CHLORINATION OF HUMAN, MONKEY-ADAPTED AND MOUSE STRAINS OF POLIOMYELITIS VIRUS» *

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1 CHLORINATION OF HUMAN, MONKEY-ADAPTED AND MOUSE STRAINS OF POLIOMYELITIS VIRUS» * BY JAMES D. TRASK,' JOSEPH L. MELNICK AND HERBERT A. WENNER (with the technical assistance of ROBERT JOINER) (Received for publication October 2th, 944) Although it is not yet possible to assess the epidemiological significance of the findings of the virus of poliomyelitis in sewage, it appears worth while to assemble new data on the efficacy of chlorine as a viricidal agent. 4 In 93, Levaditi, Kling, and Lupine () reported that an initial dose of 0.4 ppm of chlorine after an extraordinarily long contact period of 24 hours destroyed a clear, filtered solution of poliomyelitis virus, and that 4.0 ppm were necessary to inactivate a turbid suspension of infected monkey cord. Using a very small number of monkeys, Kempf and Soule (2) found that a residual chlorine concentration of 0.25 to 0.35 ppm (original concentration of 0.55 to 0.80) after 4-to-5 hour contact was required to destroy approximately one minimal infective dose (MID) of the MV strain. These investigators in a later report found that to.5 ppm of residual Ch after 25-minute contact would inactivate approximately one MID of the MV strain ( in,000 dilution of infected central nervous system tissue) and the DG strain ( in,000 dilution) (3). From the Section of Preventive Medicine and Department of Pediatrics, Yale University School of Medicine, New Haven, Conn. 2 Aided by a grant from the National Foundation for Infantile Paralysis, Inc. «Dr. Trask died on May 24th, These experiments were undertaken at the suggestion of Dr. John C. Baker, of Wallace and Tiernan Producte, Inc., Newark, N. J. Much valuable advice was given by him and the late Dr. Franz Schmelkes. 30 Kessel et al. (4) reported that for complete virus destruction of a l-in-,000 dilution of the Le strain, it was necessary for chlorine solutions with residual levels of 0.5 to ppm to be in contact with the virus solution for 3 hours. The present study is concerned with the effect of chlorine on poliomyelitis virus as present in stools of paralytic patients, and in suspensions of monkey cords after 5 to 6 successive transfers after the original isolations from human stools; and of chlorine, chloramine, and azochloramid s on the FA strain of Theiler's mouse poliomyelitis virus. Part I. Chlorination of human and monkey-adapted poliomyelitis virus Virus. Four strains of poliomyelitis virus were employed. The SK strain, isolated originally in October, 939, from the stool of an 8-month-old child with a mild febrile illness, was in the thirteenth and sixteenth generations of passage. The CR strain was isolated from the stool of a 3-year-old paralyzed patient in July, 939, and was in the fifth generation of passage. The last two strains were naturally present in human stools, viz., the RO strain, present in the stool of a paralytic patient who became ill with paralytic poliomyelitis in August, 940, and the WA strain, actually a pool of stools from 7 persons affected with poliomyelitis in the same outbreak in Azochloramid is a chlorine compound (N-Ndichloroazodicarbonamidine) manufactured by Wallace and Tiernan Products, Inc.

2 CHLORINATION OF POLIOMYELITIS VIRUS 3 Preparation of inoculum, (o) Infected spinal cord suspension: Weighed portions of lumbar and cervical spinal cord segments obtained from paralyzed monkeys were triturated with sterile sand or pyrex glass, and diluted with distilled water to make a per cent suspension. This was centrifuged at 2,000 rpm for 5 minutes; from the cloudy supernatant fluid the desired dilutions were made. (6) Stool suspensions: These were prepared from stools from affected persons who were known to have excreted virus in the sampled specimens or from stools from apparently healthy persons which were artificially contaminated with spinal cord suspensions. Samples of stool were suspended in cold distilled water to make a per cent suspension. The stool suspensions were treated with per cent ether by volume, and allowed to stand in contact in the refrigerator (6 C) overnight. After centrifugation, the middle layer was removed for purposes of chlorination, and subsequent inoculation. Chlorination., as sodium hypochlorite, was used in these experiments. The virus mixture was allowed to remain in contact with the chlorine solution for minutes. By means of a preliminary experiment the chlorine demand of an aliquot of each virus suspension was determined. Then sufficient chlorine was added to allow for the -minute demand plus enough to yield the desired residual value. values were determined by titration against standard thiosulfate solutions in the presence of 30 per cent acetic acid. Monkeys were inoculated as near to minutes after the addition of chlorine as possible. Animals. Macaca mulatto were employed as test animals in all except experiment no. 6, in which Cercopithecus aethiops sabeus (the green African monkey) was the test animal used. Inoculated animals were exercised daily, and daily rectal temperatures were recorded for 4 weeks in the intracerebrally inoculated monkeys, and for 5 weeks in animals inoculated by peripheral routes, unless the animal was killed earlier. All animals which showed symptoms of poliomyelitis, or symptoms remotely suggestive of poliomyelitis, were sacrificed at what appeared to be an appropriate time, and examined for histological lesions in the spinal cord. Results with monkeys The effect of various concentrations of chlorine on the virus of poliomyelitis was studied to determine its capacity to inactivate in minutes various concentrations of virus in (a) spinal cord suspensions from infected monkeys, and (6) naturally and artificially infective human stool extracts. These experiments were exploratory in nature to determine whether tests made on monkeys would be sufficiently clear cut to be informative. a. Effect of chlorine on cord virus suspensions. Reference to table illustrates that, in experiment no., relatively small doses of chlorine, 0.2 ppm, appeared to inactivate the SK strains of poliomyelitis virus in ~ 3 and ~ 4 concentrations of infective cord in minutes. In view of the extraordinarily high titer of the controls, at least ~ 6, in this experiment 0.2 ppm of chlorine were able to destroy a suspension of virus containing at least,000 monkey-paralyzing doses of virus per 0.5 ml. In experiment no. 4, however, a striking disparity was noted in that not one of the monkeys which had received per cent of infective SK cord, as in the first experiment, was protected following exposure of the virus to 0.5 ppm of residual chlorine for minutes. Experiment no. 2, carried out with the CR strain, illustrates the pitfalls in this type of experiment. Although 0.05 and

3 32 J. D. TRASK, J. L. MELNICK AND H. A. WENNER TABLE Effect of chlorine on monkey-adapted poliomyelitis virus after a -minvte contact * Exp. no. and date Virus strain Strength of cord suspension in per cent Added Demand Residual Result of test for virus in monkeyst Exp Exp Exp Exp SK Gen. 3 CR Gen. 5 CR Gen. 5 SK Gen _ + + +, -, - _ + +, +, - +, -, -, ~ +, +, +, + +, +, + *A monkeys received 0.5 ml intracerebrally. Only rhesus monkeys were used in these experiments. t Each symbol in this column represents the result of a test in one monkey. ppm of chlorine appeared to inactivate a per cent suspension of infected cord, 2 of 3 control monkeys which received untreated virus also failed to develop the disease. One may say that a concentration of chlorine calculated to provide 0.4 ppm of chlorine beyond immediate demand of minutes failed to inactivate a per cent suspension of infected cord. In experiment no. 3 it was demonstrated again that a concentration of chlorine calculated to give 0.4

4 CHLORINATION OF POLIOMYELITIS VIRUS 33 ppm beyond immediate demand was not effective in inactivating the virus. Again the low susceptibility of the controls does not allow one to draw any conclusions from monkeys which failed to develop the disease. b. Effect of chlorine on stool extracts. Reference to table 2 illustrates that these experiments were also inconclusive because of the poor susceptibility of the control animals to the RO strain of virus. The WA strain, following intraperitoneal inoculation of Cercopithecus monkeys, was able to withstand a concentration of chlorine of ppm after minutes of contact. Only 2 of 7 control animals developed poliomyelitis. The results obtained by suspending a ~ 3 concentration of SK infected cords in normal human stool extract indicate that such a suspension is not inactivated by as much as ppm of chlorine beyond immediate demand. The resistance of this amount of virus to ppm of residual chlorine may be explained perhaps TABLE 2 by the presence of extraneous organic matter. This topic is discussed in greater detail in the next section of this paper. In summary, such inconsistent results were obtained with monkeys as test animals that it was felt that a satisfactory answer could be obtained only by the use of large numbers of test animals. Consequently we selected mice, using the virus of spontaneous mouse encephalomyelitis (Theiler's virus) which has many of the characteristic properties of human poliomyelitis virus. Part II. Chlorination of Theiler's mouse poliomyelitis virus Virus. The FA strain 6 was used in these experiments. A per cent suspension of infected mouse brains was centrifuged at 2,000 rpm for 5 minutes and the supernatant fluid was frozen in a dry-ice chest in several tubes. For each Dr. Max Theiler, of the International Health Division of The Rockefeller Foundation, kindly supplied us with this strain. Effect of chlorine on poliomyelitis virus present in naturally and artificially infective stool extracts * ' Exp. no. ana date Exp. 5 Exp Exp Virus strain RO Paralytic patient WA Pool from 7 paralytic patients SK per cent cord in per cent normal human stools Strength of stool suspension in per cent Added Result of test for virus in monkeys» i i +, -, - +, -, " Demand Residual +,+,- +, , - i * Contact time: minutes. Rhesus monkeys were used for experiments no. 5 and 7, green African monkeys for experiment no. 6. The monkeys in experiment no. 5 were inoculated intranasally; those in experiments no. 6 and 7 were inoculated intraperitoneally.

5 34 J. D. TRASK, J. L. MELNICK AND H. A. WENNER experiment the contents of an individual tube were thawed, and enough 0.0M sodium phosphate buffer was added to make a ~ s brain concentration. This was centrifuged at 2,000 rpm for 5 minutes and the clear supernatant fluid used to make subsequent dilutions, buffer being employed as the diluent. Buffer at ph 7 was used after a preliminary experiment indicated there were no apparent differences between the results with buffers at ph 6, 7, or 8. Chlorination., chloramine, and azochloramid (N-N-dichloroazodicarbonamidine) served as chlorinators. Contact times of, 30, and 60 minutes in the dark at room temperature were employed. At the end of the contact period, a portion of the sample was quickly inoculated intracerebrally into 4-week-old Swiss mice (0.03 ml per mouse). The time required to inoculate 6 mice was minute or less. At the same time as the mice were being inoculated, the residual chlorine was determined in an aliquot of the solution by means of titration against a standard thiosulfate solution. The chlorine demand of the various solutions was determined prior to each experiment. Animals. As controls, groups of mice which did not receive chlorine were included in each experiment. All animals were examined for 2 days, and the time of the onset of encephalitic or myelitic symptoms was noted. Deaths within 3 days after inoculation were regarded as nonspecific in view of the dilutions of virus employed; deaths after 3 days were regarded as due to the virus. It should be stated that about 80 per cent of the animals showed signs of central nervous system involvement before death. Occasionally histological sections of the brains and cords of sick mice were prepared, and these were found to exhibit typical lesions of the disease. Results with mice Method of expression. The results of these experiments were calculated to show the concentration of residual chlorine which would destroy known numbers TABLE 3 Preliminary determination of chlorine residuals on addition of chloramine to Theiler's virus in brain suspensions * Cone, infected brain - 3 io-«-" Ch dose NH. dose N thio. (ml) Residual Ch * Contact time 30 minutes; sample size 5 ml; NHj solution added first, followed immediately by addition of Cl 2 solution. of ID 6 o 7 of the virus, i.e., reduce the infectivity of varied -fold dilutions of infected brains, each containing known multiples of ID 60 of virus, to one ID 60. This method of expression allows one to use the objective Reed-Muench formula JO = an infective dose for 50 per cent of the mice inoculated, or that amount of virus which produces the disease after intracerebral inoculation in 50 per cent of the mice inoculated.

6 CHLORINATION OF POLIOMYELITIS VIRUS 35 (5) for estimating the results of each experiment. To illustrate the method of recording the results, the protocols of one experiment will be presented in detail. TABLE 4 Treatment of Theiler'g virus with chloramine * Virus cone. io-* -0 -' -* -* " 6 -' Desired residual : Cl» dose required t Control NoCl a NH«dose required t Ml thio..00 N Actual Cl. residual Morbidity ratio in miee 6/6 5/5 5/6 5/6 4/6 3/6 2/6 3/6 /5 /5 3/6 0/4 /5 /5 0/6 0/5 0/5 0/6 0/5 0/6 34/34 34/36 44/56 35/56 3/ * Contact time: 30 minutes. t Clj doses interpolated from graphs constructed from data in table 3. t NHi solution added before the Clj solution. Numerator denotes number of mice which became infected: denominator denotes the number of animals tested. Total mice used as controls in experiments described in part II of this paper. Aliquots of one virus suspension, frozen in small samples, were used for each experiment. The titration endpoint varied slightly, but averaged ~'. May 8th, 942. The effect of chloramine on Theiler's virus, FA strain, after contact periods of and 30 minutes was tested. Preliminary determinations of the CI2 uptake of the various virus suspensions were made (table 3). By plotting for each brain concentration the doses of chloramine used and the experimentally determined residual levels for each of these doses and by interpolation of the curve obtained, the required chloramine dose to yield the desired residual values may be determined. The data in table 4 illustrate how closely it was possible to obtain the desired residual levels in the actual experiment. At the same time that an aliquot of the chloraminetreated virus suspension was chemically titrated, an aliquot was inoculated into 6 mice. The results of the animal inoculations are presented in the last column of the same table. By applying the Reed-Muench formula to the data in table 4 it is possible to calculate the amount of residual chlorine necessary to reduce the virus content of each infected brain concentration to one ID 50. From the control titrations the number of ID 60 in each of the virus dilutions is known, and it is thus possible to estimate the number of ID 6 o destroyed in solutions having these residual CU levels (table 5, experiment no. ; figure ). It is shown that, after a 30-minute contact with ~ 3 virus dilution, chloramine is effective in a concentration giving ppm; at ~* virus dilution, in a concentration yielding ppm; and at ~ 5 virus dilution, in a concentration giving ppm of residual chlorine since, in these concentrations, chloramine inactivates one ID 6 o of virus. Discussion of results. The results of these experiments are presented in table 5 and figure. For chloramine there was a direct relationship between the virus concentration and the chlorine re-

7 36 J. D. TKASK, J. L. MELNTCK AND H. A. WENNER TABLE 5 Chloriruition of TheUer's virus Exp. no. Chlorinator Contact time (.minutes) Cone, infected brain Number OIIDM Cli dose (ppro) Residual Cl» necessary to reduce infectivity to one ID Calculated residual Clj to destroy one ID so Chloramine Chloramine Azochloramid 30 CHLORAMINE 30 MINUTES CONTACT "0 200 NUMBER OF io-«io-«-* io- 4 io-* io- 8 IO-* io-«- 5 io OO IOOO I.0.50 DOSES DESTROYED FIGURE. Inactivation of mouse poliomyelitis virus (Theiler's FA strain) by chloramine.,000 0, ,000, <0.35 > < > < (<8 destroys ID 80 ) > < > (0.08 destroys ID 60 ) < > < >0.0032' < sidual values: to ppm of residual CI2 after a 30-minute contact was found necessary to cause the destruction of one ID 6 o with the infected brain concentration varying from ~ 3 to ~ 6. As was to be expected, a shorter contact period of minutes required a higher residual level, namely, 0.08 ppm. When chlorine itself served as the chlorinating agent, as the virus concentration was increased -fold, it was found that the residual chlorine level necessary to reduce the infectivity of the suspension to one ID 60 had to be increased 5-fold. Thus at higher concentrations, less residual chlorine was necessary to destroy one ID50 dose than at low virus concentrations. With an infectedbrain concentration of ~ 8, containing,000 ID 6 o, 8.8 ppm of residual chlorine,

8 CHLORINATION OF POLIOMYELITIS VIRUS 37 representing ppm for each ID B o, were required. As the virus suspension was serially diluted -fold, the chlorine residual level necessary for the destruction of one ID 6 o dose serially increased 2-fold, so that at a brain concentration of ~ 6, the required residual level was found to be 0.08 ppm NUMBER OF CHLORINE MINUTES CONTACT I.D. 5O DOSES DESTROYED FIGURE 2. Inactivation of mouse poliomyelitis virus (Theiler's FA strain) by chlorine. The reproducibility of the results may be seen from the data obtained in experiments no. 3 and 4 (table 5), and also from examination of figure 2, where it is shown that at ~ 4 brain concentration, chlorine residuals of.3 and.7 ppm were effective while at ~ 6, residuals of 0.33 and 0.35 ppm, destroyed the virus. With azochloramid for brain concentration of ~ 8 the necessary residual level was higher than with chloramine or chlorine, being 2. ppm to reduce the infectivity to one ID 6 o- Effect of extraneous organic matter on destruction of virus by chlorine. In the experiments presented in table 5, the proportion of extraneous organic matter in the form of mouse-brain tissue to virus remained constant. An increase of one always meant a corresponding increase in the other. In an attempt to determine the influence of such extraviral material on the required residual chlorine level, the following experiments were carried out. Virus suspensions were made up containing a ~ 8 concentration of infected mouse brain and in addition " 4, ~ 3, and ~ 2 concentrations of normal mouse brain, respectively. The normal brain suspension served to supply the same extraviral organic material as that present in infected brain. As previously described, the residual chlorine level sufficient to reduce the infectivity to one ID 6 o was determined, and this was found to be less than 0.6 ppm. An accurate figure was not obtainable in this test, as 0.6 ppm was the lowest value tried, and this destroyed all the virus present. However, two previously determined values at this virus concentration in satisfactory experiments yielded 0.33 and 0.35 ppm as the required residual chlorine level. It was found that, when the organic matter present in the ~ 5 virus dilution was increased -fold by the addition of a " 4 concentration of normal brain, a residual level of 0.85 ppm of chlorine was necessary to reduce the infectivity to one ID B o. This was times the control level (0.34 ppm). When the virus suspension contained a ~ 3 concentration of normal brain, a 0-fold increase of organic matter, the required residual chlorine level had to be increased 8 times (from 0.34 to 6. ppm). With a,000-fold increase (~ 2 normal brain) of organic matter, the required residual level was found to be 3.5 ppm, or a - fold increase. It should be emphasized that in these experiments with extraneous organic matter, additional chlorine

9 38 J. D. TRASK, J. L. MELNTCK AND H. A. WENNER TABLE 6 Effect of organic matter (normal brain tissue) on the chlorination of Theiler's virus Exp. no. Chlorinator Contact time (minute*) Cone, infected mouse brain* Cone, normal mouse brain* Number of ID,. Chdose Residual Clt necessary to reduce infectivity to one IDto Calculated residual Cls to destroy one IDu 2 Azochloramid 60 -* -* IO- 6 io-«- 6 io-* io- 6 0 ~ 4 io-* 0 io-* ^ was added in each case to meet the additional demand. The sole function of the added organic matter in these experiments was not the reduction of chlorine to the chloride form. Organic matter of this type when added to chlorine solutions involves tying up the chlorine by amino groups to form chloramines of a low order of activity. in this form is found as residual chlorine when acid is used in the titration, but it has much less activity than residual chlorine in the form of free hypochlorous acid. Azochloramid shows less interaction with organic matter than other chlorine compounds used for bactericidal purposes (6). Because it reacts with and oxidizes organic matter very slowly, it remains available for bactericidal action for long periods of time. Moreover, Schmelkes and Horning (7) were able to demonstrate that the bactericidal action of azochloramid was not as adversely affected by the addition of organic matter, such as serum, as were those of other chlorinating compounds, such as chloramine T and sodium hypochlorite. In -= o o to I.D o EDUCE <-> ildual VI UJ 6 Q_ 0.59f AZOCHLORAMID CHLORINE \ rt f / ^^ * A ~ l concentration of mouse brain contains 0.5 per cent protein. t The actual value found in this experiment was <0.85 ppm per dose and <0.6 ppm for residual chlorine. These were the lowest amounts tried and produced complete inactivation. The average S of the two previous results (see table 5, experiments no. 3 and 4) at this virus concentration is there- fore tabulated. \ our experiments with azochloramid, a,000-fold increase of organic matter necessitated a 4-fold increase of residual chlorine from 2. to 48 ppm to reduce the virus content to one ID 60. These data are presented in table 6 and figure 3. AZOCHLORAMIO CHLORINE K VIRUS-INFECTED IO" S IO" 5 IO" 5 BRAIN NORMAL BRAIN lo" 4 IO" J \ IO" S -2 FIGTJBE 3. Protective action of extraneous organic matter on the destruction of mouse poliomyelitis virus (Theiler's FA strain) by chlorine and azochloramid.

10 CHLORINATION OP POLIOMYELITIS VIRUS 39 The results of these experiments should make one hesitate to carry over laboratory findings on the chlorination of poliomyelitis virus to chlorination under natural conditions. The ratio of organic matter to virus in suspension apparently influences greatly the amount of residual chlorine necessary for the destruction of the virus. SUMMARY. A study was made of the effect of the chlorinating compounds, sodium hypochlorite (chlorine), chloramine, and azochloramid, on human, monkey and mouse strains of poliomyelitis virus. 2. Because of the technical difficulties and prohibitive cost of using large numbers of monkeys, complete answers were not obtained from the study of the chlorination of infected monkey-spinal-cord suspensions and of infective stools from poliomyelitis patients. a. A residual chlorine level of 0.4 ppm after a -minute contact was insufficient to inactivate the CR strain (~~ 2 concentration of cord); and a residual level of 0.5 ppm was insufficient to destroy the SK strain (~ 3 concentration of cord). i. The virus present in a per cent centrifuged suspension of stools from poliomyelitic patients withstood a chlorine dose of 20 ppm, leaving a residual value of 5 ppm after a - minute contact. 3. Quantitative data were obtained in a study of the chlorination of the FA strain of Theiler's mouse poliomyelitis virus. The Reed-Muench formula was found to be useful in calculating the results. a. For chloramine, a residual chlorine level of 0.57 ppm after a 30- minute contact was found necessary to reduce the infectivity of a ~ 6 infected brain concentration to one ID 8 o. As the concentration of virus was increased - and 0-fold, the required chlorine residual was proportionately increased. 6. With sodium hypochlorite, a chlorine level of 0.35 ppm for a ~ 5 brain concentration was found to be necessary. For each successive - fold increase in virus concentration, a successive 5-fold increase in the residual chlorine was required. c. With azochloramid, a chlorine level of 2. ppm for a ~ 6 brain concentration was required. 4. When extraneous organic matter was added to a ~ 5 concentration of infected brain, the chlorine residual values, after sufficient additional chlorine had been added to meet the additional demand, had to be increased as much as to 8 times to inactivate the same amount of virus as in the original suspension. This finding serves to emphasize the caution which must be exercised before attempting to carry over laboratory findings on the chlorination of poliomyelitis virus to chlorination under natural conditions. REFERENCES. Levaditi, C, Kling, C, and Lepine, P. Nouvelles recherches experimentales sur la transmission de la poliomye'lite par la voie digestive. Action du chlore sur le virus poliomye'litique. Bull. Acad. de MeU, Paris, 93, 5: Kempf, J. E., and Soule, M. H. Effect of city water on virus of poliomyelitis. Proc. Soc. Exp. Biol. and Med., 940, U'- 43.

11 40 J. D. TKASK, J. L. MELNICK AND H. A. WENNER 3. Kempf, J. E., Wilson, M. G., Pierce, M. E., and Soule, M. H. Effect of aluminum hydroxide sedimentation, sand filtration and chlorination on the virus of poliomyelitis. Amer. Jour. Pub. Health, 942, 82: Kessel, J. F., Allison, D. K., Moore, F. J., and Kaime, M. Comparison of chlorine and ozone as virucidal agents of poliomyelitis virus. Proc. Soc. Exp. Biol. and Med., 943, 58: Reed, L. J., and Muench, H. A simple method of estimating fifty per cent endpoints. Amer. Jour. Hyg., 938, 27: Guiteras, A. F., and Schmelkes, F. C. The comparative action of sodium hypochlorite, chloramine-t, and azochloramid on organic substrates. Jour. Biol. Chem., 934,7: Schmelkes, F. C, and Horning, E. S. Bactericidal action of azochloramid (N-N-dichloroazodicarbonamidine). Jour. Bact., 935, 29: 323.