Actn Medicn Scnndinavicn. Yol. CLIX, fnse. I, 1057.

From the Division of Laboratories and Research, New York State Department of Health, Albany, New York.

Eiitcric Virus Isolations froni Scwtlgc. BY

SALLY KELLY.

(Submitted for publication June 13, 1957.)

Sewage samples were collected in 1956 from the Albany treatment plant to determine the agents present in the area and to continue the study begun in 1952 of the enteric virus reservoir in this community.

liecognition of sewage as a potential reservoir of virus infection has been a long time coming. Nearly fifty years ago, Kling and coworkers (12) made the basic observations identifying poliomyelitis with enteric agents of disease. The idea was overshadowed for several years by the respiratory portal-of-entry theory. Not until Paul, Trask, and Culotta (24) applied the Landsteiner monkey test to sewage did the status of viruses in sewage rise to that of the typhoid bacilli, whose presence until Wilson detected them, had been sbased on faith and not on demon- stration)) (34). The oral route of infection became a more acceptable theory as Kling (10) and othcrs continued to associate the virus with, and isolate i t from, feces and sewage.

Isolations of enteric viruses from sewage are no longer the hit-or-miss ventures they originally were. I n our laboratory they have become a routine activity in which the absence of certain strains is as noteworthy as their presence a t other times. This change was brought about chiefly by improvements in technic, listed in table 1, which summarizes the history of virus isolations from sewage. Many of the advances are parallel to earlier experiences with enteric bacteria - the proper medium for selection and propagation, recognition of multiple serologic types, composite sampling procedures and quantitative refinements.

Methods and Materials.

The methods used in 1956 were a development of those used in previous years. Samples were taken as 48-hour swabs ( 2 2 ) , thirty in all, collected from the influent and effluent of the Albany sewage treatment plant, its outfall in the Hudson

64 SALLY KELLY.

Table 1.

Technics used in enteric virus isolations from sewage and water.

None

None & Ppt.

Evaporation None

Ppt.

Nonc Visking sac

None

Ppt.

Ppt.

Ppt.

Centrifuged

Centrifuged

None Nonc

Resin

Ppt & cen-

Resin trifuged

Enteric virus isolated

Monkeys

Monkcys

Monkeys Monkeys

Monkeys

Man Rat

Monkeya

Monkcys

Mice

Monkeys

Monkeys

Mice

Monkeys Monkeys

Mice

Mice

Mice and tissue culture

Polio

Polio

Polio? Polio

Polio

Hepatitis Polio

Polio

Polio

Coxsackie

Polio

Polio

Coxsackic A, A5

Polio Polio

('oxsackic A,

C!oxsackie A,

Coxsackie A,

Polio 1, 2, 3

€3

B

B

Frequency

Twice from 1 sample in epidemic

8 yo samples (5j66 mon- keys) in epidemic

Well water isolation Once in epidemic Once after epidemic Once in 15 months

Well water isolation Once isolated from creek

after adaptation In various stages of scwage

treatment 10 yo samples in epidemic

From several samples

Once before reported case; 5 times in epidemic

Once from creek watcr Once from sewer outlet Twice in summer

From single house Not related to diseaee

Frequently in summcr and

Frequently in summcr and

Frequently in summer and

incidence

fall

fall

fa1 1

Methods

Sampling

Catch

Catch

Catch

Pooled- Catch

Catch

Catch

Pooled- Catch

Pooled- Catch

Pooled- Catch

Catch

Pooled- Catch

Swab Swab

Swab

Swab

Swab

Concentra- Hosts tion

fear of 'ublica. ,ion

1939

1940

1939,'40 1940; 1942 1942

1945 1946

1946; 1949 1947

1949

1950

1950

1951; 1952 1952 1953

1953; 1955 1954

1957

- 3efer- ince

- 24

25

9 .3, 11

32

23 31

5, 4

17

21

27

33

1, 26

14 15

6 , 7

19

8

River, and a t several points up and down stream. Sampling took place in the sum- mer and fall when enteric viruses have been prevalent (17). Swab pressings were resin treated (6) to concentrate virus and remove toxic matter. Inoculations were made in several kinds of hosts: newborn mice (3), HeLa cell tube cultures (29), HeLa cell bottle cultures (16), monkey kidney tissue tube cultures (as), monkey kidney tissue bottle cultures (20), amnion cell tube cultures (35), and Detroit-6 cell tube cultures (30). The same volume of sample was used in each host, a total of approximately 0 . 5 ml. Observations and subsequent isolations were made as follows: The newborn mice were watched for signs of disease over a 2-week period. Paralyzed or spastic mice were sacrificed and their tissues stored in a dry-ice chest for confirmatory passage and serologic and pathologic identification. The tube cultures were examined microscopically for cell degeneration. Fluids from degenerated cells were stored at -20" C for confirmatory passage and sero-

ENTERIC VIRUS ISOLATIONS FROM SEWAGE.

Table 2.

Distribufion of isolates in virus-containing samples August-December, 1956.

1 1 - - - 2

2 2 1 4

n. d. 2 3 1

-

65

-- PIE, f. p. - - - P,

Pl P, PI PI

PI f. p. f. p.

-

Sample No.

Source

48 1 482 4x4 486 487 490 49ti 497 498 499 500 50 1 502 504 509

No. of Types isolates -

Isolation technic

RRW . . . . . . . . . . . . . . . I I d Effluent. ........... 11 Sludge 0 Outfall 3 Downstream. . . . . . . . . 1

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

Kcwborn I HeLa cells mousc

+ I + + + + I + + + + + + +

[&,opat,ho- genicity Plaque

No. -

f. p. Pl

f. p.

Monkey kidney tissue

Cytopatho- genicity

Human amnion ;issue

Zytopatho. Tenicity

+ + - -

- - - -

E6 n, n. d. n. d. n. d.

n. d. n. d.

-

logic identification. Bottle cultures were examined for plaques. Plaques were picked from the agar overlay, propagated in tube cultures of the same type, and identified.

Results.

Twenty-seven agents were isolated (table 2): nine were Coxsackie viruses, six of Group A, three of Group B. Ten were poliomyelitis viruses, nine type 1 and one type 3. Five were Echo viruses, type 6. Three agents were not identified as mem- bers of the poliomyelitis, Coxsackie, or Echo families, or mixtures of them.

The isolates were mainly from raw and effluent sewage (table 3). A few were

COXSACKlE A. COXSACKIE 8'

POLIO 1.

POLIOrn. E C H O . .

Technic

I I I

,..A. . . . . . . ..A. . . . . . . . . . . . . .A . . ,A A A '

...a,. . . . . . .a . . . . . . . . . . . . ..a

... rn rn rn

0 I . . . 88

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . @

No. of isolations

Newborn mouse .... HeLa tube ..........

bottle ........ Monkey kidney tube .

bottle Detroit-6 tube, ...... Amnion tube ........

Types

Coxsackie I Polio -- Echo Unknown -

+ + + + +

ENTERIC VIRUS ISOLATIONS FROM SEWAGE.

Year

67

No. of sampler

Fig. 2. Isolation of viruses from sewage a8 plaques: Left - Plaques formed after passage of sewage eluates in monkey kidney tube cultures. E,. Right - Plaques formed by sewage eluates; later identified as P,.

Total

No. I yo

The validity of using one of these isolation technics as an index of the total virus popuIation of sewage was tested by comparing the number of infected new- born mic,e with the total number of agents isolated from various samples. AS a rule (figure 3)) the mouse test corresponded to the number of isolations.

The isolations in 1956 were compared with those of other years. The comparison illustrates well the yearly changes in virus population of sewage. More viruses were isolated in 1954, for example, than two years later, yet Echo viruses were found only in the 1956 samples (table 5). That some of this difference may be due

Coxsackie A

No. I %

Tehle 6. Isolates in 1954 and 1956 from Albany sewage plant in newborn mice and in HeLa

cell and monkey kidney tissue tube cultures.

Coxsackie B Polio- Echo

No. 1 % No. I % No. I %

myelitis Unknown

No. I %

Strains isolated

SALLY KELLY

pig. 3. Number of infected mice vs. number of agents isolated.

B e 1 ~ - - - _ _ - , , , ,, , , , , 7, rir,--,-, , i i , , ,Be

A S O N A M J J A S O N J A S O N D A S 0

Fig. 4. Coxsackie virus types isolated from the Albany and Colonie sewage treatment plants.

ENTERIC VIRUS ISOLATIONS FROM SEWAGE. 69

to an insufficient number of isolation attempts is suggested when isolates as plaques are added to the 1956 listing. Perhaps the changes are also caused by a shift in the disease pattern of the community: a four-year record of Coxsackie viruses in sewage (figure 4) shows the changing annual patterns that were early noted in the study of these agents (2). I n 1953 type-5 and -6 strains were fre- quently isolated; one year later not a t all. I n 1954 types 4 and 8 were found to the exclusion of other Group-A strains. Two years later they were absent. The presence of dominant strains in sewage has been noted by others (19). Their absence imme- diately following a year of abundance may reflect the immunity of the population.

D i Rcussion.

Throughout these studies we have been impressed by the numbers and varieties of viruses present in sewage. Our present technics, however, may leave much to be desired in sensitivity and, of course, do not identify hepatitis virus a t all.

At present i t is quite clear that several methods of isolation are required. For example, the 1956 tests yielded viruses in only 7 per cent of the specimens when tested on HeLa cells, in 27 per cent when tested in mice, and in 43 per cent when tested on monkey kidney cells. Several methods yield more isolations than any single one. This is largely due to the selective sensitivity of each method for par- ticular enteric viruses. Multiple testing probably contributes to the yield also by providing more chances and larger samples.

When the sentisivity of a technic is judged, the volume of inoculum may be critical, as was well illustrated by the poliovirus vaccine safety testing program. To arrive a t an index of sensitivity of the isolation technics used in this study, the following formula is suggested, in which

1 x A, s = ~ . ~~~ (A, = No. agents isolated by method,) ml inoculum,

Judged in this way, mouse pathogenicity tests are about 10 times more sen- sitive for Coxsackie virus isolations than are plaque formations in monkey kidney bottle cultures for polio virus isolations, and the cytopathogenicity of polio virus in tube cultures is about twice as sensitive as plaque formation.

The application of bottle cultures to isolations of viruses from sewage has certain advantages, however, over that of tube cultures. An obvious virtue (18), the immediate resolving of mixtures of viruses, remained untried, since the number of mixtures in the 1956 samples was negligible and one of these was derived from a single plaque. The isolation of viruses as plaques permits enumeration. In the present work (a maximum of 4 plaques per 0.5 ml concentrated sewage), plaque- forming particles of polio virus (PFU) were found a t the rate of about 80/lOO ml or less in nonconcentrated sewage. By way of contrast, coliform bacteria counts in sewage run to the millions (MPN/100 ml) and coli phage has been estimated a t 800,000 or more particles/100 in1 sewage. Consequently, if potable waters (MPN/100 ml of < 2.2) were suspected of virus carriage, and the relationship of coliform numbers to virus particles remained unchanged, ten thousand fold con-

‘7 0 SALLY KELLY.

centration would be required for the isolation of viruses as plaques. The number of PFU cited is much greater than the number of polio virus infectious doses for monkeys tha t w-ere calculated in sewage in earlier studies (17).

S 11 mmary. A picture of the enteric virus population of sewage in 1956 was obtained by

examining relatively few samples more or less exhaustively. One-half of the samples contained viruses, including poliomyelitis types 1 and 3, Coxsackie Groups A and B, Echo type 6, and unidentified agents. Agents were isolated most frequently in hosts in the following decreasing order: monkey kidney and mice > amnion > HeLa > Detroit 6. Isolation of polio viruses as plaques provided an estimate tha t no more than 80 plaque forming units occurred in 100 ml of sewage.

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