Persistence of Inoculated Hepatitis A Virus in Mixed Human and Animal Wastes

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1995, p Vol. 61, No /95/$ Copyright 1995, American Society for Microbiology Persistence of Inoculated Hepatitis A Virus in Mixed Human and Animal Wastes MING YI DENG 1 AND DEAN O. CLIVER 1,2 * Food Research Institute, Department of Food Microbiology and Toxicology, and Department of Animal Health and Biomedical Sciences, World Health Organization Collaborating Center on Food Virology, 1 and Department of Bacteriology, 2 University of Wisconsin Madison, Madison, Wisconsin Received 8 August 1994/Accepted 31 October 1994 The persistence of hepatitis A virus (HAV) was determined both in mixtures of septic tank effluent (STE) with dairy cattle manure slurry (DCMS) and in mixtures of STE with swine manure slurry (SMS). HAV was consistently inactivated more rapidly in the two types of mixed wastes than in STE alone or in the control Dulbecco s phosphate-buffered saline (PBS). At 5 C, the D values (time, in days, for a 90% reduction of virus titer) were 34.6 for the mixed STE and DCMS, 48.5 for the mixed STE and SMS, 58.5 for STE, and for the Dulbecco s PBS control. At 22 C, the D values were 23.0, 17.1, 35.1, and 90.1 for the four suspension media, respectively. A comparison of HAV inactivation in mixed wastes subjected to different treatments at the same ph and temperatures showed that the virus inactivation in the mixed wastes was related, at least in part, to microbial activity. In mixed STE and DCMS, the D values at 25 C were 8.3 for raw mixed wastes, 15.1 for autoclaved mixed wastes, and 9.6 for bacterium-free filtrate of raw mixed wastes; D values at 37 C were 6.8, 10.1, and 7.0 for these three suspension media, respectively. In mixed STE and SMS, the D values at 25 C were 8.1 for raw mixed wastes, 14.3 for autoclaved mixed wastes, and 9.1 for bacterium-free filtrate of raw mixed wastes; the D values at 37 C were 6.8, 9.4, and 6.9 for the three suspensions, respectively. Mixing human wastes, in the form of septic tank effluent (STE), with animal wastes for storage and eventual disposal together on the land surface has been considered as an alternative for waste disposal on farms where geological conditions preclude disposal by conventional soil absorption systems (20). Most disease agents in body wastes are shed in feces rather than urine. These pathogens are categorized as viruses, bacteria, and parasites. Many of the most significant bacteria (e.g., Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella spp.) are more likely to be shed in animal manure than in human feces. The same is apparently true of the more commonly waterborne parasites Cryptosporidium parvum and Giardia lamblia. Therefore, the most significant public health concern in connection with mixing human wastes with animal wastes and treating both as animal wastes should be the viruses, which are quite host specific (20). Our previous study showed that poliovirus type 1 (PO1) is consistently inactivated more rapidly in a mixture of STE and swine manure slurry (SMS) than in the analogous controls under both laboratory and field conditions (4). Hepatitis A virus (HAV) is an important waterborne and foodborne virus in the United States (2) and is more resistant than PO1 to adverse environmental conditions (8, 13). We know of no previous studies of HAV inactivation under conditions likely to lead to biodegradation, and our previous mixed-waste study used only SMS. This report details the persistence and apparent microbial inactivation of HAV in human wastes mixed both with SMS and with dairy cattle manure slurry (DCMS). * Corresponding author. Mailing address: Food Research Institute, University of Wisconsin Madison, 1925 Willow Drive, Madison, WI Phone: (608) Fax: (608) Electronic mail address: cliver@macc.wisc.edu. MATERIALS AND METHODS Cell cultures. FRhK-4, a continuous line of fetal rhesus monkey kidney cells, was kindly provided by S. M. Lemon, University of North Carolina, Chapel Hill. Cells were propagated in Dulbecco s modified Eagle medium (GIBCO BRL, Life Technologies Inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, Mo.), 0.1 mm nonessential amino acids (GIBCO BRL), and 1 mm sodium pyruvate (Sigma). The same medium with 5% FBS was used for maintenance. Virus and human anti-hav IgG. HAV HM175/18f, a rapidly growing and cytolytic HAV variant (16), was also generously provided by S. M. Lemon. The virus was propagated in monolayers of FRhK-4 cells with maintenance medium, frozen and thawed three times, clarified by centrifugation at 4,000 g for 30 min, filtered with and m-pore-size filters (Gelman Sciences, Ann Arbor, Mich.), and stored at 20 C. Human anti-hav immunoglobulin G (IgG) from high-titer serum was obtained from the Wisconsin State Laboratory of Hygiene, Madison. Virus assay. HAV, in stock suspensions and inoculated samples, was detected and quantified with a plaque assay (PA) in confluent monolayers of FRhK-4 cells grown in 25-cm 2 polystyrene tissue culture flasks (Corning Glass Works, Corning, N.Y.). Virus samples were diluted serially in 10-fold steps in Dulbecco s phosphate-buffered saline (D-PBS) (Sigma) plus 2% FBS; each culture received 0.5 ml of inoculum. Three successive dilutions of each sample were tested in at least three flasks per dilution. After 1hat37 C, 5 ml of semisolid overlay medium was added. The overlay medium comprised maintenance medium plus 0.75% agarose (Agarose Type II Medium EEO; Sigma). After incubation at 37 C for 10 days, another 5 ml of semisolid overlay medium was added. The cells were incubated for 6 more days. The overlay medium was removed, the cells were fixed with a 1:2 dilution of 37% formaldehyde in distilled water at room temperature for 2 h, and plaques were made visible by staining the cell sheet with crystal violet solution for 1 to 2 min. The stain solution was prepared by dissolving 2.5 g of crystal violet and 4.25 g of NaCl in 345 ml of distilled water 130 ml of 95% ethanol 25 ml of 37% formaldehyde. Virus titers were reported as the number of PFU per milliliter of the test sample. Plaque neutralization. Plaque neutralization was done by a procedure described by Habel (10). Briefly, mixtures of virus and human anti-hav IgG (equal volumes of HAV at PFU/ml in D-PBS plus 2% FBS and human anti-hav IgG in D-PBS without FBS) were incubated at 37 C for 60 min. Each flask with a confluent monolayer of FRhK-4 cells received 0.5 ml of the mixture, after which the procedure was performed as described for the virus assay. AC-PCR for detecting HAV and analysis of AC-PCR products. Antigen capture (AC)-PCR for detecting HAV and analysis of AC-PCR products were performed as described previously (5). Wastes. STE was collected from a residence housing two adults and two children at the Arlington Experimental Farm of the University of Wisconsin, located in Columbia County. DCMS was obtained from a dairy farm in Wauke- 87

2 88 DENG AND CLIVER APPL. ENVIRON. MICROBIOL. sha County. SMS was drawn from a swine farm in northern Dane County. Mixed waste comprising STE and DCMS was prepared by combining 200 ml of STE with 800 ml of DCMS, with thorough stirring, and passing the mixture through a series of sieves with 2-, , and mm-diameter apertures. Mixed STE and SMS was prepared in the same way, except that 800 ml of SMS was substituted for DCMS. Samples of STE, DCMS, SMS, and mixed wastes were analyzed for ph, indigenous HAV, and total solids. The ph was measured with a ph meter (Corning). Testing for indigenous HAV from the wastes was done as described for inoculated HAV in other samples. The total concentration of solids in mixed waste was determined by the method for total solids dried at 103 to 105 C (1). Recovery of HAV from test samples. HAV was extracted from test samples with a sonication-centrifugation-filtration procedure previously described for PO1 (4). Inactivation of HAV in mixed wastes at 5 and 22 C. HAV stock suspension was added to mixed STE and DCMS, mixed STE and SMS, and (as controls) to STE alone and to sterile D-PBS containing 2% FBS, all at the same virus concentration ( PFU/ml). The ph of each of the suspensions was adjusted to 6.90 before the addition of the virus. Eighty milliliters of each virus-contaminated suspension was distributed into each of two sterile 250-ml narrow-mouth glass bottles closed with screw caps (Wheaton, Millville, N.J.). One was kept at 5 C, and the other was kept at 22 C. On the day the experiment began and weekly for 10 weeks thereafter, two separate 2-ml samples for virus titration and one 2.5-ml sample for ph measurement were taken from each bottle. Virus extraction and ph measurement were done immediately after sampling. The virus was quantified by the PA described above. Comparison of virus inactivation in raw mixed wastes, autoclaved mixed wastes, and bacterium-free filtrate of raw mixed wastes. Mixed STE and DCMS and mixed STE and SMS were prepared as described above, adjusted to ph 6.90, and divided into three portions. One of these portions was autoclaved at 121 C for 15 min and cooled before use; another was processed to bacterium-free filtrate by centrifugation at 10,400 g for 1 h followed by filtration of the supernatant fluid through a series of sterile 47-mm filters with porosities of 5, 1.2, 0.45, and 0.20 m (Gelman). The third portion was not subjected to any further processing and was designated as raw mixed waste. HAV ( PFU/ml) was added to each of the three suspension media. Eighty milliliters of each test mixture was distributed into each of two sterile 250-ml glass bottles (Wheaton). One was kept at 25 C, and the other was kept at 37 C. All bottles were capped loosely and covered with hospital wrap, a paper product developed for wrapping items for autoclave sterilization. On the day the experiment was set up and weekly for 5 weeks thereafter, each suspension was sampled and assayed for viral concentration. As in the previous experiment, two replicate samples were taken on each occasion. Statistical analyses. The virus inactivation in mixed wastes and other suspension media was analyzed by the general linear model procedure using the leastsquares principle (25). The virus inactivation rate was expressed as the regression for the log 10 PFU per milliliter versus time (in days). The inactivation rate was also expressed as a D value (time, in days, required for a 90% reduction of viral infectivity), which is equivalent to the inverse of the regression. The correlation between virus inactivation and ph changes in test suspensions was determined by simple correlation analysis (26). RESULTS PA and plaque neutralization. The first step of this study was the development of a classical PA, as first described by Dulbecco and Vogt (7) for poliovirus, for quantification of HAV HM175/18f, the model virus in our study. At 16 days, plaques were approximately 5 mm in diameter. The PFU were completely neutralized by human anti-hav IgG at a 1:1,600 dilution and to a lesser extent at dilutions to 1:6,400 (data not presented). These results authenticated the PA and demonstrated that the agent used was indeed HAV. Waste characterization. No indigenous HAV was detected from mixed STE and DCMS, the mixed STE and SMS, or the STE, DCMS, and SMS used for preparation of the mixed wastes. ph values and total concentrations of solids of these wastes are presented in Table 1. Inactivation of HAV in mixed wastes at 5 and 22 C. HAV inactivation in four suspension media at 5 and 22 C is shown in Fig. 1. The regression s and D values from this experiment are summarized in Table 2. At both temperatures, HAV was inactivated most rapidly in mixed wastes, the D-PBS had slowest viral inactivation, and the STE alone was more efficient in virus inactivation than the D-PBS (P for each of the suspension media). Inactivation in the mixture of TABLE 1. ph values and total concentrations of solids in waste samples Waste(s) STE and DCMS was more rapid than that in STE and SMS at 5 C but was less rapid than that of the counterpart at 22 C. It was also obvious that viral inactivation in each of the four suspension media at the higher temperature was faster than that at the lower one. At both temperatures, the mixture of STE and SMS had highest ph values and the D-PBS had the lowest ph values. The correlation between viral inactivation and ph changes in all of the suspensions at both temperatures is shown in Table 2. As indicated in the table, there seemed to be a good correlation between viral inactivation and the increase of ph in the mixture of STE and SMS. However, virus inactivation was not simply related to the increase of ph in the medium in which the virus was suspended. High ph values were not always associated with high efficiency of virus inactivation in the experiment. At 5 C, the mixture of STE and DCMS had lower ph values but faster viral inactivation than that of STE and SMS; at 22 C, the STE had higher ph values but a slower viral inactivation than the mixture of STE and DCMS. Virus inactivation in mixed wastes subjected to different treatments. Inactivation of HAV in mixed-waste suspension media at 25 and 37 C is presented in Fig. 2. The regression s and D values from this experiment are summarized in Table 3. Although the ph of each suspension had been adjusted to 6.9 at the outset, considerable changes occurred during the 5 weeks of incubation. It can be seen clearly, for both types of mixed wastes, that the virus was consistently inactivated more rapidly at both temperatures in the raw mixed wastes than it was in the autoclaved mixed wastes. Also, the virus inactivation in the bacterium-free filtrates of raw mixed wastes was much faster than that in the autoclaved mixed wastes. At 37 C, day 35 samples of both types of raw mixed ph Total solids (mg/liter) a STE ,015 DCMS ,565 SMS ,840 STE (20%) DCMS (80%) ,125 STE (20%) SMS (80%) ,375 a Values are means from two replicate samples. FIG. 1. Inactivation of HAV as a function of time in different suspension media at 5 and 22 C. Viral titers are represented by for mixed STE and DCMS, for mixed STE and SMS, } for STE alone, and { for D-PBS. Each datum point is a mean from two replicate samples.

3 VOL. 61, 1995 HEPATITIS A VIRUS INACTIVATION IN MIXED WASTES 89 Temp ( C) TABLE 2. Inactivation of inoculated HAV in four different suspension media at two temperatures Suspension medium Regression (day 1 ) a D value Final ph Correlation with ph 5 STE (20%) DCMS (80%) STE (20%) SMS (80%) STE D-PBS b STE (20%) DCMS (80%) STE (20%) SMS (80%) STE D-PBS b a Log 10 PFU per milliliter (P in each case). b D-PBS (ph 7.4) containing 2% FBS. wastes and bacterium-free filtrates gave negative results with both the PA and the AC-PCR, while virus titers of samples of both types of autoclaved mixed wastes taken at the same time remained above 10 2 PFU/ml. In addition, the inactivation in each of the suspension media was more rapid at the higher temperature. DISCUSSION FIG. 2. Inactivation of HAV in mixed-waste suspension media at 25 and 37 C. Viral titers are represented by for raw mixture of STE and DCMS, } for autoclaved mixture of STE and DCMS, F for bacterium-free filtrate of raw mixture of STE and DCMS, for raw mixture of STE and SMS, { for autoclaved mixture of STE and SMS, and E for bacterium-free filtrate of raw mixture of STE and SMS. Each datum point is a mean from two replicate samples. The results of this study indicate that HAV, which might threaten human health if spread on land, will be inactivated relatively rapidly in a mixture of STE and DCMS or STE and SMS and that antiviral activity of the microflora in the mixed wastes may play an important role in viral inactivation. We know of no previous instance in which microbial (probably bacterial) degradation of HAV has been demonstrated in environmental contexts. HAV was shown to be much more rapidly inactivated in mixtures of STE with animal manure than in laboratory buffer solution or in STE without manure. These results corroborate earlier work done in this laboratory which showed a higher rate of PO1 inactivation in mixtures of STE with DCMS and STE with SMS than in STE alone and in the D-PBS control (4, 21). The D values for HAV in STE plus SMS reported in Table 3 are generally longer than their counterparts in the study with PO1, especially at 37 C. Temperature has been well established as a primary influence in determining rates of virus inactivation (14, 19). In general, viruses survive longer at low temperatures and conversely are inactivated by heat. Work done in this laboratory earlier (19) indicates that temperature is a predominant factor in determining the inactivation rate of enteroviruses. Green (9) found a 97% reduction in 28 days when PO1 was held at 20 C, versus a 57% reduction when it was held at 7 C. Lund et al. (17) reported that the inactivation of both porcine and human enteroviruses was primarily temperature dependent. They also noted that the greater inactivation occurred in the presence of oxygen. Although our earlier reports of viral biodegradation were based on aerobic conditions (3, 12), more recent studies showed that PO1, at least, was degradable under anaerobic conditions (4). Virus inactivation at elevated temperatures may result primarily from protein denaturation (18). Poliovirus RNA becomes more susceptible to ribonuclease after the virus is heated at high temperatures, indicating structural alterations in the viral capsid. At lower temperatures, inactivation due to damage of the RNA may be more important (6). In addition to the thermal mechanism, temperature could strongly influence microbial activity, which in turn influences viral persistence. ph is another factor that influences the stability of viruses. Enteric viruses are generally most stable near ph 7 but are more stable at low phs (3 to 5) than at alkaline phs (9 to 12) (18). Hurst et al. (14) measured the effects of various environmental variations on virus persistence in soil; they found that the factors temperature and virus adsorption to soil appeared to be more important than soil ph. The effect of ph on HAV inactivation was evidently outweighed by biological factors in this study, as in our previously reported experiments with PO1 (4). Among mixed wastes subjected to different treatments, HAV was consistently inactivated, at both 25 and 37 C, more rapidly in the raw mixed wastes than it was in the autoclaved mixed wastes, in which all microbes had presumably been killed. Virus inactivation in the bacterium-free filtrates of raw mixed wastes was also much faster than that in the autoclaved mixed wastes. These filtrates likely contained microbial metabolites that acted on the virus, as in our previous study with PO1 (4). Laboratory studies have indicated that some bacteria are microbial predators of enteric viruses. These microbes may produce metabolites that adversely affect virus particles or may use the virus capsid as a nutrient source (3, 11). Sobsey et al. (22) found shorter virus inactivation times in nonsterile suspensions of wastewater. Herrmann et al. (12) found that two enteroviruses were inactivated more rapidly in a lake than in sterile lake water. Ward (23) reported virus loss in activated

4 90 DENG AND CLIVER APPL. ENVIRON. MICROBIOL. Temp ( C) TABLE 3. Inactivation of inoculated HAV in mixed-waste suspension media at two temperatures Mixed wastes Suspension medium Regression (day 1 ) a 25 STE (20%) DCMS (80%) Raw Autoclaved Filtrate STE (20%) SMS (80%) Raw Autoclaved Filtrate b 9.1 D value 37 STE (20%) DCMS (80%) Raw Autoclaved Filtrate a Log 10 PFU per milliliter. b P (P in each other case). STE (20%) SMS (80%) Raw Autoclaved Filtrate sludge due to microbial activity. Studies by Ward et al. (24) indicate that proteolytic bacterial enzymes inactivate echovirus particles in fresh water by cleavage of virus proteins (most notably VP-4 and, to a lesser extent, VP-1) thus exposing the viral RNA to nuclease digestion. Our study with PO1 demonstrated that the virus inactivation in mixed wastes is in part due to proteolytic enzymes produced by bacteria in the wastes (4). In another report, we describe the isolation, characterization, and identification of some of the bacteria in DCMS and SMS that are active against HAV (data not presented). Persistence in the environment varies with the type of virus. HAV, for example, is more stable than most enteroviruses at elevated temperatures (8). The fact that HAV no longer detectable by the PA was also undetectable by AC-PCR (e.g., after 35 days at 37 C in the experiment reported in Fig. 2) indicates that degradation of the viral particles was extensive enough either to preclude reaction of the antigen with its homologous antibody or to prevent amplification of the target sequence in the viral genome. Conversely, this finding shows that, at least in some instances, the AC-PCR does not yield positive results with inactivated virus, as is true with some other PCR methods. The survival time of viruses in wastewater is highly variable but should be considered in terms of days, weeks, or months as opposed to minutes or hours. Enteroviruses are reported to survive for 3 to 170 days in soil of various compositions at various temperatures and for 1 to 23 days on crops (15). Survival is substantially longer at cold temperatures. Detention and inactivation are the keys to the prevention of virus transmission in waste disposal (20). There is nothing particularly antiviral in STE per se. Virus inactivation is largely a function of post-septic tank treatment. Consequently, disposal of virus-laden STE after mixing with animal manure slurry and detention in a storage basin appears very attractive. A mixed-waste system entails increased detention, increased microbial load, and exposure to temperatures which may be higher than those in a septic tank all factors that should contribute to inactivation of the virus. ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences of the University of Wisconsin Madison and by the Small Scale Waste Management Project of the State of Wisconsin. We thank Jennifer C. Luks and Stephanie Bartel for providing cell cultures; Bill Enters for collecting STE, DCMS, and SMS; and Thomas Morgan for analyzing total concentrations of solids of waste samples. REFERENCES 1. Clesceri, L. S., A. E. Greenberg, and R. R. Trussell (ed.) Standard methods for the examination of water and wastewater, 17th ed., p American Public Health Association, Washington, D.C. 2. Cliver, D. O Food virology. Biotechnology 4: Cliver, D. O., and J. E. Herrmann Proteolytic and microbial inactivation of enteroviruses. Water Res. 6: Deng, M. Y., and D. O. Cliver Inactivation of poliovirus type 1 in mixed human and swine wastes and by bacteria from swine manure. Appl. Environ. Microbiol. 58: Deng, M. Y., S. P. Day, and D. O. 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