A method for detecting human enteroviruses in aquatic sediments

Jownal qf Virologicai

153

Merhods, IO (1985) 353-162

Etsevier 3VM 00368

A METHOD

FOR DETECTING

HUMAN

ENTEROVIRUSES

IN AQUATIC

SEDIMENTS

G.D.

LEWIS*, M.W. LOUTIT

and F.J. AUSTIN2

‘Microbiology Deparrmeni. and 2NZ MRC Virus Rmarck

iltrfr, Univemiry of

Ofago,

P. 0. Box 56, Drmedin.

Ives Zealand (Accepted

t 6 October

A method recovered glycol

t984)

is described

for detecting

from sediments

by elution

6000. The recovery

freshwater

sediments.

of the sediments

enter&ruses

efficiency

Although

in both freshwater

and marine sediments.

into 6% beef extract at pH 9.0 and concentration ranged

the efficiency

it was used successfully

from

6 to 55% for marine

of the method

was influenced

to detect viruses occurring

sediments

Viruses were

with polyethylene and

16 to 77% for

by the composition

in marine and freshwater

and source

sediments near

sewer outfalls.

enterovinls

aquatic sediments

virus detection

It is now widely recognised

polyethylene

gtycof

that bodies of water receiving

sewage effluents

may

become contaminated by human enteric viruses, including such disease causing agents as hepatitis A virus, poliovirus, and Norwalk agent. Enteric viruses generally survive well in this environment inactivation

in aquatic

and have been demonstrated areas than sewage associated

to be much more resistant

indicator

bacteria

(Melnick

1978; Smith et al., 1978; LaBelle et al., 198t; IAWFRC, f983). Enteric viruses in water adsorb rapidly to both organic and inorganic

to

et al.,

materiai

(Bitton, 1980), and the particfe-virus association may settle to the bottom and become incorporated in the sediments. Afthough this process removes viruses from the water column, it does not cause their inactivation but rather the viruses are protected by the presence of the solids (Schaub et al., 1975). An accumulation of infectious virus in sediments may then result which will in effect serve as a reservoir of viable pathogens at a concentration often greater than that in the overlying water (Goyal et al., 1978). Recontamination of the water column by these viruses can readily occur through disturbances of the upper sediment layers. Increased flows, currents, weather conditions, or human activities such as dredging and recreation (Grimes, 197.5) can all lead to resuspension of particles from the sediment-water interface. After resuspension particle-associated virus may be transported considerable distances from the pollu-

154

tion

source

important

and contaminate

she1lfish

beds and recreational

to be able to detect enteric viruses in sediments

bacteria

have

(LaBelle

et al., 1981).

Previously

been

shown

published

non-quantitative

to be unreliable

methods

indicators

for detection

It is therefore

and particularly of the presence

of viruses

(De Flora et al., 197.5) involve filtration,

areas.

in sediments

as faecal of viruses are either

which is often difficult with

environmental samples due to clogging (Gerba et al., 1977) or have low recoveries (Bitton et al., 1982). During the course of this study Wait and Sobsey (1983)published a method

using 3% beef extract (BE) supplemented

with a chaotropic

agent (NaNO,)

for elution of virus followed by concentration with Cat-floe (Calgon Corporation) which allowed recovery of 42% of poliovirus type 1 seeded into estuarine sediments. Most methods to date have been designed for recovery of virus from marine sediments. The method presented does not use filtration and can be used for the recovery of viruses from both freshwater and marine sediments. MATERIALS

AND METHODS

Virus assay and isolation All extracts were assayed on BGM cells (Barron et al., 1970) provided by Dr. L. Irving, Fairfield Hospital, Melbourne, Australia. BGM cells (passage 50-100) were cultured in medium containing equal parts of Liebovitz medium L-15 (Gibco) and Eagle’s minimal essential medium (MEM, Gibco) containing 10% foetal calf serum and 40 mgfl gentamycin. Extracts from naturally contaminated sediment samples were assayed using an agar suspended cell plaquing technique (Simmonds et al., 1982). Cells were suspended at a concentration of 1 X 107/ml. An 8 ml layer of agar medium (Morris and Waite, 1980) consisting of medium 199 (Gibco) containing 5% foetal calf serum

(Gibco),

1.3% Noble

agar,

0.83 g/l bicarbonate,

0.001%

neutral

red and

antibiotics (gentamycin, 50 ug/ml; kanamycin, 100 ugfml; neomycin, 50 pg/ml; penicillin, 100 U/ml; streptomycin, 100 pg/ml; mycostatin, 5 &ml; and amphoteritin B, 2 ug/ml) was poured into 9 cm diameter vented plastic Petri dishes and allowed to set. Cells (1 ml) and 1 ml of sample were mixed rapidly with 2.5 ml of agar medium kept molten at 46°C and poured at once onto the agar layer. The cultures were incubated for IO days at 37°C in a humid atmosphere containing 5% CO, and the plates examined daily for plaques. Viruses for identification were isolated directly from these plates. All other assays were carried out in Linbro flat bottom microtitre trays. Viral titres were calculated by moving averages (Meynell and Meynell, 1970) and expressed as a 50% tissue culture infectious dose (TCID,,). Virus identification Enteroviruses were identified by neutralisation tests on BGM cells grown on flat bottom micro-titre trays using the Lim Benyesh-Melnick antiserum pools obtained

155

from the United

States National

Institutes

of Health,

Bethesda

(Simmonds

et al.,

1982). Sediments Six aquatic

sediments

with different

compositions

(Table

1) were used for assess-

ing viral adsorption and for developing viral recovery techniques. Sediments were collected and stored at 4°C until used. Samples from a single collection were used for all experiments. Composition of the sediments was established by particle size analysis using a sieving technique and ashing at 600°C to determine the combustible organic matter content (Briggs, 1977). The pH of the sediments ranged from 6.5 to 7.2. Virus adsorption to sediments To assess virus adsorption to the test sediments a known quantity of poliovirus (Pl Mahoney strain) suspended in 20 ml of sterile sea water (pH 6.5, conductivity 39,000 pohms/cm) or river water (pH 6.5, conductivity 30 pohms/cm) was added to 10 g of sediment, agitated on a vortex mixer for 30 set, shaken at 4°C for 30 min and then centrifuged for 10 min at 10,000 X g. The supernatant was assayed to establish the amount of unadsorbed virus. The quantity of virus adsorbed was calculated by subtraction of this amount from the initial inoculum. Control tubes without sediment were included to allow for virus adsorption to the containers. The amount of

TABLE

1

Composition

of aquatic

sediments

Composition Coarse

sand

used for viral adsorption

and elution

experiments

(% dry weight) Sand

and gravel

Silt and

Combustible

clay

organic matter

Particle

size

>0.5

mm

0.5-0.0625

mm

<0.0625

mm

Sediments Marine 1 (Fine) 2 (Sandy) 3 (Gravelly) Fresh water

23

49

15

0

99

0

1

60

28

7

5

0.4

13

0

95.6

4

5 (Sandy)

87

12.2

0

0.8

6 (Gravelly)

68

27.5

3.6

0.9

4 (Fine)

156

virus recovered adsorption

from these tubes was taken to be equal to the initial

experiments

were carried

inoculum.

All

out in triplicate.

Elution and concentration of viruses from sediments Three eluents were tested: 6% beef extract (Difco) at pH 9.0; 2% skim milk (Difco), pH 9.0; and 4 M urea (May and Baker), 0.05 M lysine (Sigma), pH 9.0. Three different virus concentration methods were tested. Pl was added to 300 ml of 6% BE and the suspension

was divided

into three equal volumes

each of which was

subjected to a different concentration method. (I) The pH was adjusted to 3.5, the mixture stirred and left to stand for 15 min. After centrifugation at 10,000 X g for 20 min the pellet was resuspended in tissue culture growth medium containing 5% serum. (2) The same procedure as described above was used except that the BE was diluted 1 : 5 with sterile distilled water prior to pH adjustment. (3) Polyethylene glycol6000 (BDH Chemicals) was added to give a final concentration of 8% and mixed at 4°C for 1 h followed by centrifugation at 10,000 X g for 20 min. The pellet was resuspended in tissue culture growth medium containing 5% calf serum. In a further experiment 150 ml of 6% BE (pH 9.0) was mixed with 50 g of sediment, shaken for 1 h and centrifuged. Poliovirus 1 was added to the supernatant, mixed and then concentrated

by the PEG method

(3).

Virus recovery from sediments The detection method developed was based on a technique already established in our laboratory for recovering human enteroviruses from shellfish (Lewis et al., 1982). Samples (10 g) of sediments to which virus had been adsorbed as described above were mixed with three times the amount (w/v) of 6% BE (Difco) at pH 9.0. This mixture was agitated on a vortex mixer for 30 set then incubated at 4°C for 1 h to allow elution to occur. The samples were then centrifuged at 10,000 X g for 20 min and virus from the supernatant concentration

concentrated

g, the pellet was resuspended containing

by mixing

with polyethylene

of 8%, for at least 1 h. Following

in BGM tissue culture

5% caif serum and stored at -70°C

glycol 6000, to a final

centrifugation medium

for 20 min at lO,~O X (approximately

5 ml)

until assay.

Recovery of indigenous virus To test the effectiveness of the method for recovering enteroviruses from naturally contaminated sediments, 100-200 g samples from freshwater and marine areas near sewer outfalls were collected and extracted within 12 h. RESULTS

Virus adsorption to sediments The results of experiments to determine

the adsorption

of PI to the sediments

are

157

shown

in Table

adsorption

2. The mean adsorption

to freshwater

sediments

to marine

sediments

was slightly lower(67.5%)

was high (98.3%) but when 9.0 X lo5 TCID,,

of Pl was added. Method development When an initial method of virus recovery (elution using 6% BE, pH 9.0 and concentration by acid precipitation) was tested on marine sand and freshwater mud, the virus recovery was only 11% and O.l%, respectively. In order to increase the recovery rates two more eluents (2% skim milk and 4 M urea, 0.05 M lysine, Bitton et al., 1982) were compared to 6% BE. After elution (1 h for BE and skim milk and 10 min for urea-lysine) and centrifugation the eluents were assayed. The results (Table 3) show that both were inferior to 6% BE for eluting Pl from the test sediments. While acid precipitation was used initially for concentration of Pl from the beef extract supernatant, polyethylene glycol 6000 (PEG) (BDH Chemicals) was also tested. PEG 6000 has been shown to be effective for concentrating viruses from tissue culture fluid, allantoic fluid and pond water on a number of occasions (Heyward et al., 1977; Hamelin and Lussier, 1979; Markwell and Shortridge, 1982). A modified acid precipitation concentration was also tested in which the BE was diluted 1 : 10 in distilled water prior to adjustment to pH 3.5. This method was included as dilution in this manner had previously been found to enhance virus recovery from shellfish extracts by acid precipitation (Lewis et al., 1982). The results of all concentration experiments are given in Table 4. PEG appears to be superior to both of the acid precipitation methods for concentrating Pl from BE. Even at low viral input levels and in the presence of sediment factors, which may be expected to reduce the concentration efficiency, recovery is still 76-92%.

TABLE

2

Adsorption

of poliovirus

Sediment

1 to marine

PI Adsorption (% f SD)

Marine I (Fine)

99.4 f

2 (Sandy)

95.6 f

3.4

100.0 -I

0.1

14.1

3 (Gravelly)

0.5

Freshwater 4 (Fine)

69.3 f

5 (Sandy)

36.7 f

1.3

6 (Gravelly)

96.9 f

3.2

and freshwater

sediments

using an input of 9.0 X IOs TCID,,

158

TABLE Elution

3 of poliovirus

Sediment

la from marine

and freshwater

sediments

using 3 different

eluents

Eluentsb 6% Beef extract

2% Skim milk

4 M Urea-

(PH 9.0)

(PH 9.0)

0.05 M lysine W

9.0)

Marine 1 (Fine)

15.5 J:

3’

7.1 $:.5

2 (Sandy)

53.3 *

8

6.7 zk 5

3 (Gravelly)

25.5 f

5

10.9 I 5

4 (Pine)

43.1 + 16

5 (Sandy)

35.8 *

7

153.0 4 20

1.9 f

0.5

31.3 f 23 11.2 f

4

20.2 f 4

33.7 *

8

22.7 4 7

27.2 k

8

12.9 rl: 4


Freshwater

6 (Gravelly)

a Virus input level (the amount

of virus adsorbed

’ Sediment

3.

to eluent ratio = 1:

’ 9%Recovery carried

f

SE. Eiution

was measured

to each sediment

by assay of the elution

type) = I X IO6 TCID,,. supernatant,

no concentration

was

out.

It was found that recovery of viruses from freshwater sediments could be increased by the addition of 2% NaCl at the same time as the PEG 6000 giving a recovery of 60-70% for a viral input of approximately 1 X lo3 TCID,,. The addition of NaCl did not enhance the recovery of viruses from marine sediments. The elution and concentration methods as described in Materials and Methods were then tested on the six test sediments. The results for the efficiency of recovery of PI from the test sediments are shown in Table 5. Recovery of indigenous enteric viruses

The recovery method as established and which is given in the Materials and Methods was used for testing for viruses in sediments collected near seweroutfalls. The results are shown in Table 6. Virus isolates obtained from these samples were identified where possible, with poliovirus types 1, 2 and 3 and coxsackievirus B5 being recovered. Clearly enteric viruses do occur in sediments and can be concentrated and detected by this method. These results also show that enteric viruses can be detected as far as 4 km from their point of entry.

159

TABLE

4

Comparison

of concentration

methods

for recovering

PI from 6% BE after viral elution Concen-

Concentration

Virusa

Virus

Recovery

method

input

recovered

(% f SD)

(TCfD,,)

(TCfD,,)

4.65 X lo6

7.30 x 104

1.6f

1

0.1

4.65 X lo6

4.71 x 104

l.Of

1

0.5

4.65 X lo6

5.54 x 106

119.1 f

10

14.5

1.43 x 102

1.09 x 102

76.2 f

4

18.2

1.43 x to*

1.32 X IO2

92.3 +

5

17.5

Acid precipitation,

tration factorb

pH 3.5 (100 ml BE + Pl)’ Acid precipitation, pH 3.5 + 1 : 5 dilution in distilled

water

(100 ml BE f Pl) 8% PEG 6000 (100 ml BE + PI) 8% PEG 6000 (150 ml + sediment

1

+ Pl) 8% PEG 6000 (150 ml + sediment

3

+ Pl) Virus input equals Concentration

factor

Composition its volume

Overall

=

of virus added

Virus recovered Virus input

to the BE mixture just prior to concentration.

X initial volume

X final volume.

of sample to be concentrated.

Sediments

8% PEG 6000 = polyethylene

’

were incorporated

of 6% BE (pH 9.0) for 10 min and centrifuging.

BE = beef extract;

TABLE

the total amount

by mixing sediment with 3 times

The supernatant

was then concentrated.

glycol 6000 to a final concentration

of 8%.

5 efficiency

Sediment

of the virus detection Virus inputa TCID,dlOO

technique

for recovering

poliovirus

Recovery g

(% f SD)

Marine 1 (Fine)

6.78 X lo4

17.8 f 20

2 (Sandy)

6.96 X LO4

55.8 f 29

3 (Gravelly) Freshwater

6.69 X IO4

4 (Fine)

4.90 x 104

15.8 f

4

5 (Sandy)

3.39 x 105

39.5 *

4

6 (Gravelly)

8.72 X IO5

76.8 f

0

a Virus input equals

the amount

6.3f

4

of virus adsorbed

to the sediment.

1 from six different

sediments

160

TABLE

6

Naturally

occurring

viruses isolated

Site

Sediment

Silverstream Taieri River

from sediments

type

Distance

pfu/lOO

from outfall

sediment

Isolate?

and

identification

20

m

62

3/5

P2X1, P3X2

FW mud

100

m

6

1/l

CBS

km

4

l/l

P3

100

m

3

l/l

Pl

50

m

300

Marine

gravel

Head

Marine

sand

Smaills Beach

Marine

sand

Lawyers

g

FW sand

4 Oamaru

near sewer outfalls

2.5 km

1l/42 P2X6 UIX5

2400

12/30 P2X8, CBSX3,

a Plaques

subcultured

poliovirus

for identification/plaques

3; CB5 = coxsackievirus

occurring.

B5; UI = unidentified;

Pl = poliovirus

UIXl

1; P2 = poliovirus

pfu = plaque forming

2; P3 =

unit; FW =freshwater.

DISCUSSION

Consideration of the data relating to adsorption of Pl to sediments showed that some sediments appeared to adsorb virus more strongly than others, notably marine sediments and those containing a high proportion of fine particulate material, <0.0625 mm diameter (Table 2). Adsorption of viruses to solids is enhanced by the presence of cations (Wallis et al., 1967; Floyd, 1979; Bitton, 1980) and this, when the high ionic concentration of marine sediments is considered, may explain the observed difference in the adsorption of viruses to marine and freshwater sediments. This dissimilarity was adsorbed The presence enhance

has been reported to marine

previously.

sediments,

of fine particulate

the adsorption

Bitton et al. (1982) observed

while only 40% adsorbed matter

of viruses.

(<0.0625

to

that 99% of Pl

freshwater

mm diameter)

sediments.

also appears

This may be due to the greater

surface

to area

available in these sediments or the presence of clays to which viruses will readily bind (Bitton, 1975, 1980). This last factor probably accounts for the differences in viral adsorption apparent within the marine and freshwater sediment groups, particularly in the low level of association

of Pl with sediment

5, a coarse freshwater

sand.

The efficacy of any viral recovery technique involving sediments depends primarily on the initial elution step. The effectiveness of this step clearly relates to both the eluent and the type of sediment tested. Six percent BE was chosen as the best of the three eluents in this study, as it proved the most effective for all of the sediments tested (Table 3). Urea-lysine was used with some success by Bitton et al. (1982) and the poor results obtained with that eluent in this study may be due to differences in sediment composition. Six percent BE also has the added advantage that it is easily obtainable, simply prepared and stored and does not appear to have any adverse effect on the viruses. Three percent BE was also used for eluting viruses from marine sediments by Tsai et al. (1983) with some success.

161

Reconcentration ing 76-92%

of eluted virus. The resulting

low to negligible Variations results.

of virus using 8% PEG proved simple, cheap and effective returnextract

was generally

less than 5 ml and of

toxicity.

between

brands

The PEG 6000 (BDH

and batches Chemicals)

of BE and PEG may produce used in this study performed

variable

consistently

better than a second powdered type tested (Union Carbide). BE has also been shown to vary markedly even within batches from the same supplier (Wait and Sobsey, 1983; Melnick et al., 1984) and the quality and effectiveness of different batches should be assessed prior to use in a virus detection method such as that discussed here. Although the detection method for both marine and freshwater sediments was developed using Pl, results in Table 6 show that it is also effective for other virus types with poliovirus 2 and 3 and coxsackievirus B5 being isolated as frequently as Pl. The overall efficiency of the method presented in this paper is 15.8-76.8% for freshwater sediments and 6.3-55.8% for marine sediments and compares favorably with those discussed in the literature. For example, Gerba et al. (1977) was able to recover 50% of Pl from estuarine sediments by eluting with glycine-EDTA at pH 11.0 and concentrating by filtration. In this method the high pH of the eluent will cause viral inactivation with prolonged exposure so extreme care must be taken (Tsai et al., 1983), and in addition the use of filtration can prove difficult for such samples. Bitton et al. (1982) recovered 8-22% of virus added to marine sediments and 23-59% from freshwater sediments using a urea-lysine elution technique and concentration by flocculation. This method also requires care due to the ability of urea to cause viral inactivation (Tsai et al., 1983). Recoveries using the method described in this paper are similar to those of Wait and Sobsey (1983) who recovered

42% of PI seeded into estuarine

sediments

using 3% BE

and NO, at pH 5.5 as an eluent and concentration of the virus with Cat-floe. Although Cat-floe was used with considerable success by these investigators (Wait and Sobsey, 1983) it could not be tested in this study due to its unavailability. Direct comparison of methods is difficult particularly as the sediment type tested appears to have considerable effect on the overall efficiency of the technique. The method presented is considerably shorter than those cited, which may take in excess of 24 h, and requires only 3.5 h for completion; step without risk of viral inactivation.

if necessary it can be haltedat

any

Using this method up to 2,400 pfu/lOO g of sediment could be recovered from sediments in the vicinity of sewer outfalls and virus could still be detected up to 4 km from the nearest known source of contamination. The technique is currently being used to study the occurrence and the interactions of enteric viruses in marine and freshwater sediments in New Zealand. ACKNOWLEDGEMENT

This work was partially supported and Soil Conservation Organisation.

by a research

contract

from the National

Water

162

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