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Effectiveness of the organic flocculation method in concentrating echovirus 7 and coxsackievirus A9 from water
Waler Re.arch Vol. 15, pp. 13 to 15 Pergamon Press Ltd 1981. Ih.mted in Great ~'itain
0043-13M/gI~I01-0013102.00/O
EFFECTIVENESS OF THE ORGANIC FLOCCULATION METHOD IN CONCENTRATING ECHOVIRUS 7 AND COXSACKIEVIRUS A9 FROM WATER S. K ~ M I
and B. FA'I'rAL
Environmental Health Laboratory, The Hebrew University, Hadassah Medical School, Jerusalem, Israel (Received December 1979) A~ract--The possible application of the adsorption--l~/o beef extract elution and organic flocculation concentration method to non-polio enteroviruses was examined. Echovirus 7 and coxsackievirus A9 were concentrated from seeded tap water and good recovery efliciencieswere obtained,
filtered through a microfiber cartridge filter (Balston, Grade AA, 0.3/an pore size); and elutlon was conducted with 300 ml of l~g beef extract pH 9. The volume of the final concentrate was about l 0 ml. In experiments with 5 and 101. water samples seeded with a high virus input, 1.5 × 105-6.9 x 106 pfu, the concentrate was assayed by titration. In experiments with 100-1. water samples seeded with a low virus input, 1-35 pfu, all the concentrate was inoculated in replicate tissue-culture plates.
INTRODUCTION
Outbreaks of water-borne viral diseases have been reported in recent years by several laboratories. These have involved different viruses and different types of water (Goldfield, 1976). It is important to develop a concentration method that is capable of detecting low levels of a wide range of viruses in water, and that has a good recovery efficiency. A two-step concentration method involving adsorp-" tion-beef extract elution and organic flocculation (Katzenehon et al., 1976) was developed using pp.lioIllegULT$ virus type I as a modeL The method was found to be most suitable for the detection of polioviruses in Control concentration experiments using drinking water drinking water, with a mean recovery efficiency of seeded with po|iovir~ I
75%.
In order to confirm the recovery efficiency of the modified experimental procedure here described, as compared to the original method, several concentration experiments were conducted with poliovirus I (Table I). As can be seen, the mean ~o recovery efficiency for small and large volumes of water and for different virus inputs was 90 + 14. Based on the good recovery efficiency with pofiovirus I, experiments with echovirus 7 and coxsackievirus A9 were carried out with the same procedure. The final concentration factor was x I0,000. All samples, dilutions with beef extract pH 7, and the final concentrates contained 10% of the antibiotic solution and were stored at - 8 0 ° C until plaque assayed.
The present study involved the application of the method to non-polio enteroviruses commonly found in sewage. For this purpose, echovirus 7 and coxsackievirus A9 were chosen as representatives. MErHODS Virus and viral assay Concentration experiments were conducted with echovirus 7 and coxsackievirus A9 which were chosen to represent non-polio enteroviruseg Before each experiment, the stock viruses which were stored at -80°C, were thawed and diluted according to the experimental scheme. The viruses were assayed by the plaque method (Shuval et aL, 1971).and the results given in plaque-forming units (pfu). BGM cells (Dahling et aL, 1974) were used for viral assay. They were maintained with RPMI 1640 medium contain. ing bicarbonate, 4 mM glutamine, antibiotic solution (penicillin 200 g, streptomycin 200 ~g, kanamycin 200 ~ neomycin 25~g, mycostatin 100/tm1-1 final concentration) and 10% (final concentration) fetal bovine serum (Flow Laboratories). Experimental procedure Experiments conducted with poliovirus I, .echovirus 7 and coxsackievirus A9 involved small and large volumes of tap water with different virus inputs. The seeded water was concentrated according to the adsorption.clution and organic flocculation method previously described by Katzenelson et al. (1976), with two modifications: the water was
Concentration of drinking water seeded with echovirus 7 Three sets of experiments using ec~ovirus 7 involved high, int~i~iediate and low virus inputs (Table 2). These also involved small and large volumes of water, with a mean conductivity of 0,52 mMho era-1 The volume of the water sample and the amount of virus seeded did not significantly change the efficiency of the method and the mean recovery was 84 +_ 20. It is worthwhile to point out that only a very low percentage of virus (0.1-0.4~o) •was detected in the filtrates of the high input experiments.
13
14
S. KEDMI and B. FATTAL Table 1. Control concentration experiments with poliovirus I in water* Vol. of sample (1,)
Exp. no,
% Beef extract
Calculated input (pfu)
1
5
1
3.5 × 103
2 3 4 5
5 10 10 100
3 1 1 1
3.5 1,1 7.0 1.0
x x x x
% Recovery
105 l0 s 104 10~
103 86 72 83 105 90 +- 14
Mean Standard deviation
* All concentration experiments were run with microfiber cartridge filters (Balston, Grade AA--pore size 0.3 ,amL Table 2. Recovery et~ciency of seeded echovirus 7 in water Calculated total input (pfu)
Exp. no.*
2.7 x 10e
1
2 3 4 5 6 7 8 9 10 11 t2 13 14
Z5 5.2 6.9 1.5 Z0 1.7 1.5 1.5 1.0 7.0 1.4 1.3 3.5
x x x x x x x x x x x x x
I06 106 I06 103 I03 103 103 103 101 10° 10~ 101 101
Filtrate 0.2 0.3 0.1 0.4 NT~ NT NT NT NT NT NT NT NT NT
% Recovery Final concentration* 69 95 65 113 107 83 103 104 53 90 57 78 100 65 8 4 + 20
M e a n Standard deviation
* Experiments 1-9 involved 5-1. samples. Experiments 10--14 involved 100-1. samples. t Two-step concentration by adsorption-l% beef extract elution and organic llocculation. ~: NT not tested.
Concentration of drinking water seeded with coxsackievirus A 9 Table 3 summarizes experimonts using coxsackievirus A9 which was inoculated at high inputs to small volume samples, and at low inputs to large volume
samples. The m e a n % recovery was 87 + 17. During the period these experiments were conducted, the mean conductivity of the water was 0.67 m M h o c m - z In the filtrates of high input experiments, the level of virus was barely detectable (0. 1%).
Table 3. Recovery efficiency of seeded coxsackievirus A9 in water Exp. no*.
Calculated total input (pfu)
Filtrate
1 2
1.2 x 106 2.3 x 106
0 ~1
3 4 5 6
1.0 6.0 3.0 2.0
NT~ NT NT NT
Mean Standard deviation
× × x x
I0 ° 10° 10° 101
% Recovery Final concentrationt 116
70 100 83 85 65 87 +- 17
*Experiments 1-2 involved 5-L samples. Experiments 3-6 involved 100-1. samples. t Two-step concentration by adsorption--l% beef extract elution and organic flocculation. ++NT not tested.
Effectiveness of the organic flocculation method DISCU~ION
The combination of adsorption-beef.extract elution and organic floeculation has been found efficient in concentrating polioviruses from drinking water (Katzenelson et al., 1976) and wastewater (Katzenelson & Kedmi, 1979; Landry et al., 1978). It was necessary to widen the range of applications of this method to different qualities of drinking water, to other natural waters and to other common waterborne viruses. With this in mind, the effectiveness of the concentration method using echovirus 7 and coxsackievirus A9 was examined. The experiments involved a wide range of virus inputs, 1 x 10-2-1.4 x 106pfu1-1. Within this range the mean % recovery was 84 + 20 for echovirus 7 and 87 ± 17 for coxsackievirus A9. This efficiency correlated well with that of control experiments conducted with poliovirus I, as well as with similar experiments previously conducted by Katzeneison et al. (1976). The procedure involved a modification in the type of filter used and ~o of beef extract (17/o). Good recovery efficiencies in those conditions have been reported also by Landry et al. (1978). In field experiments with raw sewage and seawater using the above described procedure a wide range of enteroviruses was detected (Shuval & Fattal, 19791 The data reported in this study indicate the applicability and good recovery efficiency of the beef
15
extract organic floceulation method to commonly found enteroviruses other than polioviruses. Acknowledoement--This research was supported by the US
Environmental Protection Agency, Grant No. T-806588. REFERENCES
Dahling D. R., Berg G. & Berman D. (1974) B G M , a continuous cellline more sensitivethan primary Rhesus and African Green kidney cells for the recovery of viruses from water. Hlth Lab. Sci. 4, 275-282.
Goldfield M. (1976) Epidemioiogical indicators for transmission of viruses by water. In Viruses in Water (Edited by Berg G.. Bodily H., Lcnnette E., Mehaick J. & Metcalf T. pp. 70-85. American Public Health Assoc., Inc, Katzcnelson E. & Kcdmi S. 0979) Unsuitability of polioviruses as indicators of virological quality of water. Appl. Envir. Microbiol. 37, 343-344. Katzenelson E., Fattal B. & Hostovsky T. (1976) Organic flecculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. envir. Microbiol. 32, 638-639. Landry E. F., Vaughn J. M., McHarrell Z. T. & Vicale T. J. (1978) Efficiency of beef extract for the recovery of polioviruses from wastewater effluents. AppL Envir. Microbiol. 36, 544-548. Shuval H. I. & Fattal B. (1979) Coastal quality monitoring of recreational and shellfish areas (MED VII), pp. 54-64. WHO Workshop, Rome. Shuval H. I. Thompson A., Fattal B.. Cymbalista S. & Wiener Y. (1971) Natural virus inactivation processes in sea water, d. sanit, engng Die. Am. Soc. cir. Enos 97, 587-600.
0043-13M/gI~I01-0013102.00/O
EFFECTIVENESS OF THE ORGANIC FLOCCULATION METHOD IN CONCENTRATING ECHOVIRUS 7 AND COXSACKIEVIRUS A9 FROM WATER S. K ~ M I
and B. FA'I'rAL
Environmental Health Laboratory, The Hebrew University, Hadassah Medical School, Jerusalem, Israel (Received December 1979) A~ract--The possible application of the adsorption--l~/o beef extract elution and organic flocculation concentration method to non-polio enteroviruses was examined. Echovirus 7 and coxsackievirus A9 were concentrated from seeded tap water and good recovery efliciencieswere obtained,
filtered through a microfiber cartridge filter (Balston, Grade AA, 0.3/an pore size); and elutlon was conducted with 300 ml of l~g beef extract pH 9. The volume of the final concentrate was about l 0 ml. In experiments with 5 and 101. water samples seeded with a high virus input, 1.5 × 105-6.9 x 106 pfu, the concentrate was assayed by titration. In experiments with 100-1. water samples seeded with a low virus input, 1-35 pfu, all the concentrate was inoculated in replicate tissue-culture plates.
INTRODUCTION
Outbreaks of water-borne viral diseases have been reported in recent years by several laboratories. These have involved different viruses and different types of water (Goldfield, 1976). It is important to develop a concentration method that is capable of detecting low levels of a wide range of viruses in water, and that has a good recovery efficiency. A two-step concentration method involving adsorp-" tion-beef extract elution and organic flocculation (Katzenehon et al., 1976) was developed using pp.lioIllegULT$ virus type I as a modeL The method was found to be most suitable for the detection of polioviruses in Control concentration experiments using drinking water drinking water, with a mean recovery efficiency of seeded with po|iovir~ I
75%.
In order to confirm the recovery efficiency of the modified experimental procedure here described, as compared to the original method, several concentration experiments were conducted with poliovirus I (Table I). As can be seen, the mean ~o recovery efficiency for small and large volumes of water and for different virus inputs was 90 + 14. Based on the good recovery efficiency with pofiovirus I, experiments with echovirus 7 and coxsackievirus A9 were carried out with the same procedure. The final concentration factor was x I0,000. All samples, dilutions with beef extract pH 7, and the final concentrates contained 10% of the antibiotic solution and were stored at - 8 0 ° C until plaque assayed.
The present study involved the application of the method to non-polio enteroviruses commonly found in sewage. For this purpose, echovirus 7 and coxsackievirus A9 were chosen as representatives. MErHODS Virus and viral assay Concentration experiments were conducted with echovirus 7 and coxsackievirus A9 which were chosen to represent non-polio enteroviruseg Before each experiment, the stock viruses which were stored at -80°C, were thawed and diluted according to the experimental scheme. The viruses were assayed by the plaque method (Shuval et aL, 1971).and the results given in plaque-forming units (pfu). BGM cells (Dahling et aL, 1974) were used for viral assay. They were maintained with RPMI 1640 medium contain. ing bicarbonate, 4 mM glutamine, antibiotic solution (penicillin 200 g, streptomycin 200 ~g, kanamycin 200 ~ neomycin 25~g, mycostatin 100/tm1-1 final concentration) and 10% (final concentration) fetal bovine serum (Flow Laboratories). Experimental procedure Experiments conducted with poliovirus I, .echovirus 7 and coxsackievirus A9 involved small and large volumes of tap water with different virus inputs. The seeded water was concentrated according to the adsorption.clution and organic flocculation method previously described by Katzenelson et al. (1976), with two modifications: the water was
Concentration of drinking water seeded with echovirus 7 Three sets of experiments using ec~ovirus 7 involved high, int~i~iediate and low virus inputs (Table 2). These also involved small and large volumes of water, with a mean conductivity of 0,52 mMho era-1 The volume of the water sample and the amount of virus seeded did not significantly change the efficiency of the method and the mean recovery was 84 +_ 20. It is worthwhile to point out that only a very low percentage of virus (0.1-0.4~o) •was detected in the filtrates of the high input experiments.
13
14
S. KEDMI and B. FATTAL Table 1. Control concentration experiments with poliovirus I in water* Vol. of sample (1,)
Exp. no,
% Beef extract
Calculated input (pfu)
1
5
1
3.5 × 103
2 3 4 5
5 10 10 100
3 1 1 1
3.5 1,1 7.0 1.0
x x x x
% Recovery
105 l0 s 104 10~
103 86 72 83 105 90 +- 14
Mean Standard deviation
* All concentration experiments were run with microfiber cartridge filters (Balston, Grade AA--pore size 0.3 ,amL Table 2. Recovery et~ciency of seeded echovirus 7 in water Calculated total input (pfu)
Exp. no.*
2.7 x 10e
1
2 3 4 5 6 7 8 9 10 11 t2 13 14
Z5 5.2 6.9 1.5 Z0 1.7 1.5 1.5 1.0 7.0 1.4 1.3 3.5
x x x x x x x x x x x x x
I06 106 I06 103 I03 103 103 103 101 10° 10~ 101 101
Filtrate 0.2 0.3 0.1 0.4 NT~ NT NT NT NT NT NT NT NT NT
% Recovery Final concentration* 69 95 65 113 107 83 103 104 53 90 57 78 100 65 8 4 + 20
M e a n Standard deviation
* Experiments 1-9 involved 5-1. samples. Experiments 10--14 involved 100-1. samples. t Two-step concentration by adsorption-l% beef extract elution and organic llocculation. ~: NT not tested.
Concentration of drinking water seeded with coxsackievirus A 9 Table 3 summarizes experimonts using coxsackievirus A9 which was inoculated at high inputs to small volume samples, and at low inputs to large volume
samples. The m e a n % recovery was 87 + 17. During the period these experiments were conducted, the mean conductivity of the water was 0.67 m M h o c m - z In the filtrates of high input experiments, the level of virus was barely detectable (0. 1%).
Table 3. Recovery efficiency of seeded coxsackievirus A9 in water Exp. no*.
Calculated total input (pfu)
Filtrate
1 2
1.2 x 106 2.3 x 106
0 ~1
3 4 5 6
1.0 6.0 3.0 2.0
NT~ NT NT NT
Mean Standard deviation
× × x x
I0 ° 10° 10° 101
% Recovery Final concentrationt 116
70 100 83 85 65 87 +- 17
*Experiments 1-2 involved 5-L samples. Experiments 3-6 involved 100-1. samples. t Two-step concentration by adsorption--l% beef extract elution and organic flocculation. ++NT not tested.
Effectiveness of the organic flocculation method DISCU~ION
The combination of adsorption-beef.extract elution and organic floeculation has been found efficient in concentrating polioviruses from drinking water (Katzenelson et al., 1976) and wastewater (Katzenelson & Kedmi, 1979; Landry et al., 1978). It was necessary to widen the range of applications of this method to different qualities of drinking water, to other natural waters and to other common waterborne viruses. With this in mind, the effectiveness of the concentration method using echovirus 7 and coxsackievirus A9 was examined. The experiments involved a wide range of virus inputs, 1 x 10-2-1.4 x 106pfu1-1. Within this range the mean % recovery was 84 + 20 for echovirus 7 and 87 ± 17 for coxsackievirus A9. This efficiency correlated well with that of control experiments conducted with poliovirus I, as well as with similar experiments previously conducted by Katzeneison et al. (1976). The procedure involved a modification in the type of filter used and ~o of beef extract (17/o). Good recovery efficiencies in those conditions have been reported also by Landry et al. (1978). In field experiments with raw sewage and seawater using the above described procedure a wide range of enteroviruses was detected (Shuval & Fattal, 19791 The data reported in this study indicate the applicability and good recovery efficiency of the beef
15
extract organic floceulation method to commonly found enteroviruses other than polioviruses. Acknowledoement--This research was supported by the US
Environmental Protection Agency, Grant No. T-806588. REFERENCES
Dahling D. R., Berg G. & Berman D. (1974) B G M , a continuous cellline more sensitivethan primary Rhesus and African Green kidney cells for the recovery of viruses from water. Hlth Lab. Sci. 4, 275-282.
Goldfield M. (1976) Epidemioiogical indicators for transmission of viruses by water. In Viruses in Water (Edited by Berg G.. Bodily H., Lcnnette E., Mehaick J. & Metcalf T. pp. 70-85. American Public Health Assoc., Inc, Katzcnelson E. & Kcdmi S. 0979) Unsuitability of polioviruses as indicators of virological quality of water. Appl. Envir. Microbiol. 37, 343-344. Katzenelson E., Fattal B. & Hostovsky T. (1976) Organic flecculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. envir. Microbiol. 32, 638-639. Landry E. F., Vaughn J. M., McHarrell Z. T. & Vicale T. J. (1978) Efficiency of beef extract for the recovery of polioviruses from wastewater effluents. AppL Envir. Microbiol. 36, 544-548. Shuval H. I. & Fattal B. (1979) Coastal quality monitoring of recreational and shellfish areas (MED VII), pp. 54-64. WHO Workshop, Rome. Shuval H. I. Thompson A., Fattal B.. Cymbalista S. & Wiener Y. (1971) Natural virus inactivation processes in sea water, d. sanit, engng Die. Am. Soc. cir. Enos 97, 587-600.