Soil inactivation of DNA viruses in septic seepage

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Soil inactivation of DNA viruses in septic seepage
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  ORIGINAL ARTICLE Soil inactivation of DNA viruses in septic seepage C.M. Davies 1,4 , M.R. Logan 2 , V.J. Rothwell 2 , M. Krogh 3 , C.M. Ferguson 1,3,4 , K. Charles 1,4 , D.A. Deere 4 and N.J. Ashbolt 1,4 1 Centre for Water and Waste Technology, School of Civil and Environmental Engineering, University of New South Wales, Sydney, NSW,Australia2 Sydney Water Corporation, West Ryde, NSW, Australia3 Sydney Catchment Authority, Penrith, NSW, Australia4 CRC for Water Quality and Treatment, Salisbury, SA, Australia Introduction A considerable proportion of people living in developedregions does not have access to centralized wastewatertreatment systems and rely on the use of on-site systems.It is estimated that wastewater from more than 23% of households in the US is treated in on-site and small-scaletreatment systems (USEPA 2002). Similarly, approxi-mately 30% of the population within the drinking watersupply catchment of Sydney, Australia are served by on-site systems, equivalent to an estimated 18 000 unseweredpremises (Charles  et al.  2001). An assessment of Sydney’sfailing on-site systems (Charles  et al.  2001) revealed thatpoor design, operation and maintenance are major causesof water contamination by these systems, as is suggestedelsewhere (Borchardt  et al.  2003). In addition, humanenteric viruses have been detected in 40% of the septictank effluents (Charles  et al.  2003a), and have beenshown to cause waterborne outbreaks in various countries(Borchardt  et al.  2003).A critical issue with sewage-related viruses releasedfrom on-site systems is their rate of inactivation in soil.Key influential factors include temperature, moisture con-tent, pH, soil type, virus type (Goyal and Gerba 1979),organic matter content, electrolyte concentration (Lanceand Gerba 1984), and the presence of other biota (Yeagerand O’Brien 1979; Alvarez  et al.  2000; Jin  et al.  2000).Most research on the impact of on-site systems has Keywords Adenovirus, PRD1 bacteriophage, inactivation,septic seepage, temperature. Correspondence C.M. Davies, Centre for Water and WasteTechnology, School of Civil and EnvironmentalEngineering, University of New South Wales,Sydney, NSW 2052, Australia.E-mail: received 13 February 2005,revised 9 June 2005 and accepted 10 June2005doi:10.1111/j.1365-2672.2005.02777.x Abstract Aims:  To generate field-relevant inactivation data for incorporation intomodels to predict the likelihood of viral contamination of surface waters by septic seepage. Methods and Results:  Inactivation rates were determined for PRD1 bacterio-phage and Adenovirus 2 in two catchment soils under a range of temperature,moisture and biotic status regimes. Inactivation rates presented for both viruseswere significantly different at different temperatures and in different soil types( a  = 0 Æ 05). Soil moisture generally did not significantly affect virus inactivationrate. Biotic status significantly affected inactivation rates of PRD1 in the loamsoil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly inthe loam soil than PRD1 bacteriophage. Conclusions:  Virus inactivation rates incorporated into models should beappropriate for the climate/catchment in question with particular regard to soiltype and temperature. Given that PRD1 is similar in size to adenoviruses, yetmore conservative with regard to inactivation in soil, it may be a useful surro-gate in studies of Adenovirus fate and transport. Significance and Impact of the Study:  A better understanding of the factorsthat govern virus fate and transport in catchments would facilitate the designof barrier measures to prevent viral contamination of surface waters by septicseepage. Journal of Applied Microbiology ISSN 1364-5072 ª  2005 Awwa Research Foundation, Journal of Applied Microbiology  100  (2006) 365–374  365  examined the migration of viruses through soil and hasassessed their potential to contaminate groundwater(Schijven 2001; Jin and Flury 2002; Faulkner  et al.  2003;Maxwell  et al.  2003). Few studies have examined theirpotential impact on surface waters due to overland trans-port (Davies  et al.  2005b).While on-site sewage disposal is typically subsurface viaan absorption trench, viruses may be deposited on theland surface or in the upper soil layers by both failingsubsurface sewage disposal systems and surface (e.g. spray irrigation) disposal areas. These viruses are then availablefor transport in surface runoff. Buffer zones provide bar-riers to the migration of viruses between septic systemsand surface waters by increasing the distance to traveland thus the travel time required. An increase in traveltime may increase the likelihood of viral inactivation by environmental factors and also the likelihood of virusretardation by adsorption to soil particles. It has beenreported that adsorption of viruses to soil particles isinfluenced by soil texture, the presence of cations such asMg 2+ and Ca 2+ , pH and virus type (Gerba and Schaiber-ger 1975; Nasser  et al.  1993). The introduction of guide-lines for determining set back distances for preventingviral contamination of surface waters is limited by thelack of knowledge with regard to the capabilities of catch-ment soils to immobilize and inactivate viruses.Human adenoviruses are one of only four virusesincluded in the Contaminant Candidate List of theUSEPA (1998), a list of emerging waterborne pathogensthat may pose a risk in drinking water and therefore areconsidered of importance in the water industry. Adeno-viruses, particularly types 40 and 41 are considered to bea primary cause of gastroenteritis in children (Uhnoo et al.  1986; Allard  et al.  1990; Cruz  et al.  1990). Further-more, adenoviruses are one of the most numerous viriontypes that can be cultivated from sewage (Bofill-Mas  et al. 2000) and have a high prevalence in raw surface and trea-ted waters (Santos  et al.  2004; Van Heerden  et al.  2004).Hence, along with the known extended persistence of adenoviruses in the environment compared to otherenteric viruses (Pina  et al.  1998), they were considered apertinent virus index group to study since they areassumed to be conservative with regard to their inactiva-tion rates. Due to the fastidious nature of Adenovirus 40and 41 however, they do not grow well in cell culture.Adenovirus 2 was considered to be a suitable, more easily cultured representative of the adenoviruses. Adenovirus 2is a known enteric double-stranded DNA virus that may cause respiratory infections, and is highly resistant to UVinactivation (Gerba  et al.  2002). As to human entericvirus models, the double-stranded DNA bacteriophagePRD1 (  60 nm) has generally been shown to be morepersistent in soil than either MS2 or Poliovirus 1 (Blancand Nasser 1996). In addition, it is similar in size tohuman and animal adenoviruses (60–80 nm), whichwould potentially make it a more suitable model forstudying the migration of Adenovirus through soil (Dowd et al.  1998). The objective of the present study was toestimate inactivation rates for viruses in soil undervarious environmental conditions so that they may beincorporated into models to describe the fate and trans-port of viruses through soil in septic seepage. Materials and methods Soil types The soils utilized in the study had previously been charac-terized as a loam (49% sand, 27% silt and 24% clay) anda clay loam (7% sand, 55% silt and 38% clay) (W. Hijnenand P. Stuyfzand, personal communication). Preparation of inocula Stock suspensions of PRD1 and Adenovirus 2 were pre-pared as follows: PRD1 was replicated in host cells of  Salmonella typhimurium  strain LT2 (ATCC 19585). Theculture was diluted by 10 ) 2 in gamma-irradiated settledsewage (90 kGy using  60 Co source) to give approximately 10 7 virions per ml.Adenovirus 2 was used in place of Adenovirus 40 or41, as the latter two viruses could not be grown to highenough titres for use in the inactivation experiments.Adenovirus 2 (ATCC VR846) was grown on a monolayerof HEp-2 (ATCC CCL-23) cells. Within 3 days, the entiremonolayer displayed cytopathic effects typical of adeno-viruses. The cultures were freeze-thawed to lyse the cellsand release virions into the medium, followed by centri-fugation at 300  g   for 10 min to remove cell debris. It wasthen necessary to remove most of the culture mediumfrom the virions to minimize stimulation of bacteriologi-cal growth in the soil microcosms. The virions wereseparated from the culture medium by sucrose cushionultracentrifugation. The viral pellet was resuspended inphosphate buffered saline and the resulting virus titreafter this step was approximately 10 7 ml ) 1 . The titre wasdetermined by TCID 50  over a period of 10 days to pro-vide high sensitivity of the assay. A dilution by 10 ) 2 ingamma-irradiated sewage gave a suspension containingapproximately 10 5 virions per ml. Preparation of soil microcosms Surface soils were collected from two locations within theSydney drinking water catchment at Marulan and Robert-son, NSW and designated loam and clay loam, respect- Soil inactivation of viruses  C.M. Davies  et al. 366  ª  2005 Awwa Research Foundation, Journal of Applied Microbiology  100  (2006) 365–374  ively. The soil was air-dried and sieved using a 1200  l msoil sieve. Several hundred portions of each of the soils(2 g) were weighed into 5 ml polyethylene vials. Approxi-mately half of the vials of sieved soil from each site weresterilized by gamma-irradiation (90 kGy).Each vial was inoculated with either 0 Æ 1 ml of PRD1 or0 Æ 1 ml of Adenovirus 2 diluted suspensions to achieveapproximate final number of virions per vial of 1  ·  10 6 or 1  ·  10 4 , respectively. Irradiated (90 kGy) or nonirradi-ated settled sewage (0 Æ 1 ml) was added to irradiated ornonirradiated soil microcosms, respectively. Settled sew-age was considered an appropriate substitute for septictank effluent. A number of control vials of each soil typewere also left uninoculated to be used for moisture deter-minations. MilliQ water (0 Æ 2 ml) was added to each of these vials to ensure that the moisture content was similarto inoculated vials.Salt solutions (approximately 250 ml) containing0 Æ 08 mol l ) 1 and 0 Æ 77 mol l ) 1 NaCl were placed into thebottom of sealable jars (capacity approximately 2 l) tosimulate soil matrix potentials of field capacity and dry conditions, respectively, and designated ‘wet’ and ‘dry’(Davies  et al.  2005a). Wire mesh discs were used to ele-vate the vials above the level of the salt solution. Thevials, with caps loosened, were placed in airtight sealed jars, which were incubated at 4 and 20  C. The jars werestored in the dark and weighed before incubation. ForPRD1 microcosms incubated at 20 and 35  C, non- andgamma-irradiated soils were inoculated. Due to the highcost of analysis and labour-intensive nature of the Adeno-virus 2 assay, biotic status was not included as a factorfor adenovirus microcosm experiments. Also at 4  C forPRD1, only nonirradiated soil microcosms were preparedbecause it was assumed that biotic activity at this tem-perature would be negligible. The jars were weighedbefore and after vials were removed to confirm that they had remained sealed and that there had been no moistureleakage during incubation. Five or three replicate inocula-ted microcosms, respectively for PRD1 or Adenovirus 2,from each treatment (soil type, moisture, temperatureand biotic status) were withdrawn periodically from eachsealed jar for determination of infective PRD1 or Adeno-virus. The microcosms were sampled destructively. Inaddition, duplicate vials were removed periodically fromeach jar for per cent moisture determination by drying inpreweighed crucibles at 105  C for 48 h (APHA 1998). Enumeration of PRD1 bacteriophages Each 2 g of inoculated soil from sampled vials waswashed into a 50 ml Falcon TM tube (Becton Dickinson,Franklin Lakes, NJ, USA) using 20 ml of 0 Æ 002 mol l ) 1 sodium pyrophosphate (pH 9) and vortexing. The soilslurry was then vortexed for 2 min and allowed to standfor 30 min. After further vortexing for a few seconds theslurries were allowed to settle for 10 min after which1 ml was withdrawn by pipette from the top 2 mm. Thiswas diluted serially in sterile Milli Q water and assayed by the double agar layer technique (Adams 1959) using Salm. typhimurium  strain LT2 as a host. Concentrationsof PRD1 were expressed as plaque forming units (PFU)per gram dry weight of soil.A recovery control was prepared for each soil type by freshly inoculating 2 g of gamma-irradiated soil withapproximately 1  ·  10 6 PRD1 virions from a stock suspen-sion. The PRD1 was allowed to adsorb to the soil by stor-ing at room temperature for 2 h, before being processedas described above. The PRD1 titre of the stock suspen-sion used to inoculate the recovery controls was alsodetermined. Per cent recovery for each soil type wasdetermined as the concentration of PRD1 recovered divi-ded by the concentration spiked into the soil 100 times. Enumeration of Adenovirus 2 Soil samples were processed by adding 9 ml of carbonatebuffer (pH 9 Æ 6) to each vial, followed by vortexing for1 min. The pH was measured to confirm that the pH was9 Æ 6 and adjusted if required. The samples were then sha-ken for 10 min on a shaker and centrifuged at 2000  g   for10 min. The supernatant was transferred to a fresh, cleantube and filtered through a 0 Æ 45  l m filter. The pH wasadjusted to pH 7 Æ 0. Serial dilutions were prepared andused to inoculate 12 well-microtitre plates of HEp-2 cells.Infective viruses were expressed as tissue culture infectivedose for 50% (TCID 50 ) after 10 days (Mahy and Kangro1996).A recovery control was prepared for each soil type by freshly inoculating 2 g of gamma-irradiated soil withapproximately 1  ·  10 4 Adenovirus 2 virions from a stock suspension. Per cent recovery for each soil type was deter-mined as described for PRD1. In addition, uninoculatedvials were withdrawn for moisture determination. Data analysis PRD1 concentrations and Adenovirus TCID 50  lower thanthe detection limit were assigned the value of (detectionlimit)/2. The model used to determine inactivation rateswas log 10  N  t  /  N  0  =  ) K  T. Inactivation rates were calculatedusing linear regression of log 10  N  t   against time, where  N  t  was the concentration of viable viruses at time  t   (leastsquares technique, SAS Version 8.1, SAS Institute Inc.,Cary, NC, USA). The slope of the line of best fit wasequal to  ) K   and the intercept was equal to log 10  N  0 ,where  N  0  was the mean concentration at time zero. A C.M. Davies  et al.  Soil inactivation of viruses ª  2005 Awwa Research Foundation, Journal of Applied Microbiology  100  (2006) 365–374  367  measure of the appropriateness of this approach wasderived by assessing the  r  2 value and significance of theregression model and parameter values at the  a  = 0 Æ 05level. Results Recoveries from soils The recoveries of the PRD1 controls were 83 ± 27%( n  = 15) from the loam soil and 61 ± 15% ( n  = 9) forthe clay loam soil. Virus recovery controls were used toshow that the method employed to enumerate the virusesin the microcosm performed consistently, and that therewas no change in the efficiency of the method to detectvirions over time, i.e. as the soil aged. Therefore it ispossible to conclude that the decrease in virus concentra-tions in the microcosms was due to inactivation. Per centrecoveries were not used to adjust the virus concentra-tions.The recoveries for Adenovirus 2 from the loam soilwere 71 ± 19% ( n  = 13) and 59 ± 20% ( n  = 3) for theclay loam soil. As for PRD1, percent recovery was notused to adjust the data for recovery. PRD1 inactivation Figures 1–3 show the concentrations of PRD1 over timein soil microcosms at 35, 20 and 4 C, respectively. Theerror bars represent ±1 SD of the mean of five replicatemicrocosms. There was a general decrease in the log 10 viral concentration (PFU g ) 1 ) with time at each of thethree temperatures examined. Log 10  transformation of thedata improved the heterogeneity of the variances andthe normality of the residuals. Only inactivation in nonir-radiated soil was determined at 4  C because it wasassumed that biotic activity at this temperature would benegligible.At 35  C (Fig. 1), inactivation in clay loam soilappeared to be more rapid than in loam soil, with a5 log 10  decrease in PRD1 concentration within 5 days inclay loam soil compared to a 2–4 log 10  decrease within15–30 days in loam soil. At 20  C (Fig. 2), the inactivationof PRD1 once again appeared to be more rapid in clay loam soil than in loam soil. A 5 log 10  decrease in PRD1concentration occurred in clay loam soil within 70 dayscompared to a 2–4 log 10  decrease within 140 days in loamsoil, indicating also that inactivation was less rapid at20  C than at 35  C. At 4  C (Fig. 3), the PRD1 populationdecreased by 5 log 10  in clay loam soil, and by 4 log 10  inloam soil within 140 days.Clay loam soil had a relatively low pH of 4 Æ 5–5 Æ 0, com-pared with loam soil, which was approximately pH 6 Æ 0.The low pH in clay loam soil probably contributed to themore rapid inactivation of PRD1 in this soil. Adenovirus inactivation Figures 4–6 show the inactivation of Adenovirus 2 in soilmicrocosms at 35, 20 and 4  C, respectively. Due to theextremely rapid inactivation of Adenovirus 2 in clay loamsoil at 20 and 35  C (i.e. complete inactivation within1 day) (not shown), and the apparent interference of this 01234560 5 10 15 20 25 30Time (days) (a)(b)    L  o  g    1   0   c  o  n  c  e  n   t  r  a   t   i  o  n   P   F   U   g   –   1     (   d  r  y  w   t   )   L  o  g    1   0   c  o  n  c  e  n   t  r  a   t   i  o  n   P   F   U   g   –   1     (   d  r  y  w   t   ) 01234560 1 2 3 4Time (days) Figure 1  Inactivation of PRD1 bacteriophage in (a) loam soil and (b)clay loam soil, at 35  C. Error bars are ±1 SD of five replicate micro-cosms. ( r ) dry; ( ) wet; ( · ) dry irradiated and ( d ) wet irradiated. 01234560 20 40 60 80 100 120 140 160Time (days)    L  o  g    1   0   c  o  n  c  e  n   t  r  a   t   i  o  n   P   F   U   g   –   1     (   d  r  y  w   t   ) Figure 2  Inactivation of PRD1 bacteriophage in soil at 20  C. Errorbars are ±1 SD of five replicate microcosms. ( ) loam dry; ( ) loamwet; ( · ) clay loam dry; ( d ) clay loam wet; ( n ) loam dry irradiated; ( s )loam wet irradiated; ( e ) clay loam dry irradiated and ( h ) clay loamwet irradiated. Soil inactivation of viruses  C.M. Davies  et al. 368  ª  2005 Awwa Research Foundation, Journal of Applied Microbiology  100  (2006) 365–374  soil with the cell culture assay, it was possible only to usedata for clay loam soil from microcosms incubated at4  C. The more rapid inactivation of Adenovirus in clay loam soil compared to loam soil was thought to be dueto the acidity of the soil. At 35  C, in loam soil there wasa 3-log 10  decrease in Adenovirus 2 concentration within3–4 days (Fig. 4). The same soil at 20  C showed a 3 log 10 decrease within 20 days (Fig. 5). At 4  C the Adenovirus 2concentration in soil from loam decreased by approximately 2 log 10  within 45 days, while soil from clay loam showed a 2 log 10  decrease within 30 days. Summary of inactivation rates Inactivation rates derived from  K  , the slope of the line of best fit when log 10  N  t   PRD1 was regressed against time,are given in Table 1. In general, this relationship fittedthe data well as indicated by the  r  2 values, which were allgreater than 0 Æ 7 and were significant (at  a  = 0 Æ 05).Figure 7 summarizes the effects of the different factors onthe inactivation rates ( K  ) of PRD1 in soil. The 95% con-fidence intervals for  K   are also presented (as errors bars).There was a much greater difference in inactivation ratesbetween 20 and 35  C than between 4 and 20  C.Similarly, inactivation rates for Adenovirus 2 are givenin Table 2. Figure 8 summarizes the effects of the soilmoisture and soil type on the inactivation rates of Adeno-virus 2. Soil moisture had little effect on the inactivationrate. As is the case for PRD1, there was a much greaterdifference in Adenovirus 2 inactivation rates between 20and 35  C than between 4 and 20  C, emphasizing theinfluence of higher temperatures on virus inactivation.Soil moistures in terms of the soil moisture characteris-tic  h  are given in Tables 3 and 4 for PRD1 and Adeno-virus microcosms, respectively. The  h  values were derivedfrom the gravimetric soil moisture determinations andconverted to soil moisture potential using soil moisturecurves derived for loam and 11 soils (not shown) (Davies et al.  2005b). For PRD1 microcosms in loam soil at 20  Cand clay loam soil at 4 and 20  C, there is little differencebetween measured moistures in dry and wet microcosms.The reason for this is not known since for Adenovirus(Table 4) and the remaining PRD1 microcosms the mois-tures are appreciably different. However, even when themoistures were significantly different in dry and wetmicrocosms, moisture did not appear to affect the inacti-vation rate of either of the two viruses, with the exceptionof PRD1 in loam soil at 35  C (Figure 1). 0123450 1 2 3 4Time (days)    L  o  g    1   0    T   C   I   D    5   0 Figure 4  Inactivation of Adenovirus 2 in soil at 35  C. Error bars are±1 SD of three replicate microcosms. ( ) loam dry and ( ) loam wet. 0123450 10 20 30 40 50Time (days)    L  o  g    1   0    T   C   I   D    5   0 Figure 6  Inactivation of Adenovirus 2 in soil at 4  C. Error bars are±1 SD of three replicate microcosms. ( ) loam dry; ( ) loam wet; ( · )clay loam dry and ( d ) clay loam wet. 01234560 20 40 60 80 100 120 140 160Time (days)    L  o  g    1   0   c  o  n  c  e  n   t  r  a   t   i  o  n   P   F   U   g   –   1     (   d  r  y  w   t   ) Figure 3  Inactivation of PRD1 bacteriophage in soil at 4  C. Error barsare ±1 SD of five replicate microcosms. ( ) loam dry; ( ) loam wet;( · ) clay loam dry and ( d ) clay loam wet. 0123450 5 10 15 20 25 30 35 40Time (days)    L  o  g    1   0    T   C   I   D    5   0 Figure 5  Inactivation of Adenovirus 2 in soil at 20  C. Error bars are±1 SD of three replicate microcosms. ( ) loam dry and ( ) loam wet.C.M. Davies  et al.  Soil inactivation of viruses ª  2005 Awwa Research Foundation, Journal of Applied Microbiology  100  (2006) 365–374  369
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