Chlorine Resistance of Poliovirus Isolants Recovered from Drinking Water

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 198, p /8/ /7$2./ Vol. 4, No. 6 Chlorine Resistance of Poliovirus Isolants Recovered from Drinking Water PETER T. B. SHAFFER,`* THEODORE G. METCALF,2 AND OTIS J. SPROUL3 Electro Minerals Division, The Carborundum Company, a subsidiary of the Kennecott Company, Niagara Falls, New York 14321; Department ofmicrobiology, University of New Hampshire, Durham, New Hampshire 38242; and Department of Civil Engineering, Ohio State University, Columbus, Ohio Poliovirus 1 isolants were recovered from finished drinking water produced by a modem, well-operated water treatment plant. These waters contained free chlorine residuals in excess of 1 mg/liter. The chlorine inactivation of purified high-titer preparations of two such isolants was compared with the inactivation behavior of two stock strains of poliovirus 1, LSc and Mahoney. The surviving fraction of virus derived from the two natural isolants was shown to be orders of magnitude greater than that of the standard strains. These results raise the question whether indirect drinking water standards based on free chlorine residuals are adequate public health measures, or whether direct standards based on virus determinations might be necessary. Disinfection studies, based on the use of standard laboratory strains of enteric viruses, have led to the belief that viruses will not survive for an extended period of time in the presence of free chlorine and in the absence of solids which might physically protect the virion (5, 6). Other factors such as ph, the presence of organic matter, and temperature have been shown to have a strong influence on the rate of virus inactivation. Evidence contradictory to this belief has been obtained which indicates that at least some enteric viruses may be able to survive water plant treatment practices considered adequate for disinfection of virus. Poliovirus isolants were recovered from finished waters containing free chlorine residuals in excess of 1 mg/liter and turbidities less than 1 turbidity unit. Bacterial indicator organisms, shown as total plate counts, were absent in each case (12). Recent laboratory studies have shown that poliovirus 1, strain LSc, will exhibit progressively greater resistance to inactivation by chlorine after repeatedly exposing the virus to sublethal doses of chlorine followed by growth of the survivors in cell cultures (2, 3). The studies reported here were carried out to determine whether the polioviruses recovered from fmished drinking water might exhibit a similar increased resistance toward chlorine. Demonstration of such an increased resistance to chlorine would help to explain how enteric viruses could survive water plant treatments, including disinfection, and appear in finished waters containing adequate free chlorine residuals MATERIALS AND METHODS Natural virus isolants. An extended program to monitor the Occoquan Reservior water system in Fairfax County in Northern Virginia and the drinking waters produced from it was carried out by The Carborundum Co., a subsidiary of the Kennecott Co., and the Occoquan Monitoring Laboratory (12) Weekly samples were collected during the period from June through September. Sampling sites included three locations on the supply reservoir and upstream watershed and three sites on the finished water distribution system. Water samples examined for enteric viruses were processed by methods previously described by Shaffer et al. (18), which were essentially those described in the 14th edition of Standard Methods (1). Typically, 38-liter samples were tested. To maximize virus adsorption, ph was adjusted to 3.5, and aluminum chloride was added to a final concentration of.5 M. Where necessary, sodium thiosulfate was added to eliminate free chlorine residuals. Ten-inch (ca cm) fiberglass cartridge filters (K27RIOS, Fulflo filter, Filters Division, The Carborundum Co., a subsidiary of the Kennecott Co., Lebanon, Ind.) and 293-nm epoxy asbestos fiberglass assemblies (1.- and.45-,um porosities, type AA, Cox Filter Division, Detroit, Mich.) were used in series to adsorb the viruses. Viruses were recovered from these filters by elution with.5 M glycine buffer solutions at ph Eluates were reconcentrated by using similar Cox filter assemblies after adjustment to ph 3.5 and aluminum chloride concentrations of.5 M. Final volumes of approximately 2 ml were obtained after adjustment of the final test sample to ph 7, isotonicity, and the addition of 1% by volume of heat-inactivated fetal bovine serum (GIBCO Laboratories, Grand Island, N.Y.). Sample concentrates were frozen and shipped under dry ice to the virus assay laboratory where they were maintained at -7 C until tested for virus.

2 1116 SHAFFER, METCALF, AND SPROUL Each sample was tested in its entirety equally on Buffalo green monkey (BGM) and African green primary monkey kidney (PMK) monolayers after ether treatment to inactivate bacterial and other contaminants. Inocula of.2 ml/cm2 of monolayer were adsorbed for 1.5 h, after which each monolayer was overlaid with agar overlay medium (8). All plaques were confirmed as viral plaques, and isolants were identified by serum neutralization tests with Lim-Benyesh-Melnick antiserum pools (15). Marker tests by the rct/4 marker procedure of Carp and Koprowski (4) were used to determine whether isolants possessed vaccine- or nonvaccinetype characteristics. Known vaccine (LSc strain from J. L. Melnick, Baylor College of Medicine, Houston, Tex.) and nonvaccine (Mahoney strain, from H. Wenner, Kansas University Medical Center, Kansas City, Kans.) strains of poliovirus 1 were used as controls. Water quality assessment. Indicators of water quality, measured at the time each virus sample was collected, included ph, turbidity, chlorine residuals, and bacterial indicator organisms. Turbidity was measured with a Hach Turbidimeter, free chlorine residuals were measured by the N,N-diethyl-p-phenylenediamine method (1), and bacterial indicator organisms were measured as total plate counts. All tests were carried out in accordance with methods described in Standard Methods (1). In each case associated with a virus recovery, turbidity was less than 1 turbidity unit, and bacterial indicators were absent. Virus preparations. Pure, high-titer concentrates were prepared from two selected poliovirus isolants and two reference strains (LSc and Mahoney) by procedures described by Guskey and Wolff (11) and by Sharp (19). Virus suspensions grown on roller tube monolayers were concentrated by hydroextraction with polyethylene glycol, Freon extracted, and banded at 13, x g with cesium chloride. Each preparation was divided into 1-ml aliquots and stored at -7 C. Disinfection. All of the water used for dilution, preparation of chemicals, buffers, etc., and final rinsing of apparatus was chlorine demand-free triple-distilled water. To prepare chlorine demand-free water, 3 mg of free chlorine per liter was added to triple-distilled water, which was then stored overnight in the dark. The active chlorine residual was eliminated by irradiating the water with ultraviolet light. All glassware was exposed to 5 mg of free chlorine per liter for at least 12 h and then rinsed several times with demandfree water. The dilution water used in the disinfection study was a solution of.1 M CaCl2 in demand-free water. The ph was adjusted to 7.1 ±.1 with.25 M NaHCO3. The temperature was 21 C. A typical experiment was carried out as follows. (i) Sufficient virus was added to 75 ml of.1 M CaCl2 solution at ph 7.1 to yield an initial titer of approximately 1 x 15 plaque-forming units per ml. This required the following amounts of stock virus:.75 ml of test I,.15 ml of test II,.25 ml of LSc, and.25 ml of Mahoney. (ii) The virus suspension was mixed for 45 s at 8 rpm with a laboratory stirring machine with paddles the size of a standard glass microscope slide (25 by 1 APPL. ENVIRON. MICROBIOL. mm). An aliquot (5 ml) was taken for initial virus determination. (iii) Sufficient volume of 1 mg of free chlorine stock solution of sodium hypochlorite per ml was added to provide the desired free chlorine concentration. (iv) The chlorine-virus preparations were mixed for 3 s at 8 rpm. Contact time was measured from the end of this 3-s mixing period. (v) Aliquots (5 ml) were taken after 2, 1, and 3 min and immediately transferred to solutions of excess Na2S23 to destroy active chlorine residuals. Suitable dilutions were made before plating. (vi) Residual chlorine measurements were made at the end of the 3-min contact by the amperometric titration method (1). Virus assays were made by the plaque procedure method described by Dulbecco and Vogt (9) with LLC- MK2 monolayers. Duplicate assays of each of three virus dilutions were made. Each datum point determined was the average of plaque counts from all dilutions assayed. Electron microscopy. Electron microscope examinations to determine the state of dispersion of virus test preparations were carried out by the kinetic attachment to aluminized film method (19). RESULTS Natural virus isolants. During the Occoquan study, 11 poliovirus isolants were recovered from finished waters. The details are shown in Table 1. rct/4 marker tests for eight of the isolants showed that each had an rct/4 value different from the LSc control (Table 2). The rct/4 values, which suggested a nonvaccine virus character, indicated the eight isolants might be considered representative of a spectrum of nonvaccine types possessing slightly different temperature marker characteristics. These observations were confirmed by M. Hatch (Center for Disease Control, Atlanta, Ga.) who examined six isolants by means of rct/39.5 tests and antigenic characterization tests using a modified TABLE 1. Poliovirus recoveries from finished water (11) Date Free chlorine Plaques residual (mg/liter) BGM PMK 3 June June July August a Each water sample consisted of 378 liters, concentrated to yield a final concentrate of 2 to 4 ml, all of which was placed equally on the two types of tissue cultures. Bacterial indicators, measured as total plate counts, were negative in every case. Free chlorine residuals were measured at the end of the experiment. Bacterial and chlorine determinations were by procedures described in Standard Methods (1).

3 VOL. 4, 198 Wecker antigenic differentiation procedure. rct/ 39.5 ± reactions were obtained with three isolants. Chlorine demand. To rule out complicating effects of free versus combined chlorine inacti- TABLE 2. rct/4 data on polioviruses recovered from finished water Isolant r rct/4 5 June la 1.2 x 15/1.2 x 16 = x 14/5.9 x 15 = July x l4/1.1 x 16 = x 14/7. x 15 = x 15/3.1 x 16 = August BGM-la 1. x 14/6.5 x 15 = PMK-1 3. x 14/1. x 16 = PMK-2 9. x 14/2.7 x 1W = LSc /7.5 x 1 = /1.2 x 1c = Mahoney 4.4 x 16/6. x 16 = x 16/4.2 x 16 =.67 a Isolants selected for chlorine resistance study were 5 June -1 and 18 August BGM-1. r values were determined by comparing plaque counts developed by incubation at 37 C after 18 h at 4 C with those developed after the same total incubation time but maintained at 37 C throughout. rct/4 is defined as [(X - Ao)/(Vo- Ao)]-1, where X is the r value of the unknown, Ao is the r value of the avirulent control and Vo is the r value of the virulent control. rct/4 for the LSc control is, whereas rct/4 for the virulent Mahoney control becomes 1. CHLORINE-RESISTANT NATURAL POLIOVIRUS 1117 vation kinetics, the chlorine demand of the stock virus preparations was measured. Both total and free chlorine residuals were measured initially and after 1 and 3 min of contact. The results showed that residual chlorine was present in the form of free chlorine and that combined chlorine was present at concentrations less than.1 mg/ liter and did not increase significantly during the experiments. This confirned that the procedures used for the preparation, purification, and concentration of the virus suspension, as well as other related procedures, were adequate. Disinfection. The disinfection data for the four virus preparations measured at three levels of free chlorine residual are shown in Table 3. During the experiments, ph was monitored and shown to remain constant within the range of Temperatures remained within the range of 22 ± 3 C. These data show clearly that under similar conditions the surviving fraction of test I and test II was orders of magnitude greater than the surviving fraction of either the LSc or Mahoney. For example, a 3-min exposure to free chloride residuals of.1 to.15 mg/ liter reduced the concentration of LSc and Mahoney strains by 9 x 13 and 1 x 15, whereas test I and test II were reduced by 7 x 11 and 4 x 11, respectively. At higher free chlorine concentrations,.9 to 1.3 mg/liter, Mahoney could no longer be detected after 3 min, whereas LSc, test I and test II, were reduced by factors of 6 X 17, 5 x 11, and 7 x 13, respectively. The data for the Mahoney strain compare well with the data reported earlier by Kelly et al. (14). Portions of the disinfection data are shown graphically (Fig. 1 and 2) for greater ease of visualization. TABLE 3. Resistance of test viruses to chlorine disinfection Chlorine residual PFU after exposure tine (min)': Virus strain (mg/liter) Free Total Mahoney x x x x 1' x x x x x x x 12 ND LSc x x x x x X x x x x x 1 Test I x x x x x x x x x x x x 14 Test II x 1" 1.47 x x x x x x x x x X x 12 a Numbers presented show poliovirus 1 concentrations expressed in plaque-forming units (PFU) per milliliter surviving after 2-, 1-, and 3-min exposure to chlorine concentrations determined after 3 min. ND, No plaques developed.

4 1118 SHAFFER, METCALF, AND SPROUL APPL. ENVIRON. MICROBIOL. z 2-2 z 5 li &i -(9 -x x I.\ 3 \\ z cc z cc (I) D -J o *\M E) ita TIME (MINUTES) FIG. 1. Exposure of suspensions of several strains of type 1 poliovirus to free chlorine in the range of.1 to.15 mg/liter shows significantly greater inactivation of Mahoney (circles,.12 mg/liter) and LSc (triangles,.13 mg/liter) than of the two natural isolants (I, squares,.11 mg/liter; II, crosses,.15 mg/liter). Electron microscopy. Portions of each of the virus preparations sent to Sharp were examined by electron microscopy. None of the preparations showed significant aggregation. Less than an estimated 5% of the virions were present in aggregates. No aggregate of sufficient size to encapsulate and protect a virion was found. Neither were there indications of gross particle contamination. Figure 3 shows an electron photomicrograph of the test II virus preparation with a preparation of Mahoney strain prepared by Sharp's group inserted for comparison. This photomicrograph is typical of all four preparations. DISCUSSION For decades finished potable waters have received chlorine treatments to destroy or inactivate various disease-causing microorganisms. The complete effectiveness of such treatments has been assumed. This opinion has been based on the destruction of indicator bacteria in the waters themselves or on evidence from laboratory studies in which neither bacteria nor viruses could be shown to be capable of surviving prolonged periods of exposure to free chlorine in the absence of particulate matter which might encapsulate and physically protect the organisms. The adequacy of water treatment is monitored routinely by total coliform bacterial tests. The TIME (MINUTES) FIG. 2. Disinfection behavior of suspensions of strains of type 1 poliovirus to free chlorine residuals in the range of.9 to 1.35 mg/liter shows significantly different behavior between Mahoney (circles, 1.12 mg/ liter) which was not detected after 1 min and LSc (triangles, 1.34 mg/liter) compared with the two natural isolants (I, squares,.92 mg/liter) and II (crosses, 1.35 mg/liter). absence of these bacteria is presumptive evidence that viruses and other pathogens are also absent. The proposed drinking water regulations contain standards for residual chlorine and low turbidity. The detection of low numbers of viruses in water has only recently become technically feasible through the introduction of equipment and methods suitable for this purpose. The effectiveness of the new recovery procedures is still far from satisfactory for a number of viruses. Some enteroviruses such as the poliovirus can be recovered and demonstrated satisfactorily. For example, it has been reported that under ideal conditions, one cell culture infective unit per 38 liters can be demonstrated successfully with a high probability of success in samples of 1,9 liters (13). Other enteroviruses are recovered with varying degrees of effectiveness, ranging from fair to zero. Nothing of substance is known about the effectiveness of current water plant disinfection practices for hepatitis A, rotaviruses, or Norwalk-type viruses since these cannot be handled conveniently by laboratory recovery and assay methods. A number of reports of virus recoveries from disinfected tap waters have been published (7, 1). The small numbers of viral isolations combined with the difficulty of collecting field sam-

5 VOL. 4, 198 CHLORINE-RESISTANT NATURAL POLIOVIRUS L OrAk A:p FIG. 3. Electron photomicrograph of dispersed chlorine-resistant poliovirus strain (test II) showing the absence of major aggregates. Inset photograph is an ideal dispersion of Mahoney strain ofpoliovirus 1 for magnification comparison. ples under completely aseptic conditions had led to the assumption in some quarters that few if any of the viral isolations reported from properly treated, finished water have been legitimate isolations. According to this viewpoint, virtually all of the reported isolations have been considered to have been the result of contamination, either during the collection or processing of the test

6 112 SHAFFER, METCALF, AND SPROUL samples, or cross-contamination in the laboratory during the conduct of cell culture tests. Recent evidence, however, suggests that such isolations may in fact be correct and that laboratory data obtained with stock virus strains may not be directly applicable to water treatment practices in which indigenous viruses are involved. For example, virus recoveries have been reported from several finished tap waters containing free chlorine residuals and free of significant turbidity (16). Carefully conducted examinations of two such isolants recovered during the Occoquan study has shown that their chlorine inactivation behavior differs significantly from that of two of the more commonly used stock strains of the same virus type. These findings were similar to those reported previously in which different viruses, as well as bacteria and bacteriophages, were shown to exhibit a wide range of inactivation rates when subjected to chlorine disinfection (17). Different strains of poliovirus 1 have been shown to exhibit different rates of chlorine inactivation (14). Compared with the results of the present study, however, these differences among the test stock virus strains were small. The results of the present study, when considered along with the recently published works of Bates et al. (2, 3), showed that differences in resistance of natural poliovirus isolants to disinfection can result in a survival probability several orders of magnitude greater than the resistance of stock strains of virus of the same type. The work of Bates et al. (2, 3) showed that repeated exposures of type 1 poliovirus (LSc strain) to sublethal doses of chlorine, alternating with virus propagation in cell cultures, would lead to a significantly more resistant strain in as few as five cycles and that additional repetitions increased this resistance still further. The present study shows that at least in the case of type 1 poliovirus, wide differences in chlorine inactivation rates can exist between laboratory virus strains and natural isolants of the same virus type. Based on these studies, a reevaluation of the effectiveness of present disinfection practices as well as the proposed chlorine-turbidity standard for drinking water should be considered. Tacit acceptance of the results of laboratory studies with stock virus strains may not be justified. Data from the present study furnish further evidence that natural viruses may survive modern water treatment practices. They also suggest a plausible reason for survival, namely a chlorine resistance of a degree which enables them to withstand present disinfection treatments. APPL. ENVIRON. MICROBIOL. ACKNOWLEDGMENTS We thank D. G. Sharp and his staff in the Department of Microbiology and Immunology, University of North Carolina in Chapel Hill, for their assistance in providing the electron microscopy of these virus preparations. The disinfection study was funded by a grant from The Carborundum Co., a subsidiary of the Kennecott Co. LITERATURE CITED 1. American Public Health Association Standard methods for the examination of water and wastewater, 13th ed. American Public Health Association, Washington, D.C. 2. Bates, R. C., P. T. B. Shaffer, and S. M. Sutherland Development of resistant poliovirus by repetitive sublethal exposure to chlorine, p In R. L. Joiley (ed.), Water chlorination-environmental impact and health effect, vol. 2. Ann Arbor Press, Ann Arbor, Mich. 3. Bates, R. C., P. T. B. Shaffer, and S. M. Sutherland Development of poliovirus having increased resistance to chlorine inactivation. Appl. Environ. Microbiol. 34: Carp, R. I., and H. Koprowski A new test of the reproductive capacity temperature worker of poliovirus. The limited thermal exposure test. Virology 16: Clarke, N. A., G. Berg,. C. Liu, T. G. Metcalf, R. Sullivan, and L. T. Vlassoff Viruses in water, J. Am. Water Works Assoc. 61: Clarke, N. A., and S. L. Chang Enteric viruses in water. J. Am. Water Works Assoc. 51: Coin, L., M. L. Menetrier, J. Labonde, and M. C. Hannoun Modern microbiological and virological aspects of water pollution, p In. Jagg (ed.), Advances in water pollution research, vol. I. Pergamon Press, New York. 8. Dahling, D. R., G. Berg, and D. Berman BGM, a continuous cell line more sensitive than primary Rhesus and African Green Kidney cells for the recovery of viruses from water. Health Lab. Sci. 11: Dulbecco, R., and M. Vogt Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99: Foliguet, J. M., L. Schwartzbrod, and. G. Gaudin La pollution virale des eaux usees, de surface, et d'alimentation. Bull. W.H.O. 35: Guskey, L. E., and D. A. Wolff Concentration and purification of poliovirus by ultrafiltration and isopycnic centrifugation. Appl. Microbiol. 24: Hoehn, R. C., C. W. Randall, F. A. Bell, and P. T. B. Shaffer Trihalomethanes and viruses in a water supply. J. Environ. Eng. Div., Am. Soc. Civil Eng. 13: Jakubowski, W., W. F. Hill, Jr., and N. A. Clarke Comparative study of four microporous filters for concentrating viruses from drinking water. Appl. Microbiol. 3: Kelly, S., and W. W. Sanderson The effect of chlorine in water on enteric viruses. Am. J. Public Health 48: Melnick, J. L., V. Rennick, B. Hampil, N. H. Schmidt, and H. H. Ho Lyophilized combination pools of enterovirus equine antisera: preparation and test procedures for the identification of field strains of 42 enteroviruses. Bull. W.H.O. 48: Metcalf, T. G., P. T. B. Shaffer, and R. Mooney Incidence of virus in water-case histories. 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7 VOL. 4, 198 CHLORINE-RESISTANT NATURAL POLIOVIRUS 1121 tivation of viruses in water by chlorine. Water Res. 6: 19. Sharp, D. G Physical assay purified viruses, partic ularly the small ones, p In C. J. Arceneaux 18. Shaffer, P. T. B., R. E. Meierer, and C. D. McGee. (ed.), Proceedings of the 32nd Annual Meeting, Electron Virus recovery from natural water. J. Am. Water Microscopy Society of American. Claitor's Publishing Works Assoc. 69: Div., Baton Rouge, La.