Multiyear Study of Sludge Application to Farmland: Prevalence of Bacterial Enteric Pathogens and Antibody Status of Farm Families

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, My 1987, p /87/ $02.00/0 Copyright C) 1987, Americn Society for Microbiology Vol. 53, No. 5 Multiyer Study of Sludge Appliction to Frmlnd: Prevlence of Bcteril Enteric Pthogens nd Antibody Sttus of Frm Fmilies ABRAMO C. OTTOLENGHIl* AND VINCENT V. HAMPARIAN12 Deprtment of Medicl Microbiology nd Immunologyl nd Deprtment of Peditrics,2 The Ohio Stte University, Columbus, Ohio Received 6 Februry 1986/Accepted 17 Februry 1987 We describe our experience with the isoltion of slmonelle from sewge sludge from four tretment plnts in different geogrphic res of Ohio. Over 3 yers, we isolted slmonelle 50 times from 311 sludge smples. Most isoltions were mde fter enrichment in Selenite broth (BBL Microbiology Systems, Cockeysville, Md.). The lrgest proportion of isoltions cme from the plnt serving the popultion of Columbus, lrge metropolitn re. A significntly greter number of isoltions from this plnt were mde during the first qurter of the yer. Twenty-one different serotypes were isolted, long with five untypble strins. The most frequently isolted serotype ws Slmonell infntis. Five of the strins were multiply resistnt to ntibiotics. We lso describe the prevlence of ntibodies to slmonelle in members of the fmilies residing on the frms in the study. It ws found tht ntibodies to group C slmonelle predominted. As prt of study involving the ppliction of wstewter sludge to frmlnds in Ohio, the presence of bcteri nd viruses known to cuse enteric disese in humns ws monitored. The study ws designed to monitor frm workers nd their fmilies to ssess the risk, if ny, resulting from the ppliction of wstewter sludge to lnd under controlled conditions. In this study, we describe the results of testing sludge smples for the presence of Slmonell spp., Shigell spp., nd Cmpylobcter spp. We lso describe the serologicl sttus of frm workers nd their fmilies t the beginning nd end of the study. In ddition, stool smples tken from the prticipnts t regulr intervls throughout the study were tested for the presence of Slmonell spp. The epidemiologicl design is described in detil in the full project report (8). Briefly, frms in the study were ssigned rndomly either to receive sludge or to be controls, nd the dt were nlyzed on the bsis of mtched frm pirs. MATERIALS AND METHODS Sewge plnts nd sludge smples. A totl of 311 sludge smples were collected nd tested for slmonelle nd shigelle over 36 months. The smples were from four wstewter tretment plnts in three geogrphic res of Ohio. Detils of the types of plnts s well s of the physicochemicl chrcteristics of the sludge produced t the different sites cn be found in the complete project report (18) nd hve been summrized in previous publiction (13). Sludges. Sludge smples were collected biweekly from the M300 nd M500 plnts in Medin County, the Jckson Pike Plnt of Columbus, nd the sewge tretment plnt of the city of Springfield, Ohio. Smples of 1.5 to 2.0 liters were collected in sterile plstic bottles t the plnt site from which trucks were loded. The bottles were tightly seled, lbeled, nd stored t 4 C. The next morning, the smples from Medin nd Springfield were plced in insulted boxes on ice nd shipped to the lbortory vi commercil crrier. The Columbus smples were picked up by lbortory personnel * Corresponding uthor on the dy of collection. Attempts to isolte bcteri were done on the dy of rrivl of the smples to the lbortory. Stool smples. Stool smples were obtined three times per yer both from unexposed controls nd from subjects from sludge-receiving frms. The smples were obtined t pproximtely 4-month intervls with t lest one smple obtined during the summer or erly fll. The smples were collected in plstic continers which were seled nd either plced in cooler with ice nd delivered to the lbortory the sme dy or refrigerted overnight nd delivered the next dy. The smples were tested for bcteril pthogens on the dy of rrivl. Humn ser. A bse-line serum smple ws obtined from ech subject before the initition of lnd ppliction of sludge in given re. Subsequently, three serum smples per yer were obtined from ech individul throughout the course of the study. When blood smples were obtined from subjects outside the Columbus re, they were centrifuged on the sme dy by locl commercil lbortory, nd the ser were trnsferred septiclly to sterile vils nd trnsported on ice to our lbortory. Isoltion of slmonelle nd shigelle from sludge. Unless otherwise noted, ll medi were obtined s dehydrted bses from Difco Lbortories, Detroit, Mich. (Fig. 1). (i) Dy 1. Sludge ws swbbed directly onto pltes of Hektoen Enteric gr (HEK), McConkey gr (MAC), nd xylose-lysine deoxycholte gr (XLD). The pltes were then streked for isoltion. Impregnted swbs were dded to tubes contining 10 ml of Selenite or GN broths (BBL Microbiology Systems, Cockeysville, Md.). In erly smplings, Tetrthionte broth (BBL) tubes were lso inoculted. This procedure ws lter bndoned becuse recovery ws no better thn with the other two enrichment medi. Pltes nd enrichment broths were incubted t 37 C for 24 h (pltes) or for 24 nd 48 h (broths). (ii) Dy 2. Colonies producing H2S were picked from HEK nd XLD grs nd tested on biochemicl medi. Lctosenegtive colonies were tested for oxidse ctivity, nd those without ctivity were tested on biochemicl medi. The enrichment medi cultures were streked on HEK, XLD, nd MAC grs nd incubted for 18 to 24 h t 37 C.

2 VOL. 53, 1987 BACTERIAL PATHOGENS IN SLUDGE APPLIED TO LAND 1119 The biochemicl medi used for screening were Triple Isoltion of slmonelle nd shigelle from stool specimens. Sugr Iron gr, lysine-indole gr, nd ure gr. Colonies A 10% suspension of the stool specimen (less thn 24 h old) were lso suspended in 0.3 ml of sline contiifning disk of ws cultured nd hndled s described bove for sludge. A o-nitrophenyl-,3-d-glctopyrnoside. All tube-s were incu- totl of 1,821 stool smples were tested. When specimen bted for 18 to 24 h t 37 C. ws positive, repet cultures were tken every 3 weeks until (iii) Dy 3. All pltes streked from the enric:hment broths two consecutive negtive cultures were obtined. were exmined bove, nd suspected colonie: s were inocu- Recovery of slmonelle nd shigelle from seeded sludge lted into biochemicl test medi s describe-d bove. All smples. Three different strins of slmonell were used. For tubes of biochemicl rections were exmineod, nd where the erly experiments, stock of Slmonell typhimurium the rections indicted the possible presence off slmonelle, from our lbortory collection ws used. For lter experi- API 20E strips (Anlytb Products, Plinview, N.Y.) were ments, strins of S. typhimurium nd of Slmonell sint- pul, both isolted from sludge, were used. A Shigell inoculted ccording to the directions of the rmnufcturer. The enrichment cultures were gin strek ed on HEK, flexnerii strin from our deprtmentl collection ws used. XLD, nd MAC pltes. Two methods were used to test the effectiveness of our (iv) Dy 4. Pltes streked for isoltion or dy 3 were recovery system. exmined s described bove. Biochemicl tiests nd API (i) Seeding of smples t plnt. Two 1,200-ml smples of inocultions gin were performed on ll suspe( cted colonies. sludge from the Medin 300 plnt were seeded with 2 x 107 Any colony which ws identified s Slmonell sp. ws shigelle nd 1 x 107 slmonelle to give finl concentrslnts for use tion of c. 1.6 x 104 shigelle per ml nd 8 x 103 slmonelle trnsferred to three Trypticse soy gr (BBL) in serologicl identifiction. All slmonelle iisolted were per ml. These smples were then hndled s described bove tested by gglutintion for group ntigens by using group- for sludge smples. specific ser (Difco) nd then submitted to the Ohio Helth (ii) Seeding of smples in the lbortory. The slmonell Deprtment Lbortory, Columbus, Ohio, for species iden- strin to be used ws inoculted into three 9-ml smples of tifiction. freshly received sludge to give finl concentrtions of c. 2 x Antibiotic sensitivity testing nd biochemicl [confirmtion 103, 2 x 102, nd 20 CFU/ml. These three smples were then were done in the Clinicl Bcteriology Lboritories of The processed together with the routine smples. Experiments Ohio Stte University Hospitl. All confirme( d slmonelle were conducted three times with sludge from ech site in the were repurified nd freeze-dried in skim milk nd stored t study. -700C. Isoltion of Cmpylobcter spp. from sludge. For ll recovery experiments nd for control purposes, we used n isolte of Cmpylobcterjejuni from ptient, which ws obtined Fecl or Sludge Suspension from the bcteriology lbortories of The Ohio Stte University Hospitl. Sludge ws swb inoculted in triplicte on J, Cmpy Blood Agr Medium pltes (Remel, Lenex, Kns.) Selenite nd streked for isoltion. Pltes were incubted t 25, 37, 4- HEK XLD MAC Dy 2 or 3 TSI LIA 'I- GN Broth Broth nd 42 C in stinless steel jrs in n tmosphere consisting of 5% 02, 10% C02, nd 85% nitrogen. Control pltes of C. jejuni were included in ech jr. The cultures were exmined t 24 h nd 48 h, nd colonies were picked nd tested for t 1~ 24 nd 48 ho WurS >, ctlse nd oxidse ctivity. Positive colonies were further tested for motility, H2S production, nd resistnce to nli- HEK dixic cid nd cephlothin. XLD XLD Recovery of C. jejuni from seeded sludge. C. jejuni ws MAC MAC grown for 48 h on Cmpy Blood Agr Medium. The org-, nisms were suspended in 3 ml of freshly boiled thioglycolte broth. Tenfold dilutions were mde, nd clibrted loop counts were done by inocultion in triplicte with ml clibrted loop. Approprite volumes were inoculted in Suspected colonies duplicte into 9 or 9.9 ml of sludge suspension to obtin from ech plte suspensions rnging from c. 2 x 106 to 2 x 102 CFU/ml. Mixing of the suspensions ws ccomplished by bubbling the mixture with the gs used for incubtion s described bove. OCidse neg. The seeded sludge suspensions were tested s described bove for the isoltion of C. jejuni. To test for survivl of the UREA orgnisms in sludge, the suspensions were stored t 4 C for ONPG 1 h, 24 h, 4 dys, nd 7 dys before being tested s described bove. Becuse I~ of the viscosity of the sludge obtined from the Columbus Jckson Pike Plnt, this sludge ws diluted 1:4 API 20E* strips with sterile isotonic sline before the ddition of bcteri. These experiments lso were repeted three times. 'I Detection of ntibodies to slmonell ntigens. The lst vilble serum from ech individul ws tested for ntibodies to Slmonell groups B, C, D, nd E by the rpid slide Serology FIG. 1. Flow chrt for the isoltion of Slmon ell spp. from test by using Bcto-Widl Slmonell 0 ntigens (Difco). wstewter sludge. Abbrevitions re s defined in tihe text. *API is Conditions were set so s to run the test t 1:20 correlted trdemrk of Anlytb Products. dilution (0.04 ml of serum). Results were scored from

3 1120 OTTOLENGHI AND HAMPARIAN APPL. ENVIRON. MICROBIOL. TABLE 1. Slmonell recoveries from seeded sludge from the Columbus plnt Recovery fter incubtion for: Finl concn (CFU/ml) of 24 h 7 dys dded orgnism HEK MAC XLD HEKb MACb XLDb HEK MAC XLD HEKb MACb XLDb 0 / - - / -- -I/ I- _ - -I- -I- _ / 1.1 x _ - +/+ +I_ +/ /+ +I /_ 1.1 x _ - - +/+ +I /I+ - - _ +/+ +I_ +I_ Approprite numbers of orgnisms were dded to 10 ml of sludge (in duplicte) nd incubted for 24 h or 7 dys t 4 C. Isoltions were done ccording to the protocol. -, No slmonelle recovered; +, slmonelle recovered. bfrom Selenite enrichment broth/gn broth. negtive to 4+, depending on the degree of clumping observed. If the most recently obtined serum of n individul ws positive t level of 1+ or more, the bse line (obtined before ppliction of sludge in the re) nd subsequent ser were tested until positive serum ws found. If the bse-line serum of n individul ws lso positive for the given group ntigen(s), no dditionl ser were tested. RESULTS Recovery of shigelle, slmonelle, nd C. jejuni from seeded sludge. Two smples of sludge seeded with slmonelle t the plnt (Medin 300) were tested. Both yielded the strin seeded (dt not shown). Testing of sludge seeded in the lbortory resulted in the detection of the strin inoculted up to 7 dys fter seeding even when s few s 11 CFU/ml were present. This concentrtion of orgnisms ws detected only by enrichment techniques. A typicl result is TABLE 2. Medi which yielded the recovery of Slmonell spp. from sludge nd humn stool specimens Enrichment Smple Species Tetrsource Primry GN Selenite thionte Humn S. muenchen Humn S. jv Medin 300 S. sint-pul + + S. newington Medin infntis Znesvilleb S. hinshwii Columbus S. derby Medin sn diego Humn S. muenchen Medin 500 S. ornienburg + - Columbus Untypble Columbus S. gon (3 isoltes) Columbus S. infntis Columbus Untypble Columbus S. infntis Columbus S. ntum Columbus S. thompson Columbus S. ohio Columbus S. enteritidis (2 isoltes) Medin 300 S. sint-pul Medin 500 S. sint-pul - + Springfield S. infntis Primry isoltion is mrked + if recovery ws mde on HEK, XLD, or MAC pltes. If recovery ws seen fter enrichment in GN, selenite, or tetrthionte broths, the corresponding column is mrked. b Becuse of high cdmium content in the sludge, this plnt ws dropped from the lnd ppliction prt of the study. shown in Tble 1. Direct recovery by plting ws seen regulrly only when the orgnisms hd been in the sludge for no more thn 24 h nd were present t 10 times the concentrtion required for isoltion in enrichment medium (dt not shown). In one instnce when we used for seeding experiments sludge tht lredy contined group C slmonell, both the seeded group B slmonell nd the group C slmonell were recovered. The frequency of isoltion of slmonell fter direct plting or enrichment procedures is shown in Tble 2. Twenty-two recoveries from sludge smples nd from three stool smples were nlyzed to determine on which medi the slmonell colonies were detected. In only five cses were the orgnisms recognized on primry isoltion pltes. Two were humn isoltes, nd the others were one ech from the two Medin plnts nd the Columbus plnt. No shigelle were recovered in our seeding experiments, even when orgnisms were dded to finl concentrtion of 1.6 x 104 CFU/ml. Cmpylobcter sp. ws recovered when c. 150 CFU/ml were present. Recovery ws successful, even when orgnisms hd been held in sludge up to 7 dys t 4 C. Results of typicl experiment re shown in Tble 3. Attempts to isolte shigelle nd Cmpylobcter spp. No Shigell spp. were isolted from ny of the sludge or stool specimens tested. No Cmpylobcter spp. were isolted from 99 smples of sludge tested between September 1980 nd June Of the smples, 41 were from the Columbus plnt, 18 were from ech of the Medin plnts, nd 22 were from the Springfield plnt. Isoltion of slmohelle from sludge. A totl of 50 isoltions (16%) of Slmonell spp. were mde from the 311 smples of sludge tested from ll sources. The frequency of isoltion vried by yer nd by site (Tble 4). Isoltion ws most frequent from sludge smples obtined from the Columbus TABLE 3. Contct time C. jejuni recovery from seeded sludge from Medin 300 plnt No. of positive tests with inoculum (CFU/ml) ofb: 15 x x x 101 lh h hc dys C. jejuni ws inoculted in sludge to the finl concentrtion indicted nd held for the specified time t 4 C before plting. btwo tests were done for ech inoculum level nd contct time. No orgnisms were recovered from sludge to which no orgnisms hd been dded. " Subcultured in Cmpy Thioglycolte broth fter holding for 24 h t 4 C.

4 VOL. 53, 1987 BACTERIAL PATHOGENS IN SLUDGE APPLIED TO LAND 1121 Site TABLE 4. Slmonell isoltion by qurter of yer nd by site Qurter Totl positive/totl tested Positive Medin 300 5/19 3/15 1/13 1/17 10/ Medin 500 2/18 2/14 1/13 0/18 5/ Columbus 14/37 5/28 3/25 9/36 31/ Springfield 1/18 1/12 2/11 0/17 4/ Totl positive/totl tested 22/92 (24) 11/69 (16) 7/62 (11) 10/88 (11) 50/311 (16) (% positive) Dt re presented s number of positive smples/number of smples tested. tretment plnt (25%) nd lest frequent from smples from the Springfield plnt (7%). The distribution of isoltions from given plnt did not follow ny pttern, except tht there ppered to be n incresed isoltion rte during the first qurter of ech yer (Tble 4). An overll chi-squre test of the dt showed borderline P vlue (0.1 > P > 0.05), so the dt were subjected to n nlysis of residuls (11) which indicted tht significnt devition existed in the number of isoltions mde during the first qurter (2.43, stndrd devition). A 2 x 2 chi-squre nlysis of the isoltions mde during the first qurter versus those of ll other qurters combined yielded P of <0.02, indicting tht substntilly more isoltions of slmonelle were mde during the first qurter thn would hve been expected. It ppered tht the increse ws due primrily to the isoltions from the Columbus plnt. To test this hypothesis, the first-qurter dt from Columbus ws tested in 2 x 4 chi-squre test ginst first-qurter dt from the other sites. The overll test gve P of <0.05. When the first-qurter dt from Columbus s shown in Tble 4 were compred in 2 x 2 chi squre test with the combined dt from the other three qurters from Columbus, significnt difference ws lso noted (P = <0.05). Twenty-one different serotypes nd five untypble strins were isolted from the sludge which ws pplied to lnd TABLE 5. Slmonell serotypes isolted from sludges Serotype Frequency % S. delide 1 2 S. gon 4 8 S. ntum 1 2 S. binz 1 2 S. derby 1 2 S. enteritidis 2 4 S. hvn 1 2 S. infntis 8 16 S. jv 1 2 S. mnhttn 1 2 S. montevideo 3 6 S. newington 1 2 S. ohio 2 4 S. ornienburg 3 6 S. pnm 1 2 S. reding 2 4 S. sint-pul 4 8 S. sn diego 1 2 S. tennessee 1 2 S. typhimurium 4 8 S. thompson 2 4 Nontypble 5 10 Only isoltions from sludges obtined from plnts used s source of mteril pplied to lnd re listed. (Tble 5). The most frequently encountered serotype ws Slmonell infntis, isolted on eight occsions; S. sintpul, S. typhimurium, nd S. gon were the next most frequently isolted serotypes (four times ech). Antibiotic sensitivity ptterns of the slmonelle isolted from sludge. The ntibiotic sensitivity pttern of the slmonell strins isolted is importnt for epidemiologicl resons, nd Tble 6 presents the distribution of the MICs of given ntibiotics for most of the strins isolted from sludge. A few strins were lost before ntibiotic sensitivity could be tested. Most strins showed low MICs of most ntibiotics. Five strins (10%) showed multiple resistnce ptterns nd re described in Tble 7. Isoltion of slmonelle from individuls. The isoltions mde from individuls re indicted in Tble 8. One individul tested positive fter repeted cultures. In none of the cses ws clinicl disese correlted with the isoltion of slmonelle from the stool. Antibodies to slmonelle in the frm popultion. The serologicl sttus of the subject popultion t the end of the study is shown in Tble 9. We were interested in determining possible infection reflected by ntibody increses; thus, individuls with no ntibodies t the end of the study were ssumed to hve been negtive t the strt of the study. Sttisticl nlysis of the dt by the chi-squre test indictes tht there ws significntly greter (P s 0.001) incidence of ntibodies to group C slmonelle thn to the other groups. DISCUSSION Enteric bcteri in sludge. The procedure we used for the isoltion of slmonelle from sludge is similr to tht described by Dudley et l. (9), in tht we too used Selenite broth s one of our enrichment medi nd incubted the enrichment t 37 C rther thn t 42 C s suggested by vrious uthors (4, 14, 20, 21). Edgr nd Sor (10), in study compring methods for the isoltion of slmonelle, reported tht severl methods described in the literture produced different results not only becuse some of the medi used were inhibitory for the orgnisms but lso becuse, in some instnces, some were inhibitory when used t higher incubtion tempertures. These uthors reported tht brillint green gr ws the best plting medium tht they tested. Although we did not use brillint green s the plting medium, we obtined good growth nd differentition with HEK nd XLD plting medi, with the former giving better results. The sensitivity of our isoltion system ws sufficient for our purposes in tht we were ble to detect 11 CFU/ml of seeded sludge (1.9 to 6.0% solids). Dudley et l. (9) reported detection limit of >2 to <24 CFU/g of totl solids. In studies directed to the determintion of the most probble

5 1122 OTTOLENGHI AND HAMPARIAN APPL. ENVIRON. MICROBIOL. TABLE 6. Distribution of MICs for Slmonell spp. isolted from sludge % of isoltes t MIC (p.g/ml) Antibiotic ' ' Ampicillin Crbenicillinb Ticrcillin Cephlothin Cefmndole Cefoxitin Gentmicin Tobrmycin Amikcin Tetrcycline Chlormphenicol n = 43 isoltes tested for ntibiotic sensitivity. Totl isoltes, n = 50. Percentges re rounded to nerest integer. b n = 21 isoltes. number of slmonelle in sludges from different sources, rte of 8% reported by Dnielsson (6) in her studies in Jones et l. (15) were ble to find orgnisms in sludges Sweden. contining s few s 0.3 slmonelle CFU/100 ml. The We re unble to relte the higher number of isoltions method used by Jones nd his collegues (15) ws much from the Columbus plnt during the first qurter of ech yer more extensive thn ours in tht multiple enrichment sys- to ny specific condition t the plnt. Crrington et l. (3) tems were used. showed tht the inctivtion of slmonelle in nerobic As with the results reported by Dudley et l. (9), we lso sludge ws ffected by temperture during digestion, so we were unble to detect Shigell spp. in sludge smples. Our subjected the frequency of isoltion dt to correltion recovery experiments suggest tht shigelle do not survive nlysis, resoning tht in the first qurter of the yer, well in sludge. This would decrese the probbility tht this tempertures would tend to be lower nd thus the orgnisms orgnism would be present in the sludge pplied to lnd. might be protected. Sludge temperture dt (not shown) However, we cnnot exclude the possibility tht the method vilble to us indicted tht the temperture of the digestors used by us nd other workers is not sufficiently sensitive to vried between 30 nd 35 C during the course of the study permit isoltion. Becuse of this lck of isoltion of Shigell nd tht isoltion of orgnisms ws not correlted with the spp. from originl sludge or from seeded sludge, we re temperture of the digestors. unble to rech ny conclusion bout the presence or To ssess the reltive risk of infection with slmonell, the bsence of this pthogen or bout the reltive risks involved. concept of infective dose under nturl conditions must be Our inbility to recover Cmpylobcter spp. from smples considered. It is difficult to estblish such concept becuse of sludge despite recovery of the orgnism from our seeded vrious estimtes hve been mde bout the number of experiments even fter 7 dys t 4 C indictes tht the vible slmonelle required for infection. Cliver (5) reported orgnism, if present in sludge, ws undetectble by our tht slmonell gstroenteritis occurred in dults nd chilmethods. The high sensitivity of this orgnism to oxygen dren who hd eten chocolte contminted with <1 to 100 might mke its presence unlikely in erobiclly digested Slmonell estbourne orgnisms per 100 g. D'Aoust (7) sludges. reported infection doses of 0.7 to 6.1 cells in series of cses The different rtes of isoltion of slmonelle from the resulting from the ingestion of S. typhimurium-contminted different sites (Tble 4) is not surprising. Similr results hve cheese. In nother cse, Lng et l. (17) reported tht 15,000 been obtined by Dnielsson (6), Dudley et l. (9), nd Jones cells of Slmonell cubn were required for illness in et l. (15). The rte of isoltion depends on the initil number ptients ingesting contminted crmine dye. In study of of slmonelle present in the rw sewge nd on the effects the nturlly occurring infection with S. typhimuriumof the prticulr tretment. Our lowest frequency of isol- contminted drinking wter in Riverside, Clif., in 1965 (2), tion, 7% from the sludge from Springfield, ws similr to the it ws indicted tht the wter contined 17 CFU/liter. The TABLE 7. Multiple resistnce to ntibiotics in slmonelle isolted from sludge MIC (,ug/ml) of: U Source Serotype C Medin 500 S. ornienburg 2 4 -<2 -<2 -< >128 >16 Columbus S. infntis >32 >128 > < >128 8 Columbus S. typhimurium >32 ND >) >128 4 Columbus S. montevideo 8 ND > Springfield S. gon 2 ND ND, Not done.

6 VOL. 53, 1987 BACTERIAL PATHOGENS IN SLUDGE APPLIED TO LAND 1123 TABLE 8. Isoltion of slmonelle from humn stool specimens tested, 1978 to 1982 Subject Source of Dte Species no. sludge Medin 12/5/78 S. muenchen /11/78 S. jv Columbus 7/24/79 S. ornienburg Columbus 11/18/81 S. enteritidis Columbus 12/3/81 S. enteritidis Non-sludge-receiving frm. uthors of tht report, however, dvise cution in interpreting their dt for the determintion of infective dose becuse of some of the procedures used in smpling the wter. Aynwle et l. (1) reported feeding gots for 17 months on corn silge grown on lnd fertilized with sludge from which Slmonell newport C2 hd been isolted. No increses in ntibodies to the orgnism were seen. Under controlled experimentl conditions, infective doses for Slmonell spp. other thn Slmonell typhos hve been reported to vry between 105 nd 108 cells per host, lthough in some experiments, 106 cells did not cuse either disese or infection (summrized by Kowl in reference 16). Becuse of these considertions, we believe tht the effort nd expense involved in routinely determining the most probble number of slmonelle in sludge smples is not justified, since no ssessment cn be mde of the potentil of given strin to cuse disese in the numbers likely to be consumed. On the bsis of our recovery experiments from seeded sludge smples, the reltively low rte of slmonell isoltion, nd the fct tht most of our smples required enrichment for isoltion, it is likely tht the number of orgnisms in the sludges used in this study ws usully less thn 11 to 40 CFU/ml. When cells were present in higher concentrtions, there probbly were not more thn 280 CFU/ml, since in only few cses were we ble to recover orgnisms directly from plted mteril without previous enrichment. These levels of recovery re in line with the findings of Dudley et l. (9). There is lso the concern tht the lck of isoltion of slmonelle from sludge might not be sufficient criterion for the ssessment of risk since there is lwys the potentil for repopultion of the sludge while it stnds before being delivered to the fields. Russ nd Ynko (19) reported tht in composted sludges (not exctly nlogous to conditions of the present study), the pthogen repopulted redily if the moisture content ws t lest 20%, if the temperture ws in the mesophilic rnge, nd if the crbon-nitrogen rtio ws in excess of 15:1. The first two conditions existed in our study, but the crbon/nitrogen rtio ws well below 15:1 (18). Serologicl exmintion for ntibodies to slmonelle. Becuse gglutinting rections to slmonell ntigens my be TABLE 9. Subjects with gglutinting ntibodies to slmonell serotypes No. of subjects with ntibodies in county Slmonell (totl no. of subjects in county) serologicl group Medin Frnklin Pickwy Clrk (50) (39) (111) (62) B C D E Subject TABLE 10. Fmily ptterns of ntibodies to slmonell 0 ntigens Results for serologicl groupb B C D E c Rised - - Rise Conversione The first four digits of ech designtion indicte loction nd frm. The lst two digits indicte fmily members. b +, Agglutintion t serum dilution of 1:20; -, no gglutintion seen. c Rises followed infection with S. enteritidis (two isoltions from stools). No seril dilutions were done, so the negtive result with group D ntigen might be the result of prozone effect. d Rise, Increse by two grdes of rectivity in pired consecutive smples nytime during the study. e Conversion, Serologicl conversion from negtive to positive in pired consecutive smples nytime during the study. reflection of cross-rectivity with ntigens from other members of the Enterobctericee, we did not conduct s rigorous survey of gglutinting ntibodies s we did for neutrlizing ntibody increses for enteroviruses (12). Our method ws not designed to detect smll increses nd decreses in ntibodies to the different groups of slmonelle; rther, it detected ntibodies on n ll-or-none bsis with titer of 1-20 s our bse line. By using these criteri, it cn be seen tht there is significnt difference in the ntibody ptterns detected in different loctions (Tble 9). Different fmilies showed distinct serologicl ptterns, possibly showing different ptterns of intrfmily spred or common source of infection (Tble 10). We did not hve enough dt for sttisticl nlysis of ny reltionship between the occurrence of ntibodies to slmonelle nd the presence of nimls on frm. Similrly, becuse of the reltively low number of individuls with ntibodies, it ws not possible to determine whether there ws significnt difference t the end of the tril between the individuls on frms where sludge ws used nd individuls on control frms. The smll number of pprent conversions (Tble 11) TABLE 11. Conversions nd rises No. of chnges in serologicl rectivity between Slmonell bse line nd first serum smples in: serologicl Pickwy group Medin County County B 0 1 rise 2 conversions C 2 conversions 0 D 0 1 rise E 0 2 conversions For definitions of rise nd conversion, see Tble 10, footnotes d nd e, respectively. For Frnklin County nd Clrk County, no chnges were recorded for ny serologicl group. Bse-line ser were obtined before ny ppliction of sludge to lnd in the re.

7 1124 OTTOLENGHI AND HAMPARIAN indicted low risk of infection but precluded vlid sttisticl nlysis. Thus, it would pper from our studies tht the frequency of isoltion of Slmonell spp. in wstewter sludge vries from plnt to plnt nd seson to seson nd tht the risk of infection, s mesured by the rpid gglutintion test, of the popultion exposed to such slmonelle is miniml nd no different from tht of the nonexposed popultion. ACKNOWLEDGMENT This study ws supported by Ohio Frm Bureu Federtion-U.S. Environmentl Protection Agency Coopertive Agreement CS LITERATURE CITED 1. Aynwle, L. F., J. M. B. Kneene, D. M. Shermn, nd R. A. Robinson Investigtion of slmonell infection in gots fed corn silge grown on lnd fertilized with sewge sludge. Appl. Environ. Microbiol. 40: Boring, J. R., III, W. T. Mrtin, nd L. M. Elliott Isoltion of Slmonell typhimurium from municipl wter, Riverside, Cliforni, Am. J. Epidemiol. 93: Crrington, E. G., S. A. Hrmn, nd E. B. Pike Inctivtion of slmonell during nerobic digestion of sewge sludge. J. Appl. Bcteriol. 53: Cheng, C. M., W. C. Boyle, nd J. M. Goepfert Rpid quntittive method for Slmonell detection in polluted wters. Appl. Microbiol. 21: Cliver, D Infection with miniml quntities of pthogens from wstewter erosols, p In H. Phren nd W. Jkubowski (ed.), Wstewter erosols nd disese. U.S. Environmentl Protection Agency publiction no. EPA-600/ U.S. Environmentl Protection Agency, Wshington, D.C. 6. Dnielsson, M.-L Slmonell in sewge nd sludge: serologicl profiles of isoltes, their removl nd/or survivl in reltion to potentil helth hzrds to mn nd nimls. Act Vet. Scnd. Suppl. 65: D'Aoust, J.-Y Infective dose of Slmonell typhimurium in cheddr cheese. Am. J. Epidemiol. 122: Dorn, C. R., C. S. Reddy, D. N. Lmphere, J. V. Geumn, nd R. Lnese Helth effects of municipl sewge sludge ppliction on Ohio frms, p In Demonstrtion of cceptble systems for lnd disposl of sewge sludge. U.S. Environmentl Protection Agency publiction no. EPA/600/ 2-85/062 PB U.S. Environmentl Protection Agency, APPL. ENVIRON. MICROBIOL. Wshington, D.C. 9. Dudley, D. J., M. N. Guentzel, M. J. Ibrr, B. E. Moore, nd B. P. Sgik Enumertion of potentilly pthogenic bcteri from sewge sludges. Appl. Environ. Microbiol. 39: Edgr, D., nd M. S. Sor Evlution of culture medi for the isoltion of slmonells from sewge sludge. J. Appl. Bcteriol. 47: Everit, B. G The nlysis of contingency tbles, p Chpmn & Hll, Ltd., London. 12. Hmprin, V. V., J. H. Hughes, A. C. Ottolenghi, F. A. Kprl, M. L. Moeschberger, nd R. Lnese Sludge disposl on frm lnd: n epidemiologic evlution of the risk of infection, p In Demonstrtion of cceptble systems for lnd disposl of sewge sludge. U.S. Environmentl Protection Agency publiction no. EPA/600/2-85/062-PB U.S. Environmentl Protection Agency, Wshington, D.C. 13. Hmprin, V. V., A. C. Ottolenghi, nd J. H. Hughes Enteroviruses in sludge: multiyer experience with four wstewter tretment plnts. Appl. Environ. Microbiol. 50: Hrvey, R. W. S., nd T. H. Price Elevted temperture incubtion of enrichment medi for the isoltion of slmonells from hevily contminted mterils. J. Hyg. 66: Jones, P. W., L. M. Rennison, V. H. Lewin, nd D. L. Redhed The occurrence nd significnce to niml helth of slmonells in sewge sludges. J. Hyg. 84: Kowl, N. E Helth effects of lnd tretment: microbiologicl. U.S. Environmentl Protection Agency document no. 600/ U.S. Environmentl Protection Agency, Wshington, D.C. 17. Lng, D. J., L. J. Kuntz, A. R. Mrtin, S. A. Schroeder, nd L. A. Thompson Crmine s source of nosocomil slmonellosis. N. Engl. J. Med. 276: Logn, T. J., C. R. Dorn, nd C. S. Reddy Generl description of the study re, p In Demonstrtion of cceptble systems for lnd disposl of sewge sludge. U.S. Environmentl Protection Agency publiction no. EPA/600/ 2-85/062 PB U.S. Environmentl Protection Agency, Wshington, D.C. 19. Russ, C. F., nd W. A. Ynko Fctors ffecting slmonelle repopultion in composted sludges. Appl. Environ. Microbiol. 41: Spino, D. F Elevted temperture technique for the isoltion of slmonell from strems. Appl. Microbiol. 14: Yoshpe-Purer, Y., S. Ricklis, nd M. Pist A convenient method for the isoltion of slmonelle from sewge nd contminted se wter. Wter Res. 5: