULTURE
H
1
I

East
Lansing

10

Lansing

Total

Echo
3

2

Echo
6

Echo
7

1

3

3

3

Echo
8

Echo
9

u

o
OH
W

Location

Echo 1
and/or 13*

Polio
type
1

3
1

Polio
type
3

h
1

35

*Echo 1 and 13 grouped together since cross reactions were so
frequently encountered that all Echo 1 strains may be Echo 13 and vice
versa. This has also been reported by the Committee on the Entero­
viruses (1957)#

37
III and one sample contained poliovirus Type I.

In Table VII is

presented information on the source from which the viruses were iso­
lated, the titer and growth rate in tissue culture, and the month and
year of isolation.
Sewage specimens from East Lansing accounted for 27 of the
viruses that were identified as Echo or polioviruses. However, by
comparing the number of isolations obtained from the gauze pad and
dip samples from the two cities, during similar periods of collections,
little difference was noted between them.

Ten Echo virus identifi­

cations were made from East Lansing and eight from the Lansing samples.
It can be seen from Table VI that Echo virus

types 1or 13, 3* 8, and

U 4 were isolated only from East Lansing.

Lansingone specimen

In

yielded Echo virus type 9, while this type was not found in East
Lansing.

Poliovirus Type I was found once in Lansing and poliovirus

Type III vias found only in East Lansing and on four occasions.

Echo

virus types 6 and 7 were found in both locations.
The seasonal incidence of these enteric

viruseswasagain noted,

with two isolations obtained during the month of January, one Echo 9
isolated in April and one polio Type I virus was found in a sample
collected in March.

The remainder of the isolations was accomplished

during the period June through November with a concentration during
the peak months of September and October.
In Thble VIII are listed the cytopathogenic agents that were not
identified as being members of the Echo 1-lU group, poliovirus group
or the Coxsackie group of viruses.

Of the 17 isolates that composed

this "unknown11 group, 12 had a growth rate classified as being

38
TABLE VII
ANTIGENIC TYPES AND SOME BIOLOGIC CHARACTERISTICS OF VIRUSES
ISOLATED IN TISSUE CULTURE FROM SEWAGE SAMPLES
Sample
number

Source*

139
527
536
571
90l+
906
933
1016
1063
1111+

ELP
ELS
ELS
ELS
ELS
ELP
ELP
ELP
ELP
ELS

1+07
1+90

ELS
ELP

126
157
1+09
1+36

Echo
type

Titer**
TCD50 per ml

Growth
rate***

Month and
year isolated

9.3
9.5
9.0
7.5
9.0
8.5
9.5
8.5
8.5
6.8

Fast
Fast
Fast
Fast
Fast
Fast
Fast
Fast
Fast
Fast

9-56
11-56
12-56
1-57
9-57
9-57
9-57
9-57
10-57
10-57

3
3

8.0
8.3

Moderate
Fast

8-56
10-56

LD
LP
LD
ELP

6
6
6
6

8.3
9.5
9.6
9.5

Fast
Fast
Fast
Moderate

11-55
12-55
8-56
9-56

71+
1+1+6
1+62
750
801
81+5

LP
LP
LP
ELS
ELP
ELP

7
7
7
7
7
7

8.6
8.5
9.6
8.5
9.5
7.8

Fast
Fast
Fast
Fast
Moderate
Fast

11-55
9-56
9-56
6-57
7-57
8-57

205
1+76
897
926

ELP
ELP
ELS
ELP

8
8
8
8

8.5
1+.2
8.0
7.5

Slow
Slow
Fast
Fast

2-56
10-56
9-57
9-57

675

LP

9

7.2

Fast

+-57

191
1+87
921+

ELS
ELS
ELS

lit
11+
11+

7.6
8.5
8.5

Moderate
Slow
Slow

1-56
10-56
9-57

799

LP

Polio I

8.6

Fast

7-57

1
1
1
1
1
1
1
1
1
1

or
or
or
or
or
or
or
or
or
or

13
13
13
13
13
13
13
13
13
13

39

TABLE VII (continued)
Sample
number

Source*

hbo
U68
6U7
657
Total

ELS
ELP
ELP
ELD

Echo
type
Polio III
Polio III
Polio III
Polio III

Titer **
TCD50 per ml

Growth
rate***

Month and
year isolated

9*3
9.5
9.5
9.3

Fast
Fast
Fast
Fast

9-56
10-56
3-57
3-57

y3

*East Lansing Pad (ELP) ; East Lansing Dip (ELD); East Lansing
Sludge (ELS); Lansing Pad (LP); Lansing Dip (LD) •
**Expressed as the reciprocal of the TCD5CL
*":HfFast: marked CPE in U days or less; Moderate: marked CPE seen
by the sixth day; Slow: CPE seen in 7 or more days after inoculation

ho

TABLE VIII
VIRUSES ISOLATED IN TISSUE CULTURE NOT IDENTIFIABLE
AS ECHO VIRUSES 1-li;, POLIOVIRUS TYPES I, II,
OR III OR COXSACKIE VIRUSES
Sample
number

Source*

97
195
365
l*2l*
l*5i
1*52
1*86
551*
797
833
870
877
912
1035
1057
1066
1165

ELS
ELP
ELP
ELS
ELP
LD
LP
LP
ELP
LP
ELP
ELP
ELP
ELP
ELS
ELP
ELS

Total

Titer*^
TCD50 per ml

8.6
Not done
Not done
8.6
9.3
Not done
5.7
8.6
9.3
8.5
8.3
7.5
7.5
7.0
8.6
Not done
7.3

Growth
rate***

Month and
year isolated

Moderate
Moderate
Slow
Fast
Fast
Slow
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Fast
Moderate
Fast
Slow
Fast

10-55
1-56
7-56
9-56
9-56
10-56
10-56
12-56
7-57
8-57
8-57
8-57
9-57
9-5 7
10-57
10-57
11-57

17

East Lansing Pad (ELP); East Lansing Sludge (ELS); Lansing Pad (LP) ;
Lansing Dip (LD)•
^Expressed as the log of the TCD50.
***Fast: CPE manifested in U days or less* Moderate: CPE visible by
the sixth day; Slow: CPE not seen until the seventh day or later.

Ul
moderate or slow; a marked cytopathogenic effect was not evident for
at least six days following inoculation in the case of those classi­
fied as moderate and seven days or longer for those classified as
having a slow growth rate.

This was in contrast to the group of

identified viruses in which only eight of the 35 were classified as
possessing a moderate or slow growth rate.

This group of "unknowns"

was negative upon passage of the original specimen and subsequent
tissue culture passage material in infant mice and did not infect
21-day old mice when inoculated intercerebrally.
Of the 110 tissue culture cytopathogenic agents, 36 were also
found to be positive when inoculated into suckling mice.

Nineteen

of these viruses were positive when the original sewage specimen was
inoculated into the mice.

An additional 17 specimens, which were

positive in tissue culture, failed to infect infant mice when the
original specimen was tested.

However, when the tissue culture

passage material was inoculated into one-day old mice, paralysis and
death occurred.

This information

is in

Table IX.

Twenty-two tissue culture cytopathogenic agents lost their
ability to produce visible CPE in monkey kidney cells after their
original isolation.

In Table X are presented the data which showed

that the growth rate of these agents was either slow or moderate.
These agents were passed at least twice in tissue culture after
original isolation and one was passed an additional seven times.
When it became evident that the latest passage material had lost
its ability to produce a CPE in tissue culture, blind passages were
made using the older passage materials, which had been stored at

U2
TABLE IX
DUPLICATE ISOLATIONS OF CYTOPATHOGENIC AGENTS IN TISSUE CULTURE
AND COXSACKIE VIRUSES IN SUCKLING MICE
Specimens positive in tissue culture and suckling mice
upon original isolation procedures
Specimen number
Date of collection
Source*
LD
ELS
ELP
ELD
ELP
ELP
ELP
ELD
ELP
ELP
ELP
ELP
ELP
ELP
ELS
ELS
ELS
ELS
ELS

17
63
369
393
395>
1*05
U27
72li
7Ul
807
811
825
831
81*0
869
876
931
1093
1121

9-55
9-55
7-56
8-56
8-56
8-56
9-56
5-57
6-57
7-57
7-57
8-57
8-57
8-57
8-57
8-57
9-57
10-57
10-57

Specimens positive in tissue culture and negative in suckling mice upon
original isolations procedures. Tissue culture passage material
positive in mice
813
6L9
952
960
967
97U
980
981
1001
1009
1022
10U3
1057
106U
1073
1079
1157
Total

ELS
ELS
ELS
ELP
ELP
ELP
ELS
ELS
ELS
ELP
ELS
ELS
ELS
ELS
ELP
ELS
ELS

7-57
8-57
9-57
9-57
9-57
9-57
9-57
9-57
9-57
9-5 7
9-57
10-57
10-57
10-57
10-57
10-57
11-57

3/5

*East Lansing Pad (ELP) ; East Lansing Dip (ELD); East Lansing
Sludge (ELS); Lansing Dip (LD).

13

TABLE X
TISSUE CULTURE CYTOPATHOGENIC AGENTS RECOVERED FROM
SEWAGE BUT LOST ON SUBSEQUENT PASSAGE OR STORAGE
Specimen
Number

Source*
LD
LD
LD
LP
ELP
LP
LP
ELP
ELS
LD
LP
LD
LP
ELP
ELD
ELD
ELP
ELD
LP
ELP
LP
LD

Ui
57
59
120
132
198
221
228
233
2Ul
25o
278
307
327
333
366
385
U37
U56
h9h
501
699
Total

Date of
collection

Growth
rate**

Number of
passages

9-55
9-55
9-55
H-55
11-55
n-55
2-56
2-56
2-56
3-56
3-56
U-56
6-56
6-56
6-56
7-56
8-56
9-56
10-56
11-56
11-56
U-57

Slow
Moderate
Slow
Moderate
Moderate
Moderate
Slow
Slow
Slow
Slow
Slow
Slow
Slow
Slow
Slow
Slow
Moderate
Slow
Slow
Slow
Moderate
Slow

7
3
5
6
6
h
3
5
h
3
5
3
3
h
3
2
2
2
2
2
2
3

22

*LD (Lansing Dip); ELD (East Lansing Dip); LP (Lansing Pad); ELP
(East Lansing Pad): ELS (East Lansing Sludge).
"^Moderate: CPE manifested in 5-6 days; Slow: CPE manifested in 7
or more days.

hh
-20 C.

Blind passage techniques, although successful in restoring

the CPE in some instances, did not result in success with the 22
agents listed in Table IX.
An attempt was made to demonstrate the effects of sewage treat­
ment in removing viruses present in the influent*

To demonstrate this,

samples were taken from seven locations in the East Lansing treat­
ment plant; influent, raw sludge, primary tank effluent, activated
sludge tank, activated sludge return, final settling tank, and the
final, unchlorinated, effluent.
Daily samples were collected from each of the seven sampling
points from August 27 through November 6, 1957.

However, due to

lack of funds only 2lU specimens were processed either in monkey
kidney tissue culture and/or suckling mice.

There was a progressive,

significant decrease in the number of viruses isolated from stages
of treatment followed through the plant (Table XI).

The influent

yielded lL viruses from the U3 specimens tested (32.6 per cent).
From bZ samples of raw sludge 16 viruses were isolated (38.1 per cent).
Samples from the primary tank effluent were positive on 5 of 21
occasions (23.8 per cent).

The number of isolations obtained from

the activated sludge tank dropped to two of 18 samples (11.1 per
cent) • This low incidence continued when only 2 of 30 samples from
the activated sludge return were positive (6.7 per cent).

Samples

from the final settling tank yielded virus only once in 19 attempts
(5.3 per cent).

The final, unchlorinated, effluent contained virus

on four of Ul occasions (9.8 per cent).
During the first half of the Salmonella study, using tetra-

hs

TABLE XI
ISOLATION OF ENTERIC VIRUSES FROM VARIOUS
LOCATIONS IN A SEWAGE PLANT
DBS

Sampling
location

August

(27-31)

September

October

(1-30)

(1-31)

(1 -6 )

Total

Per cent
positive

November

Influent

U/5*

9/23

i/ili

oA

1UA3

32.6

Raw sludge

2/U

8A 9

UA6

2/3

16A 2

38.1

Primary tank
effluent

2/5

2/9

i/5

0/2

5/21

23.8

Activated sludge
tank

o/5

0/8

iA

1A

2/18

11.1

Activated sludge
return

O/li

1/19

1/6

oA

2/30

6.7

Final settling
tank

o/5

0/7

1/6

oA

lA 9

5.3

Final,
unchlorinated
effluent

o/5

1/20

3 /lit

0/2

UAl

9.8

^Number of samples positive out of the total number tested*
Figures represent the combination of tissue culture and/or mouse ino­
culation tests. Samples positive in both instances were counted only
as a single positive isolation for this table.

U6
thionate broth as the enrichment medium, 32 of ll*2 gauze pad and
sludge samples were positive for Salmonella (Table XII)• From these
32 positive specimens, \x9 salmonellae representing lh species were
isolated.

It should be noted that from approximately the same

number of gauze pad samples taken in the two cities, there was a
marked difference in the recovery rates.

In Lansing, six of the 59

specimens were positive and in East Lansing, 1? of 56 specimens were
positive for Salmonella. Since sludge samples were collected only
from the East Lansing plant, no comparison can be made with Lansing.
Nine of 27 raw sludge samples were positive (33 per cent) and the
per cent recovery was comparable to that from the gauze pad samples
in East Lansing (30 per cent)• However, the nine positive sludge
samples yielded 16 salmonellae that included nine species whereas,
the 1? positive gauze pad samples yielded 23 salmonellae representing
six species (Table XIII)•
The number of Salmonella isolated in the July through December
period is also shown in Table X U .

Fewer samples were taken during

this phase of the study (92) and a greater number of isolations was
made; 62 positive samples yielded 83 salmonellae representing 17
species (Table XIII).

A new species of Salmonella was isolated from

a sludge sample during this period.

It has been named S. Iansing

and its antigenic characteristics will be published by A. P. Juenker
and H. Bilanow of the Michigan Department of Health Laboratories.
Of the 21 sludge samples tested, 19 yielded 20 salmonellae which
included 12 different species.

The gauze pad samoles were positive

on h3 of 71 occasions and included 5U salmonellae representing 13

U7

TABLE XII
.COMPARISON OF TETRATHIONATE BROTH AND SELENITE
BRILLIANT GREEN MEDIA IN THE ISOLATION
OF SALMONELLA FROM SEWAGE
Tetrathionate Broth
October 195b - June 1957
Gauze pads
Sludge
Lansing
East Lansing

Totals

Selenite Brilliant Green Medium
July - December 195?
Gauze pads
Sludge

6/59*(l2?)
17/56 (30?)

32/11*2(23?)

9/27 (33?)

1*3/71 (60?)
62/92 (67?)

Number positive isolations/total specimens tested.

19/21 (90?)

ue

TABLE XIII
ISOLATION OF SALMONELLA FROM GAUZE PADS AND
RAW SLUDGE ACCORDING TO LOCATION

Species

S.
S.
"3.
3*
S.
S.
S«
S,
S.
S.
S.
S.
S.
S.
S.
S.
S*
S.
S.
*3.
S.
S.
S.

muenchen
montevideo
derby
anatum
termessee
typhiinurium
Worthington
infantis
thompson
reading
paratyphi B
heidelberg
san diego
lansing
typhl
kentucky
berta
bareilly
give
California
cerro
cnbana
blockley

Totals (133)

Tetrathionate Broth
Lansing
East Lans ing
Pad
Pad
Sludge

1
2
3

10
6
2
2
3

S
2
1
1
2
1
2
1
1

Selenite Brilliant Green
East Lansing
Pad
Sludge
2
3
S
9

h
3
S
1
2

1

2

8
3
18

2
1
2
1
1

1
1
1
1
1

1

1
2
1

10

2h

16

Sh

29

U9
species.

Eight species were found frequently in both types of

samples whereas , the pad samples were the only type of samples
positive for S. berta, S. bareilly, S. kentucky, S. give, and S.

California, Only the sludge samples were positive for S. typhi, S.
lansing, S. tennessee, and S. typhimurlum.
An attempt was made to see if sampling at different points in
the treatment plant had any effect on the recovery of Salmonella.
Although the number of samples was relatively small at some of the
sampling points (Table XIV), salmonella was recovered at every
point in the treatment system tested.

Emphasis was placed on the

sampling of the final, unchlorinated, effluent, since this material
was discharged into the Red Cedar River which flows by the plant.
A high recovery rate was obtained from this type of sample (22 of
35) indicating that although the sludge was removed at the early
stage of treatment, the supernatant fluid contains considerable
numbers of Salmonella as it passes through the plant and even in the
effluent which was discharged into the river.
Difficulties were encountered in the first phase of the study,
using tetrathionate broth, in that Proteus was frequently encountered
in such large numbers that discrete salmonella-like colonies were not
always observed.

In addition to proteus overgrowth on the bismuth

sulfite agar plates, paracolons, simulating Salmonella in the triple
sugar iron agar tubes, were found to be prevalent.

With the change

to SBG enrichment medium, little difficulty was experienced with
these groups of organisms.

In addition to inhibiting undesired

organisms, SBG medium seemed to enhance the growth of Salmonella,

5o

TABLE XIV
ISOLATION OF SALMONELLA FROM GAUZE PADS LOCATED AT
VARIOUS POINTS IN A TREATMENT PLANT

Sampling location

Number
of samples

Species

Influent

3/8*

S. san diego (2), S. derby

Primary tank effluent

6/7

S. san diego (2), S. para­
typhi B (2), S* anatum (2),
S. muenchen, S. berta, S.
kentucky

Activated sludge tank

1/6

S. san diego

Final settling tank

3/6

S. san diego (2), S* derby,
S. muenchen

Activated sludge return

8/9

S. san diego (2), S. para­
typhi B (?}, S. anatum (3),
S. montevideo, S. give,
S* California

Final, unchlorinated
effluent

Totals
o a B m

a e o B n a B n B c a a c ta M

M

22/35

S. san diego (9)* S. para­
typhi fe (b)s S. anatum (U),
S. heideiberg (3 ), S. monte■
video 12), S. derby (3)*
S. bareilly, S. infantis

U3/71

5h Salmonella isolated

B M

H

a a s a B B n a it M

E is B g

^Number of samples positive/total number tested.

51
since in a large number of samples a relatively pure culture of
salmonella-like colonies were observed on the plates, which were
subsequently confirmed as Salmonella. With the majority of samples,
typical colonies were observed on the bismuth sulfite agar plates
after only

2h

hours of incubation.

52
DISCUSSION

The presence of enteric viruses in sewage over a long sampling
period has been confirmed by the results of this study.

Since sewage

samples represent, in part, large numbers of stool specimens, the
findings of Honig et al., (1956) and Gelfand et al., (1957) should
be considered.

In Table XV is presented a summary of the results

obtained by the two previously mentioned groups and the data obtained
in this study.

The work of Gelfand represented the number of polio

and non-polio virus isolations in tissue culture, utilizing stool
samples from a group of normal healthy children over a 2h month
period.

Honig and his associates studied a group of healthy pre­

school children over a 29 month period.

The stool samples, collected

at monthly intervals from each child, were tested in tissue culture
and suckling mice to detect enteric viruses.

Table XV combines the

results for each month of the years that specimens were collected in
each of the three studies.

This type of presentation was chosen to

help compensate for the yearly changes that occurred on a month to
month basis in all three studies.

The primary difference between the

three studies was essentially the number of isolations.

However, the

basic concept that the incidence of enteric virus infections is at
a maximum during the summer and fall months is firmly established.
In the present study this peak was reached in August, which was
directly comparable to Honig*s results and appeared one month later
than the peak incidence as reported by Gelfand.

The decline from

this summer peak was gradual and the months of lowest recoveries in
the three studies were December through April.

A comparison of the

53
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51
results of this study with the other two revealed a more direct corre­
lation to the work of Gelfand in Louisiana than to the work performed
in West Virginia by the Honig group.

It should be kept in mind that

the Louisiana workers did not inoculate infant mice with any of their
specimens and the total number of isolations would have undoubtedly
been increased if this procedure would have been included in the ex­
perimental protocol.
The similarity of the results between this study, and the two
other studies, are quite interesting.

It would appear that samples

from a point in the sewage system, receiving wastes from the whole
or any particular section of a community, would serve as an excellent
source of material to determine the enteric viral flora of that area.
Of course, the work of Gelfand and Honig served other important
purposes such as obtaining information on multiple infections and the
persistence of a particular virus or viruses in any member of the
study.
From relatively few samples, 30, taken in the December through
May period, Coxsackie viruses were found on five occasions by Kelly,
Clark and Coleman (1955).

In the present study, only four Coxsackie

isolations were obtained from 22f? samples collected in the December
through May period, with isolations made in each of four different
months.

Only one Coxsackie virus isolation was reported out of the

725 specimens that were processed during the winter months by Honig
et al., (1956).

During the period when enteric virus isolation is at

a maximum, the number of Coxsackie virus isolations was dramatically
increased.

Kelly et al., (1955) reported positive isolations from

5?

66 of 88 samples (75*5 per cent) of sewage during the months of June
through November.

In the present study, Coxsackie viruses were found

in 72 of the 63U (ll.U per cent) samples tested during the June
through November period.

These results in sewage again compared

favorably with the findings of Honig*s group who recorded 28 positive
Coxsackie virus isolations from 833 stool specimens that were tested
(3*U per cent).
During the 27 months that sewage samples were collected polioviruses were only detected in five of 951 tissue culture isolation
trials.

Type I poliovirus was identified on one occasion from a

gauze pad sample collected in Lansing during the month of July 1957.
Type III poliovirus was isolated from four samples collected in the
East Lansing sewage plant.

One positive isolation was obtained in

September 1956 and one in October 1956.

Two additional type III

polioviruses were isolated during the month of March 1957.
The overall recovery rate of polioviruses, 0*5 per cent, was con­
siderably lower than those reported by Gelfand (U.6 per cent of 3667
specimens) or by Kelly, Winsser and Winkelstein (1957), who reported
that 21 per cent of 308 samples from various upper New York State
communities contained polioviruses.

It should be noted that Kelly*s

group sampled sewage coming from areas having reported cases of
paralytic poliomyelitis.

Although Gelfand*s study was based on the

findings in a group of normal children, the fact that these subjects,
for the most part, came from urban New Orleans and urban Baton Rouge,
Louisiana, may have accounted for the higher incidence of poliovirus
isolations than reported by Honig or from the results of the present

56
survey of sewage.

No information regarding the incidence of clinical

cases of poliomyelitis during the course of the study was reported
by the Louisiana researchers or by the West Virginia group.

A higher

incidence of poliovirus recoveries was reported by both groups, from
stools of children coming from the lower socio-economic areas.

In

the study by Honig, 10 of the 15 poliovirus isolations came from
children living in a section of the city where the environmental
sanitation and the economic status was generally low.

Gelfand et al.,

(1957) make the statement that "there is a definite consistency in
effect of race and socio-economic status in each year and for both
polio and non-polioviruses. The Negro group bears the greatest
burden of intestinal virus parasitism, followed in order by the whitelower economic and white-upper economic groups.

This order is in

inverse relation to the levels of personal and environmental sani­
tation in these three populations."
The lack of more poliovirus isolations in the present study may
stem from the absence of any reported clinical cases of poliomyelitis
in the city of East Lansing during the period of the study and also
that the East Lansing community represents an upper middle class
socio-economic status with high standards of environmental sanitation.
The area of Lansing that was sampled also represented an area where
good sanitation facilities existed.

Another point to consider is

that a low level of poliomyelitis incidence might affect the sensi­
tivity of the sampling methods.
Table VII contains information regarding the distribution of
Echo types 1-lh viruses identified during the study.

If the

57
assumption is made that all 10 of the viruses grouped under the head­
ing of Echo 1 or 13 represent a single type, then it may be said that
this strain, when combined with the six Echo 7 viruses accounted for
53.3 per cent of the total number of Echo viruses identified during
the study.
The high incidence of the Echo 7 virus type (20 per cent) was
similar to the findings of Ramos-Alvarez and Sabin (1956).

However,

Echo 2 virus which was identified in 1U; per cent of the viruses iso­
lated in Ohio was not found in the present work.

A comparison of the

present findings with those of Ormsbee and Melnick (1957), who identified the viruses isolated by Honig et al,, (1956), revealed that
there was a similarity between the two studies in that Echo 7, 8 and
lU accounted for 12 of the 20 Echo viruses isolated in West Virginia,
Again, no correlation between the incidence of Echo 1 or 13 virus
existed between the two studies.

In West Virginia, these strains

were reported only twice in comparison to the 10 identifications
obtained in the present study.
identified in both studies.

Echo 3 and Echo 9 viruses were also

Echo 11 virus was identified from five

of the samples in West Virginia but was not seen in samples from the
Lansing area.
Logically, an explanation for these geographical differences in
the Echo virus strains found in Ohio, West Virginia and Michigan
should be brought forth.
ble.

Unfortunately, this is not currently possi­

Perhaps, the climatic conditions, environmental factors and

geographical locations of the three sampling areas account for the
differences?

All that may be said is that carriers of a particular

58
Echo strain were present in some of the areas and were absent in the
others.

How many infected individuals need to be present to ensure

the spread of a particular strain in a large oroportion of the sus­
ceptible population is another unknown factor.
The finding of 17 tissue culture cytopathogenic agents, reported
in Table VIII, that were not identifiable as Echo viruses 1-lU, polio­
viruses or Coxsackie viruses presents an area for further speculation.
Perhaps these viruses are members of the Echo 15-19 group, the proto­
type strains not being available until late in the study precluded
their inclusion in the identification procedures.

Another possibility

was that these isolates represented mixed cultures which might have
evaded the serum neutralization screening tests.

Still another expla­

nation is that they may represent new enteric viruses not previously
classified.

Cross-neutralization tests would reveal how many of these

unknown viruses were similar to one another.

Ramos-Alvarez and Sabin

(1956) reported finding 25 unknown viruses in their study and they
were later established as being the prototype strains for Echo 7> 8,
10, 11, 18 and 19 viruses.

Ormsbee and Melnick (1957) reported that

10 of their isolates could not be identified.

Three of these were

found to be similar and have been accepted as the prototype strains
for Echo 15.
Another point that must be considered in dealing with the un­
known viruses reported in the present study is that their origin can
not be determined with certainty.

Perhaps they represent enteric

viruses from animals other than man?

The orphan viruses present in

animals have been reviewed by Hsiung and Melnick (1958).

Enteric

59
cytopathogenic bovine orphan viruses (Ecbo) and enteric cytopathogenic
swine orphan viruses (Ecso) are found in apparently normal animals and
their relationship to disease is also being investigated.

Needless

to say, it would be interesting to label the unknown viruses found in
this study.

One must not overlook the possibility that these viral

agents may be members of a group not normally considered as belonging
to the enteric virus classification, such as the adenoviruses and
herpes virus which have been found in feces.
During the late summer and early fall of 1957, a group of 17
viruses was isolated from the East Lansing plant that did not exhibit
the usual characteristics of the Echo or Coxsackie viruses.

No visi­

ble evidence was seen that indicated pathogenicity for suckling mice
when the original specimens were inoculated.

However, when the

infected tissue culture fluids were inoculated into suckling mice
there was a very marked pathogenic effect manifested by paralysis and
death of almost 100 per cent of the inoculated mice.

The control

mice in each litter remained normal throughout the test.

Serum

neutralization tests in tissue culture revealed that these viruses
were not members of the Echo 1-lU group, or poliovirus types I or III.
If these isolates belong to the Coxsackie B virus group, which
can be determined by neutralization tests in mice or tissue culture,
then some explanation should be offered as to why these 17 strains
exhibited such bizarre signs.

Hsiung and Melnick (1958) have claimed

that they have isolated a number of Coxsackie A9 and Coxsackie B
strains that failed to infect infant mice unless these agents were
first passed in tissue culture.

These authors offered no explanation

60
for this behavior and none is apparent in the present study.
one interesting epidemiological point can be brought forth.
these viruses was isolated in July 1957 and one in August.

However,
One of
In Sep­

tember, nine of these agents were isolated and in October the number
fell to five.

During the first week in November one was isolated.

From this type of data, contained in Table

IX,

small scale"epidemic" occurred in East Lansing.

it appeared that a
The lack of a

hospital in East Lansing precluded obtaining information on aseptic
meningitis admissions for these months.
In Table XI are presented the results obtained in an effort to
determine the effects of sewage treatment, in an activated sludge
type treatment plant, on the removal of enteric viruses before the
final effluent was discharged into the Red Cedar River.

It was

evident that there was a considerable reduction in the number of
viruses isolated from progressive stages of treatment.

However, the

finding of virus in the final effluent on U of Ul occasions indicated
that complete removal was not being accomplished by the treatment
process.

Further discussion of the problems that these findings may

lead to is included in the section that follows on the effect of
treatment on Salmonella.
Another difference that this study showed was the number of Echo
viruses found in this study as compared to the results of Kelly et
al., (1957) which showed that Echo viruses were only isolated on nine
occasions from 308 samples tested in tissue culture.

Mack and Fields

(in press) have reported that the ultracentrifugation method, employed
in this study, was more sensitive in detecting Coxsackie viruses from

61
sewage than the ion-exchange method used by Kelly and her co-workers.
Perhaps the ultracentrifugation method is also more sensitive in
detecting Echo viruses from sewage than the ion-exchange method?
Of the 22 tissue culture cytopathogenic agents listed in Table X
that were lost on subsequent passage or storage, all that may be said
is that there were definite indications that these agents produced
a CPE in monkey kidney cell cultures that was similar in many respects
to the enteric viruses that were identified.

There was no evidence

that these agents belonged to the indigenous monkey viruses reported
by Rustigan, Johnson and Reihart (l955).
One of the major problems that exists in enteric bacteriology is
the lack of a completely suitable enrichment medium that would serve
to enhance the growth of Salmonella and inhibit the growth of other
non-pathogenic organisms.

In an attempt to determine if a new medium,

SBG, was capable of providing the results desired in an enrichment
medium, a comparison was made with tetrathionate broth.
A marked increase in the recovery of Salmonella from sewage was
observed when the enrichment medium was changed from the tetrathionate
broth to the SBG medium of Stokes and Osborne (1955).
A comparison between the two time periods showed that the number
of recoveries of Salmonella from gauze pad samples increased in East
Lansing from 30 per cent (17 of £6) to 60 per cent (U3 of 71) when
SBG medium was used.

Even greater differences in results were ob­

tained when the number of recoveries from sludge were compared; 33
per cent (9 of 27) when tetrathionate broth was used to 90 per cent
(19 of 21) when SBG medium was employed.

The seasonal distribution

62
of enteric infections must be considered in the comparisons made in
this study.

It is generally accepted (Maxcy, 1956) that cases of

salmonellosis occur throughout the year but are at a minimum during
the winter and spring months, rising to a peak in the late summer and
early fall.

One cannot say whether this seasonal variation influenced

the findings reported in this work.

The qualitative nature of the

experimental procedure did not allow such comparisons to be mace,
since the number of positive samples indicated only that Salmonella
were present in the sewage, and did not reflect on their overall
concentration.

It was not practical to run parallel studies using

the two enrichment media.

By using a variety of differential agar

media the number of isolations might have been increased since there
is no one perfect selective medium and the type of medium employed
depends on what particular organisms are being looked for (Edwards
and Ewing, 1955).
The results obtained indicated, that the raw sludge specimens
were an excellent source of material for the isolation of Salmonella,
and higher recovery rates were obtained from this source than from
the gauze pad samples.

The high recovery rate from the final effluent

showed that the gauze pad method of sampling serves its purpose well
in monitoring supernatant fluid.
In comparing the recovery rates from the pad samples taken in
East Lansing and Lansing during the first part of the study, the fact
that Lansing is a highly industrialized community while East Lansing
is largely a residential area should be taken into consideration.
Large amounts of industrial wastes, deposited in the system, may have

63
contributed to the lower recovery of Salmonella by the gauze pad
method of sampling.
Ylhether the Salmonella isolated originated from human or animal
sources cannot be stated.

However, the results of the study serve to

strengthen the conclusion of Greenberg et al., (1957), that "typhoid,
and presumably other enteric infections, should be recognized as a
definite occupational hazard for treatment of plant operators."

In

addition to the direct threat to the plant operators, there exists
a potential health hazard to the community.

Even if the water is not

directly used for drinking or swimming purposes, the return of un­
chlorinated final effluent, containing viral and bacterial pathogens,
to a river flowing through a populated area should not be condoned.
It Is reasonable to suspect a contaminated water source as one of the
many links in the transmission of disease.

Proper chlorination of

final effluents would serve as good preventive medicine.

61
SUMMARY

1*

A total of 1018 sewage samples were tested for the presence of
enteric viruses in monkey kidney tissue culture and/or suckling
mice.

2•

Thirty-five of the isolates were identified as Echo viruses or
polioviruses.

Seventy-six were identified as Coxsackie viruses,

36 samples were positive both in tissue culture and in suckling
mice, an additional 1? viruses were not identified and 22 tissue
culture cytopathogenic agents were lost in passage or storage.

3.

Selenite brilliant green enrichment medium (62 of 92 samples
positive) was superior to tetrathionate broth (32 of lU2 samples
positive) in the isolation of Salmonella from sewage.

I4.. A new Salmonella was identified (S. lansing), among the 133
salmonellae isolated, along with 22 other species.

5.

Raw sludge as well as gauze pad samples provided the best source
of material for the isolation of enteric viruses and Salmonella„
However, large amounts of industrial wastes present in a sewage
system seem to have a detrimental effect on enteric virus re­
covery from gauze pad samples.

6 . Enteric viruses and Salmonella were found in all stages of
sewage plant treatment tested, including final effluent.

7.

Sewage samples provide a source of material from which the
enteric viral flora of a population may be determined.

65
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