Persistence of enteroviruses in sewage sludge*

Bulletin of the World Health Organization, 55 (4): 431-434 (1977) Persistence of enteroviruses in sewage sludge* T. P. SUBRAHMANYAN' Sewage from residential areas often contains viruses pathogenic for man and significant amounts are probably associated with solids in sewage sludge. Information on the survival of viruses in sewage sludge is necessary in order to develop guidelines for recycling programmes that involve spreading the sludge on land. In the present study, a number of enteroviruses were added to sewage sludge and the artificially contaminated sludges were testedfor viruses at intervals over a 12-week period. Most of the viruses survivedfor many weeks at room temperature. It is clear that sewage sludge destined for land application should be adequately treatedfor virus inactivation. In interpreting these results, it should be borne in mind that the survival of hepatitis A virus might be similar. Recent reports about the reappearance ofpoliomyelitis in regions with immunization programmes should also be taken into consideration. The faecal excretion of over 100 antigenic types of virus and their frequent occurrence in sewage from residential areas is well documented (1). Enteric viruses, which include enteroviruses, hepatitis A virus, reovirus and reo-like viruses as well as adenoviruses, are generally resistant to physical and chemical agents, and Akin et al. (2) have shown that their survival times in sewage can vary from 6 to 130 days depending on the virus type and ambient tempera- ture. Viruses in sewage are likely to be adsorbed to faecal solids and other particulate materials. It is therefore reasonable to expect that the solid phase of sewage, namely sewage sludge, will contain consider- able amounts of viruses. When suitable conditions prevail, the adsorbed viruses could be released from the sludge and pose a health hazard. Information on the persistence of viruses in sewage sludge is, there- fore, of practical importance. The present report deals with the survival of a number of enteroviruses when added to sewage sludge. MATERIALS AND METHODS Virus-negative sludge Digested sludge specimens were obtained in a well-mixed state from the digestor of a sewage * This work was supported by the Canada Ontario Agreement Contract No. 72-5-1 and was part of the Research Report No. 52 of the programme. 1 Head, Enteric Viruses Laboratory, Laboratory Services Branch, Ontario Ministry of Health, Toronto, Ontario, Canada. treatment plant carrying out activated sludge treat- ment. They were individually tested for viruses. A number of sludges in which no viruses were detected were pooled and the pool divided into 1-litre amounts. In virus survival studies, approximately 107 TCID50 of each virus under test were homogen- ized with separate 1-litre volumes of the pooled sludge. Virus-positive sludge 1 litre of a digested sludge specimen containing reovirus type 2 was provided by Dr S. A. Sattar of the University of Ottawa. Specimen processing. 100 ml of the sludge were homogenized in a Sorvall Omnimix with 50 ml of Earle's balanced salt solution (EBSS, pH 9.5) containing 2 %Y fetal calf serum. Homogenization was carried out in three 2- minute bursts, cooling the vessel in an ice bath throughout. The pH was checked each time after allowing aerosols to settle and readjusted to 9.5 with 1 mol/litre NaOH as necessary. The homogenate was centrifuged at 12 000 g for 20 minutes and the supernatant filtered through a sterile Millipore mem- brane (type HA: 0.45 ,um) to remove bacteria. Virus titration Sludge extracts and appropriate virus controls in medium (CMRL 1969) were titrated in parallel in roller tube cultures of African green monkey kidney cells, using at least 4 tubes per dilution. 3625 - 431- T. P. SUBRAHMANYAN RESULTS Optimum conditions for enterovirus elution from sludge Approximately 107 TCID50 of attenuated poliovi- rus type 1 (Sabin) were added to 50 ml of virus- negative sludge. The mixture was homogenized cold in a Sorvall Omnimix and divided into five equal portions. The portions were then homogenized cold with EBSS at different pH values, care being taken to maintain the selected pH throughout the extraction procedure. Aqueous extracts obtained by centrifuga- tion were treated with equal volumes of chloroform. The bacteriologically sterile extracts were readjusted to pH 7.4 with 1 mol/litre HCl and titrated for virus. The results are presented in Table 1. In this experiment, the largest amount of virus was eluted at pH 9.5, the highest pH level tested. In subsequent experiments, it was found that elution at pH 10.5 or higher by this procedure gave poorer virus recoveries. It was also found, in other experiments, that the lowest pH at which partial virus elution occurred varied from sludge to sludge but elution was always maximal at pH 9.5. Table 1. Elution of enteroviruses from sewage sludge Specimen pH Virus recovered Sludge preparation 1 unadjusted (6.0) 0% Sludge preparation 2 6.5 0% Sludge preparation 3 7.5 0 % Sludge preparation 4 8.5 3.2 % Sludge preparation 5 9.5 32 % The infectivities of different dosage levels of atte- nuated poliovirus type 1, coxsackievirus B5, and echovirus type 6 were not affected by chloroform treatment but a gentler procedure was considered desirable. Fetal calf serum at a final concentration of 2% was added to a sewage sample artificially con- taminated with attenuated poliovirus type 1. One portion was treated with chloroform while another was filtered through a sterile Millipore membrane (type HA: 0.45 ,um). On titration, both preparations gave identical titres of 1000 TCID50 per millilitre. Other experiments showed that there was no advan- tage in using higher serum concentrations. Virus survival in sewage sludge As the virus added to sludge is at least partially recoverable, it was considered important to deter- mine the long-term fate of these viruses. In an initial experiment, poliovirus type 1 was added to virus-negative sludge which was then divided into two parts; each part contained approxi- mately 200 TCID5' of virus. One part was stored at 4°C and the other part at room temperature (22°C). Virus titres were determined at 0, 2, 3, 7, and 14 days. The results are presented in Table 2. At 0 hours, 28% of the virus was recovered. The virus recovery was undiminished at either temperature for at least 7 days. Viruses were found even after 14 days but in smaller amounts; the smallest amounts were found in the samples stored at room temperature. Table 2. Poliovirus survival in sludge No. of TCIDso detected at: Day 40C Room temperature 0 56 2 56 100 3 56 56 7 56 56 14 32 10 The next step involved studying the persistence of a number of different enteroviruses when added to digested sludge, as described above. The viruses were homogenized with separate portions of virus-nega- tive sludge. The mixtures were kept at room tem- perature (22°C) and titrated for virus at various times from 0 to 84 days. The results of these and appropriate virus control titrations are given in Fig. 1. Compared to the virus controls, artificially con- taminated sludges yielded less virus; however, most of the virus types were detected for several weeks. Survival times were not the same for all viruses. Coxsackievirus A9 survived less than 2 weeks under these conditions and coxsackievirus B4 could not be detected after 4 weeks. Coxsackievirus B2 became undetectable by 5 weeks while polioviruses could be detected for longer periods. The vaccine strain of poliovirus type 1 disappeared at 8 weeks while the virulent strain was still present at 10 weeks. Survival 432 ENTEROVIRUSES IN SEWAGE SLUDGE i J 3.0 3.0 2.0 2.6 1.6 1.3 2.0 E 0 C, 0 -0 0.6 3.5 2.6 3.5 2.0 3.6 3.0 3.0 3.S* ECHO 9 -, * - _ ECHO 6 COXSACKIE B5 COXSACKIE B4 COXSACKIE B2 COXSACKIE A9 POLIO 3 VA C CINE POLIO 3 VIRULENT POLIO VACCINE POLIO VIRULENT I ,- .- 1 2 5 8 10 12 WEEKS AT ROOM TEMPERATURE SLUDGE -*- MEDIUM *- * Fig. 1. Enterovirus survival in sludge at room tempera- ture. did not, however, appear to be related to virulence since virulent poliovirus type 3 did not survive as long as the corresponding vaccine strain. Echovirus type 6 was detected until 10 weeks after the start of the experiment while coxsackievirus B5 and echovi- rus type 9 were still present when the experiment was terminated at 12 weeks. The differences between the viruses did not appear to be related to initial titres. In a further experiment, a sludge sample contain- ing reovirus type 2 was received from Ottawa and stored at room temperature. When the specimen was tested for virus at 2-week intervals, reovirus was detectable up to 6 weeks but not after 8 weeks. The results clearly show that many viruses persist in sewage sludge for several weeks at room tempera- ture. DISCUSSION Faecal excretion of large amounts of virus for several days is common in many enteric virus infec- tions even when they are clinically inapparent. Sub- stantial amounts of enteric viruses are, therefore, likely to enter the sewage systems in large centres oj population. These viruses are not easily inactivated and may persist in sewage for long periods (2). Strains of poliovirus apparently unrelated to the vaccine currently in use have recently been isolated from sewage in southern and eastern Ontario (3, 4). While this might be a reflection of the periodic importation of these viruses from areas where they are endemic, the circulation of potentially virulent polioviruses there has to be viewed with concern since an immunity gap to polioviruses appears to be developing in Ontario (5). This is similar to what has been observed in other vaccinated communities (6, 7). In the absence of adequate precautions, sewage could serve as a source of virulent polioviruses and infect unimmunized or partially immunized indivi- duals. In this connexion, it should be noted that the reappearance of clinical poliomyelitis in Ontario has been reported (5, 8). Factors governing the partition of viruses between the solid and liquid phases of sewage are not clearly understood. However, the tendency of viruses to adsorb to particulate materials favours their associa- tion with the solids in sewage sludge. Phosphate removal and other operations involving precipitate formation would be expected to increase the virus content of sludge. It is therefore important that suitable virus inactivation procedures are applied to both sewage effluents and sludge if accidental con- tamination of the environment is to be avoided. The daily per capita production of wet sludge in urban centres has been estimated to be 10 litres (9). In other words, large amounts of sludge accumulate in cities and towns, and proper disposal of sewage sludge should be an important public health concern. A method for the large-scale disposal of sludge is to spread it on poor agricultural land. It is economical and, since the plant nutrients in the sludge are recycled, it is ecologically desirable; however, undesirable consequences, including virus pollution, have to be taken into account and guide- lines for sludge application on land are necessary. The recommendations should ensure virological safety. It has been demonstrated that viruses are present in sludge (10, 11, 12) but there is little information on virus survival in sludge. The present study at- tempted to fill this gap. The efficacy of fetal calf serum as a virus eluent reported here has also been observed by Sattar & Westwood (13). In almost all the experiments carried out in the present study, the recovery of the added virus was partial; it could thus I 1; I.- - 433 434 T. P. SUBRAHMANYAN not be determined whether the virus remaining in the sludge was infectious because the sludge was invaria- bly toxic to cell cultures. The infectivity of " solids- adsorbed " viruses has, however, been demonstrated (12, 14). The recovery of poliovirus added to sludge was unchanged for 7 days indicating that viruses present in sludge will survive for several days under field conditions. This was found to be true of several enteroviruses and a reovirus. For obvious reasons, the survival of infectious hepatitis virus could not be studied. In setting safety standards, until experi- mental data become available, it will be prudent to assume that this virus is capable of prolonged survival. Lund (12) has reported that untreated sludges yield 10-1000 TCID50 viruses per millilitre and that digested sludges may contain up to 10% of this concentration. The present study shows that many viruses persist for considerable periods in sludge. The available evidence thus supports the view that sludge should not be spread on land unless it has been adequately treated and shown by sensitive detection techniques to be free of pathogenic viruses. Otherwise, run-offs from treated land might pollute nearby water sources. ACKNOWLEDGEMENTS I am very grateful to Mr P. C. Horwood and Mr P. Bos for their excellent technical assistance, and to Dr A. J. Rhodes for constant encouragement and valuable advice. The generous cooperation of many staff members of the Ontario Ministry of the Environment, in particular Mrs A. H. Vajdic and Mr S. A. Black, was invaluable in carrying out these studies. RESUME PERSISTANCE DES ENTEROVIRUS DANS LES BOUES DES EAUX DIEGOUTS Des echantillons preleves de boues digerees, dans lesquels aucun virus n'etait detectable, ont ete melanges; des fractions separees de 1 litre chacune ont ete homo- geneisees avec approximativement 107 DICT^O d'un des virus suivants: poliovirus type 1 et type 3 (souches attenuees et virulentes), coxsackievirus types A9, B2, B4 et B5, ainsi qu'echovirus types 6 et 9. Ces echan- tillons contamines artificiellement, de meme qu'un spe- cimen de boues contenant du reovirus type 2, ont et conserves A 22°C (temperature du laboratoire) pendant 12 semaines et les virus y ont e recherches periodique- ment. La survie de la plupart des virus a et longue: elle etait inferieure A 2 semaines pour le coxsackievirus A9 alors que le coxsackievirus B5 et 1'echovirus type 9 etaient encore decelables au bout de 12 semaines. La souche virulente de poliovirus type 1 a ete trouvee pendant 10 semaines et la souche correspondante de type 3 est restee prdsente pendant au moins 5 semaines. La dur6e de survie ne semblait determinee ni par le titre initial, ni par la virulence du virus. L'importance de ces obser- vations est encore accrue si l'on pense que le virus de l'hepatite A pourrait presenter une survie prolong6e similaire. Des rapports recents sur le declin de l'immu- nite a l'egard des poliovirus dans de nombreuses regions et la reapparition de la poliomyelite doivent aussi etre prises en consideration lors de l'interpretation de ces resultats. Les constatations ci-dessus montrent bien l'importance d'effectuer un traitement suffisant des boues des eaux d'egouts destinees a 1'epandage sur le sol. REFERENCES 1. MELNICK, J. L. ET AL. American journal of hygiene, 59: 164 (1954). 2. AKIN, E. W. ET AL. In: Proceedings of the Thirteenth Water Quality Conference. University of Illinois Bul- letin, 69: 59 (1971). 3. SUBRAHMANYAN, T. P. Canada-Ontario agreement on Great Lakes water quality, Research Report No. 52, Ministry of Supply and Services, Canada, 1977. 4. SATTAR, S. A. & WESTWOOD, J. C. N. Canadian Medical Association Journal, 116: 25 (1977). 5. SUBRAHMANYAN, T. P. ET AL. Canadian journal of public health, 65: 60 (1974). 6. MELNICK, J. L. ET AL. Journal of the American Medical Association, 209: 1181 (1969). 7. REIm, D. ET AL. Lancet, 2: 809 (1973). 8. SUBRAHMANYAN, T. P. ET AL. Bulletin of the World Health Organization, 49: 245 (1973). 9. Health hazards of the human environment. Geneva, World Health Organization, 1973, p. 48. 10. BERG, G. Bulletin of the World Health Organization, 49: 451 (1973). 11. LUND, E. & RONNE, V. Water research, 7: 863 (1973). 12. LUND, E. Viruses in water, Washington DC, Ameri- can Public Health Association, 1976, p. 196. 13. SATTAR, S. A. & WESTWOOD, J. C. N. Canadian journal of microbiology, 22: 1586 (1976). 14. MOORE, B. E. ET AL. Water research, 9: 197 (1975).