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1 Distribution of this document is unlimit
Distribution of this document is unlimited N TECHNICxL REPORT 66-44-FD INCIDENCE OF CIPSTVIOI ?-0j J'O.I:M IZ\ ACCESSION for C IN RAW MEATS CMI WNDPIsi SuCE by R .Tompkin *iSTuIi.TION/AYAILUILITV CODES 1111. AVAIL 0 w LWCAA Swift & Company Chicago, Illinois / Contract No. DA19-129-ANC-161(N) Project reference: Series- FD-50 1-K-0-12501-A-033 June 1966 Food Division U. S. ARMY NATICK LABORATORIES Natick, Massachusetts 01760 FOREWORD A radiation dose of 4.5 Mrad is commonly accepted as the minimum sterilizing dose for moats. This beliefSis based upon the assumption that it is necessary to destroy 6X10 spores of C. botulinum to make the food safe for human consumption. No definitive published evidence exists of the actual numbers of botulinal spores occ.rrir..g in. raw meats. The small amount of published material and indirect unpublished data indicate that the total indigenous mes~philic, putrefactive, anaerobic spore population is very low, ond among these, the presence of C. betulinum is even lower. Hence, a thorough survey for clostrbI0al spores, particularly C. botulinur, in raw fooog, is essential, so that correct radio- sterilization processes can be established. This contract consiste of two phases. The first phase was concerned with the developent

2 of a sensitive method for detecting and
of a sensitive method for detecting and enmnerating 'botulinal spores overwhelmed by competitive clostridial spores and heat tolerant vegetaitive bacteria. This was successfully accomplished. The next phase involved the application of this method to the most extensive survey reported to date on the natural incidence of vesaphilic clostridial spores, including C. botulinum, i. raw beef, chicken and pork, taken during the four seasors of the year, at seven different geographic locations in the US.A. arnd Canada, a&7d under the werst possible handling and processing conditions in slaughtering plants. Results in.icate that mes:,pni!ic clo.striLiai spotzes are naturally present 4m very small numbert, a&n that the presence ef botulinal spores is a relatively .arA event. Th4 activities under this Loncrr.ct was moftitez-d by Mr. Abe Anellis as the Project O:fi'er, ane by Dr. Durwoed B. Rowley as the Alternate Project Officer, FERDINAND P. MEHRLICH, PhD Diraztor Food Divisien APPROVED: DALE H. SIELINC, PhD Scientific Directar w. M. MANTZ Colonel, QMC Commandcn'.rg 'ACBLF Or CONTENT.; SUMWARY 1 .INTROCUCTION 2 MATERIALS and METHODS 3 I. Phase .. Selection of Procedure 3 A. Pasteurization 3 1. Pouch technique 4 2. Botulinal toxin evaluation 4 II. Phase II. Incidenc

3 e of C. botulinum 4 A. Sample collection
e of C. botulinum 4 A. Sample collection 4 I. Sample preparation 5 2. Enumeration and isolation of putrefactive anacrobes 5 3. Botulinal toxin evaluation 5 RESULTS 6 DISCUSSION 8 LITERATURE CITED 10 TABLE 1 Reduction of standard plate count microflora in raw r.eats at various pasteurization temperatures II T. 2 .'t:erncoccu, :urvi-.,' in uninoeulat:ed mcant! it 55 arld 6OC 12 TABLE 3 Ftitrefactive anaerobic sporc! AULVial i. it.ini(',Lc11.0111] ma; '.3 TABLE 4 Detection of P.A. 3679 spores by the Peptone Colloid I MPN method 14 STABLE 5 Detection of C. botulinum 62A spores in meats inoculated with P.A. 3679 spores by the Peptone Colloid MPN method J5 TABLE 6 Comparison of pouch and MN rccozery methods for detection of indigenous r.A. .pores in raw meats 16 iv TABLE OF CONTENTS -Cont'd. Page TABLE 7 Comparison of pouch and MPN recovery methodi for detection of C. botulinum spores in raw meats. 17 TABLE 8 Recovery of C. botulinum from chicken by pouch method 18 TABLE 9 Recovery of C., botulinum from pork by pouch 'tiibhod 19 TABLE 10 Recovery of C. botulinum from beef by pouch'method.. 20 TABLE 11 Summaticn of C. botulAinum detection resultsbTpouch method 21 TABLE 12 Incid~cc of aiesophilic putrefactive anaerobic. spores, iricludinL C botulinum, in raw be

4 ef, ;,ork and chicken 22 TABLE 13 Distri
ef, ;,ork and chicken 22 TABLE 13 Distribution of raw beef, pork and chicken samples according to level of contamination with putrltactive anaerobes , 23 TABLE 14 Tncidence of putrefactive anaerobes in raw beefi.pork, ,'ad chicken 27 TABLE 15 Distribution of samples and concentrations of "p ltrefactive anaerobes according to seasons 28 ABSTRACT Two thousand three hundred and fifty eight (2,358) raw meat samples were analyzed tor putrefactive and botulinal spores. Of 19,727 P.A. isolates, only one was botulinal (Type C). 77% of the samples had 3 P.A.'s/g or less. The overall mean was 2.82 P.A.'s/g. !It vi SUMMIARY The anerobic film pouch was demonstrated to..be an effective device for the primary isolation of Clostridiumnbutulilnum types A and B sporea from inoculated raw park, beef, andc1'Aicken. Optimal pasteurization of these =eats ?'.for red-oction of-ponwrspore microflora without affecting irndigeno-js putrefa-'tive anaerobic spore levels) was 50 min. at 60C. Clostridium botulinum spores-ware recovered with good precisien from meat samples inoculated, with mixtures of C. botlinum and putrefa.-tive anaerobe 3679 at l1;tand at 1-.99 ratios. Th* anaerobic film pouch was used in a survey to quantitate and tialate t it naturally occurring clostridial and b

5 otulinal spores in 2,358 samples of raw
otulinal spores in 2,358 samples of raw meat (k,,078 chi~kfn) 624 beef, 656 pork). One of the 19,727 putrefactive anaerobic'.sporeformers isolated was confirmed by the w,:*e protection.pt~st to be Clostridium botulinurn type C. This iselats was from a popte!io sapeo chicken from Western Canada -which contained 5.33:clostridia per g. These data ind-icate a vtry low incidence of botuliail. contamination in raw meats (0.042% ef 2,358 samples) and suggest,a twenty thousand-to-one ratio of nr~cbotulinal putrefactive anaeerbes to mesophilic C. bouiir spor-es,. Sevienty-seven percent of the 2,358 samples had only three or less putrefaztive anaerobes (P.A.'*) ' g. The meani P.A. contamina- tion'levls. for beef, pork, *nJ chicrken were 3.O3,3.O3, and 2.05/g. respectively. Samples fron tha bloody neck area had higher levels of P.A. ccntamination than did samples of triumings of beef and pork. Posteri~or chicken samples had higher levels of P*;A.'s than did anterior chicken samples er giblets. Statistical-analysis of all the beef, pork, and chicken samples ceombiaed iditcated a significant seasonal affect. Autumn, vinter, suwmmr, end sprintg were the highest to lowest levels of P.A. contaziastion, respectively. The order of P.A. corstamination by geographic region

6 s weas, from highest to lowest: far west
s weas, from highest to lowest: far western U.S., a*%ut-hwest central UIJ.S..western Canada, southern U.S.0 eastern Ca#& eastern G4., and north central U.S. INTRODUCTION Investigators who hAve attempted to nioantifv putreiactive anaerobic spores in meats have al1 coeenie6 on the aceJative scarciv of these organiscs. Harritan, Oei Giudice, Shini. and Hansen t1948) reported an average of 2-4 putrefactive annerobic spores per % in pork sampled in a Chicago packing plant. Burke, Stetnkraus, and Ayres (1950). in a similar study in Iowa, found the average putrefactive spore level to be less thAn 1/g. Comparable results were reported with beef (Avers and .dams, s'i'i; 'vers, 191)4). 5-chack. Greenberg, Blodgett .na Silliker 04igi), found less than I putrefactive anaerobic spore per a i'% I0 i aw hnsn sampled at plants in Minnesota and Alberta. iteceotly. Steinkraus and Ayres (1964) conducted another survey in Iowa and, again, found very low putrefactiva anaerobic spore populations in'pork and beef. No attempt was made to isolate botulinal spores in most of these surveys, While this omission was predicated usually on the very low total putrefactive anaerobic spore'load actually found, it must be admitted that the surveys have been far too limited, both geographi

7 cally and in numbers of samples, to perm
cally and in numbers of samples, to permit generalization on the actual occurrence of C. botulinum spores in meats. Also, isolation techniques In these surveys have varied in such basic considerations as pasteutization protocol, eat ablishment of anaerobiosis, culture medium, and requisites for declaring a culture or colony a putrefactive anaerobe. Wheaton and Pratt (1961) reported that media exerted a profound influence on the recoverability of severely heated putrefactive *n.-.robic spores. Freshly prepared media were much superior to dehydrated medLA for this purpose. However, this difference was. not as apparent with mildly heated spore suspensions, (Wheaton, Pratt and Jackson, 1959). The "severe" treatment was defined as 150 second oxiont-re at 121 C; the "mild" as 27 second at 106.5 C. Greenberg, Silliker, and Bass (1958) suggested that dehydrated media such as Peptone Colloid Broth (Difco) might be superior to "home made" media in recovering putrefactive anaerobes from foods likely to contain thermoduric streptococci. The research reported herein consists of: (a) the seleetion of a procedure for the enumeration and isolation of mesophilic putrefacttve anaerobic sporetormers, particularly S. botulinum types A, B, and C, in raw meats and (b) a :ompre

8 heusive survey of raw beef, pork, and ch
heusive survey of raw beef, pork, and chicken using the selected method. 2 MATERIALS AND METHODS Phase I : Sele--tion of Proee ura Pasteurizat .n The following procc:l uas empliyed in establishing optimal pasteurization tim,-.terperature relationships: Approximately I liter cf a 1:10 suspensain ,f ground raw meat in 0.015 M phrsphate b-ifferad dihution water was blended in a 2 liter Erlenmayer flask. The flask wa2 tbwn stoppereJ with a cork, pierced with a centigrae.: tharm :rar so that the Iu!b of the thermometer was at the center of the r-nat bleri 41dr1rg thi heating ptocess. The flark was constantly agitateS ard ma!ntALnf.d withir , 1 C of the desired temperature. Ten ml samples of thi. blend (4pproxi~ately-- g nrat) ware removed as soon as the desired pasteurization tfEmplrature was reached and at 10 or 15 min intervals theremfter. Bacrrorogical analyscs were run immedi, tely upon sampling. In ea-.h experimient ,i con.trolled r.ferenct rample was obtained as the flask contants rea:he= 30C. The following three analyses were made: A. Total Plate Counts Samples were plat.-e with Tryptons 01uccose Yeast Extract Agar (Difco). Plates were countae aft-Br 48 hr incubation at 37 C. B. Enteroc.cci Samples were place4 in Azide Dextr-_e Srcth (Difco) and incuba

9 ted 24 hr at 37 C. Grcwth was considsrtd
ted 24 hr at 37 C. Grcwth was considsrtd presumptive for enterococci. Positive tubes vere .cnfir.ned by placing a 0.1 ml aliquot into TrypticasE Soy Broth BSaltimore 'iological Laboratories) fortified witt a 6.5% sdAium chloride. After 24 hr incubation at 45 C, those tubes showing growth were examine. by gram stain for the presence of streptoco-ci. Carsaase prciuction was demonstrated by placing 0.5 ml of th,- culture on a spot plate and adding a few drops of 5% hydrogen percxide. Lack of bubbles after 3 min was considered regative for catrlast. Confirmed enterococci were ccnsiderei r' b,-*. treptococci !apable of growth in Azide Dextrose Broth and at 45 C in 6.5% scdium chloride, but incapable of producing natalase. C. Putrefactive Anaerobes Putrefacctve anaerobic spore formers were detected in Peptone Colloid Broth (Difco) modified by the addition of 1 g of dertrose, 0.2 g FeSO4, and C.3 g sodium thiosulfate per liter. Tubes were incbated 7 days at 37 C and examined for odor, hydrogen sulfide production (biackening), antd bacterial growth. All tubes showing growth, whethe.,r or not they were putrid or hydrogen sulfide positive, wh.-re pasteurized 15 min at 70 C. One-tenth ml was then transferred to fresh po-.ptone colloid tubes which were subsequently

10 incubated 48 hr at 37 C. Pouch Technique
incubated 48 hr at 37 C. Pouch Technique Pouches were prepared as described by Bladel and Creeiberg (1965). Media ubed in pouch tests were: A. Brewer's Anaerobic Agar (Difco). B. Angelotti Agar. A modification of SPS Agar (Angelotti, Hail, Foter, and Lewis, 1962) consisting of 1.5% Bacto-Tryptone (Difco), 1% Bacto Yeast Extract (Difco), 3. Bacto-Agar (Difco), 0.06% sodium thioglycollate, and 0.01% L-cystine. The medium was adjustf :o pH 7.0 / 0.2 and sterilized 15 min at 121 C. "ceshly prep.r d, seitz filtered solutions of FeSO4. 7H0,. and S03 (anhydrous) were each added to the medium at 0.0257 fi. ncentration. Large quantities of the basal medium were prepares advance. Sufficient medium was melted each day and the Seitz filtered solutions were added while the medium was at 60C in a water bath. C. Peptone Colloid Agar was prepared by adding 3.0% Bacto-Agar to modified Peptone Colloid Broth. Following pasteurization (50 min, 60C), aliquots of 1:10 suspensions of raw meat in buffered dilution water were pipetted into pouches. Agar (fempered to 60C in a water bath) was then added and the pouch flexed to mix its contents. After 72 hr incubation at 37C, colonies were counted and picked from the pouches as described by Bladel and Greenberg (1965) into tubes of

11 modified Peptone Colloid Broth and incub
modified Peptone Colloid Broth and incubated. Botulinal Torin Evaluation Presumptive evidence of toxicity was established by means of intraperitoneal Injectiqn of 0.5 ml of the peptone colloid subculture, originating from colonies picked from the pouch. Swiss strain white mice (15-20L) were used. Cultures causing death within four days were confirmed as containing botulinal toxin by protection testing against trivalent ABC anti-toxin (Fort Dodge Laboratories, Fort Dodge, Iowa). When both protected and unprotected animals died within four days, the cultures were further evaluated by dilution and by pasteurization at 99C for 15 min. Heat stable toxins and those which were indistinguishable in activity in protected and unprotected mice, were considered non-specific and non- botulinal. Phase 11: Incidence of C. botulinum Sample Collection Beef, p6rT, , and poultry samples were received during the summer, autumn, winter, and spring from plants in the following geographical regions: Eastern Seaboard, South, North Central, Central and Southwest Central, Far West, Eastern Canada, and Western Canada. Wherever possible, each region was represented by two plants, preferably one old and one new plant. The saJiPl13 wtfa collecteli by in-plant personnel, packgagd in cl

12 ean jars or plastic bags, and shIppod I1
ean jars or plastic bags, and shIppod I1- dry tee to the laboratory. U1pon raceipt, the ~sampls were inspectsd and bald frazen until analyzed. Bach *-.-a and season was represented by 12 an* pound samples of the following mesat iteums: lear, btef from the bloody neck area, beef trimings ,for dry sausage, loan-fat pork sixtuzre from the bloody neck area, and pork triu'iings for sausage. Likawwise tach arta and season was represented by 36 chicken samples taken from "parts misering!" birds after diressing, chilling, and before boxing. Tht samplea :onzisted of,12 anterior portions (wing and breast), 12 posterto-- pr,:tians (.eS and thigh) and onis pound of giblets rtpresenting appralxiwmhtely 12 birds. Whensver possible# thA s~amples congist'Ld -,f 907. brollars, 37. roasters, mad 3%. fowl.. SAM2sk- ro, t ion Each of th#. b*ef anI pork Auszplts were. hsnd-chopped separately while frozAn until a tcixture of hembitger *&aa reathed. Ch~cken samwples were. bontad pr~or t, -+opping. Elaver. g .*ro a. i.h samples was placed into a sterile fou:r o'in-. #crew-capped bociLl contardng brAk~n glass chips. iThe chcppl.ng knifox, usc.?litt auttinS bc-trd, and rubboer gloves were sanitized between samples by scrubbirt; with het water, followed by 200 ppm hypochlczr.te so

13 luti~on, and a fins!. hot water rinse. I
luti~on, and a fins!. hot water rinse. Inumo Ltin an,!. Ieo,,ticp~ of Putt a~fAt i Ansqer'ibtt Ninety nz riP'mq tz-j.VFni so-.ticrn "0.0O0036 IM2P4) were addid to -.wh saawcd '6tCLt. Mt ge ss-2as wirt rApi~ly shakor. 5 minutes on a reciprocacing ri,:ti&anL rb~aki (Mechariica1 Shaker, HXArae'. Paint Rejuvetator C6.- St. !'v.J, Th -ne ~at 9%:spo~nseirf7 we! hte~tti in a water- bath fat 50 =in~ &: 60C. Faae-. izatLi,r timp.rature was verified by a glass thierm..mtter Aituatd edst tho ginxstric .Zenter .f a bottle contain- ing a corre~poading -ote as pipetctd ltzt each o! six plastic pouches, thertby pr-viiting 0.5 g %Aat per pou~ch anxd a total of three g meet per sraiplt. aralyzoed. '7he pouteis -.sid ward. twice !h.A are& of a Petri dish. Approxim.ztely 45 ml Me-ifiesd krigelo~tti's meft=m w&S added' to each pouch. The contenits of th* povcho.e ver't mixned and tbol pouches were placed between rnarrouly tpact-Y uoo~fn *latoi in it fcrming disvieea. After solidifi- cationr of the a~gat. the po~uches wazt re-mevaid from tne form and incubated ai 37C fo'r 72 hr. Ibe p~v;.tuaj were. examirsd an! all bl.c'v., sulfide produc- ing colonits were transferzod into teat tubbe (20 x 150 m) containing 20-25 ml modified ptptone colloid weix.The col~onies were transfer

14 red from the pouches by cuttivg away one
red from the pouches by cuttivg away one sid. of the pou~ch and removing individual coloniqe with an aZcohol-flained s:alpel. The peptone colloid tubes werp. incubated at 37C for 72 hr; a!ter which, all proeumptive cultures turning the maditm black and/or having a putrid c-lor were sutjaected to mouse inotulAtion tnsts. Le.:h papton4A c~ollotd colture vas cOqAd so that a covmpleis history *,,' its sc-urce an4 �t.xcicl-gitel antlyses zould be correlated. Botulinr Tnxi~ PEvae 1.i aSEwTS One of the important considerations in this study was selection of an optimum pasteuri2ation procedure for the meat samples. The objective was Co reduce non-spore contamination within a reasonably short rime period, without destroying indigenous mesophilic putrefactive anaerobic spores. A review of the literature iound the lowest temptrature in routine use to be 80C. The data presented in Table 1 suggesta4 that 50 and 55C were below thq temperatures required for effective redaction of ncn-sporing contaminants within 60 min. Fifty degree pasteurization, for example, reduced the total count of raw pork only 70% in 60 nin. In arother typical result chicken, pasteurized at 55C, still retained about 4% of its original population after 60 min exposure. Aotc 99.99% reductio

15 n :as obtained in 15-45 min at 60C. Ente
n :as obtained in 15-45 min at 60C. Enterococci, the most prevalen: non-sporing thermoduric bacteria in raw meats, appeared to withstand 55C treatrent quite readily. In beef, no enterococcus reduction oc~.urree within 45 min at the temperature. In chicken and pcrk, 60 min exposure at 55C resulted in only 90% reduction. At 60C the enterococ,;us population was reduced in beef at least 99%. Forty-five min at that temperature reduced the enterococcus levels in raw chicken 99.99% and the same treatment reduced the enterococcus count in pork 99.9%. These data are presence itn Table 2. The interococus and toral acrobic c-unt results 4er.-nstrated that a pasteurization tempera:'ure of at leart 60C would be requirid to effect meaningful reductions in extranscus microfiora. Thirty min at 60C has been used routinely for pasteurization isolation cf Type E C. botulinum spores from marine :uturiais. Since Type E spores are appreciably less heat resistant than are typas A, B, ard C, it shculd follow that a 60C treatment would not be excesstvely injuricus to merophili: botulinal spores in meats. In order t4 etermive a "cut-off" point, beyond which indigenous spores would be expected to be !cst, several uninoculated raw meat-buffer blends were sampled at 15 mir interva

16 ls at 50, 55, 60, 65, and 70C. The resul
ls at 50, 55, 60, 65, and 70C. The results shown in Table 3 are "-rivd from single one g meat samples. While the data do not estkbiish a ninimum ncn-injurious pasteurization process, it is evident that IOC is unacceptable. Putrefactive anaerobes could be recovertd frcm beef held 45 min and from chicken 60 min at 65C, but not from etbher product after it rea:hed 70C. Taking all 1autors Into conrsideration, 50 min at 60C was selected as the "optimal" meat pasteurization protocol f:r subsequent work. Peptone Colloid Broth fDifco), supplemented with dextrose, ferrous sulfate and sodium thios.Ifate. has bien advocatad as an ideal method for quantification of small numbers of ntr-injurad putrefactive anaerobic spores (Greenberg, Silllker, and Basa, 1958). Detection of putrefactive anaerobic 3679 spore inocule from beef, pcrk, and chicken by means of Peptone Colloid Brcth most probable number e.tnmrinations was evaluated as a possible reference techniquc. The res6lts vf six experiments (2 each with chicken, 6 pork, and betf, pasteurized 50 min at 69,C) tis{ng a 3 tube MpN system, nro summarize~d in Table 4. The indigenous PA level averaged 0.37 rer ; Thus, populations of 0.47, 1.37, 10.4 and 100 would be expected from sample3o inoculated with 0.1, 1.0, 10 and 10

17 0 I-er ~.Tho actual mean recovery valiin
0 I-er ~.Tho actual mean recovery valiins for these systeM3 were 0.82, 1.32, 10.8 and 16, reepct~ively. 1These dt demonstrated the Peptone Colloid YXPU procedure to he a reasonably accurate method for deteraninitn, putrefactive anaerobic spore populations ini raw meat3. The peptone c-ol!.old system wasa next tested for detection of C. botlilnum spores in mixed culture with putrefactive anaercbee in beef, pork, and chicken. Apptoximately equal. n':-mber of spores of C. botulinum typ 62 A and r.A -_ 370 wcere acddad to bzef, pork, or chicken and paste rzd at (60C for 50 mrain Po~itive peptone colicid tube&1 were tested for botulinal. toxin. Tho results, liat~d in Table 5, again showed t~ood overall preciaicn. The figures ch:urn in th. I'lotal P.A." columns include both P.A. 3679 and C. botilinurn re--overiQ3. aincq bcth organiams aprear au putrefactive &narosrbes in peptanu colloid tv'bz. These values should be roughly ona-half thooo of the eorrespondine "Total Putrefactive Anaerol?.e" figures. IMhere one PI.A. 3679 and one C. botulinurn spore were added per g the t-jrj'l !11 va 2.3 ancl confirmed C. hottulntim, 113. VThere 10 of each .Yoer klda~d, 14i wcre datectod, with 7.6 toxic. Vie 100-100 inoculation avarrq-.cd' out at 54 totul P.A.'s and 21 hotuliria

18 l spores. The anaerobic f~lo pouch techn
l spores. The anaerobic f~lo pouch technique was compared with the Poptono Colloid M4l' sy'stem i cr ability t3 datect indigisnoita putrefactive anacrohic sporn3 in beef, pork, an~d chizk&n. Counts cbtAined in pouches containing Angelotti agar, Brewer's agar and t'eptone Colloid agar were coripared with !4PN valuies obtained with fPeptonc-,Collo~d. broth. The dlata, li. 4 in Table 6, show that the pouch system, uuing-'all thret media, was at least as sensitive as was the ?4PN procedure. Angelotti agar gave consistently higher counts than the other two ratedia, but the difierance was not. statistically significant at the 957. level. 'The methodsa .ere then co'nparei by testing btsf, pork, and chicken which hiad been in',culateJ with P.A. 3679, C. botu'Ainum 33 A and C. botuliirzm 113B. The 1nocuium consisted of tw:) r.A. 3679 speres per g of meat and one spopre per a of each of the two C. bot'.linum strains. Three experiments were run on each type of r.aut. The data demonstrated again that the pouch system wa3 as senzvitive 3a the most probable number technique. Again, the Angelotti agar shotwed a~ tendency to prodo.ce higher counta. Thee summiarizod data soh'w a mean total peptroe colloid MPN of 2.9 per g, 1.3 of which (or 45%) were confirmed C. bottilinu

19 m. Aragelotti agar pouches averaged 5.8
m. Aragelotti agar pouches averaged 5.8 P.A. spores per C with 2.7 %1477o) as C, botulirnUM,(Table 7). C. botulinum spores undoubtedly constttute an exceedingly small per- centagt. of the rotal putrtfactive anaerobic spore population. It was,there- fore, essential to confirm the. utility of the Bladel pou*.-h-Angelotti agar 3ystem In recovering small numbers of C. botulinum spores from chicken, beef, and pork bearing a heavy P.A. spore load. A beries of tests were conducted, utilizing irocaila consisting of a 99-1 ratio of P.A. 3679 to C. botulinum. Six individual tests ware conducted on all three meat substriates. The data are listed for t~he individual. meats i&i Tables 8, 9, and 10, and are stma~rtad in T7able 1E. A. total of almost 4,400 individual colonies ware evaluat-d in these experiments, 103 of which wore Identified as C,~ i.ouLnvzz. The Approxim~ate 2,5%. recovery, when compared to the #xPected 1075., 7is Texeedingly precise for a nicrobiological procedure. On the baeis of the proceeding results, the anaerobic plastic fils pouch was employed in the phase 11 survey to analyze 2,1358 samples of beef, pork~, and chicew. ,'he results in Table 12 show that 19.727 mesophilic putrefrttiv~r aiusarobic sporeformers (A0)were isolated. The overAll mean cu

20 sicentyat~fu of P.A. spores was Z,82 per
sicentyat~fu of P.A. spores was Z,82 per g of seat. The variatlin in thp lave~rcO.p.Apontamination of the neat samples is given in Table 13, Seve-a-. ,zs-even par cent of the 2,358 smuples had three F~,&,. spec~es pier s a- 1#6..us The most heavily contaminated sample had 115 P.A.. eporea/g. On, C -ouiu r.~I: ' apore wat detected among the 19.727 P.A. itolatesTi war ise'Rre-4, ftom a posterior sasple of chicken from 'Jezntern Canada conta~ining 5,33 clostridia per g.' The data suggests tharefore, thsit thit incidence: of bo'tvtinal contamination in raw seats is very low (0.042%. of 2,,i$8 samples) 'rhe results further indictite anm BpproYvimate ratio ftatg hbsdt sk fP..t eohlc These data were anal~yzed statistically and the following differences woedatected attp4%o etrconfidence level using chi-square and 1. Te Ivela.,r~k sprtswas si~gntficantly lower in chicken tha inbut orpark, wildle. the levels tf P.A. spores in beef and pork wtere equ.~l.. 2 5 amples from thi blooedy nrrk ares hl bw ign~ificantly higher P.A.. spore lw~vels th-r. did the criimaingi *r,- the beef and pork samples. Tb. pr'st,.rior chiWktn siimplvat had aignifie*rntly higher levels of P.A., sporea tt~an t,P onterior chickon samples or the giblets, 4, The leve'l of 1-A. sports varizd

21 vith the season (Table 15), The order of
vith the season (Table 15), The order of P A,. spore cont~inination from highest to lowestt v., aut~tmn, winter, sunwr., and spring, respectively. The :ca~onal 4ifIferieracen were significant at the 99%. confidence lovtl, 5 Lhmre were signiftcaont differences in the levels of P.A., spore tontlominvtion of *eits from the various geographic area* riu'fwyeii. The oider of contamination,, from highest to lo*west W9511 fs- west-ern U.S.,, southwest, tontra&1S, western Canada, sjuthern U.S.. eastern Canada, eastern 1JS., and north central V'S. The phase I data supportt the utility of comerciall~y available dehydra- ted edi (a ilustrtedby odiiedPeptone Colloid Broth) isi recovering putrfat~vaanarobc sptesfro midlypasteurized raw meata. La proabe umersytoman sbjctngpositive tbsto toi asyI Lc* roattributes of the n~u..r~~eunmber approach tend to di~scourata its se or routine bvatulin.A *part aabay,. First, the probability of a 8 noCopy 5in-le C. botulinuir. spore entering an individual culture tube it; c::trcmel,. remote, particularly at the low lcvels expected in fresh meats. Mixed cultures would be the rule. Crisley and Helz (1961) have reported inhibi- tion of C. botulinum spore germination by filtrates of S., faecalis. The question of "missed" positives would

22 thus consistently cloud results obtained
thus consistently cloud results obtained by any M1PN procedure. The second problem is the tremendous glassware and incubation space requirements involved In conducting apylarge scale studies. Indeed, space and equipment requirements tend to be rohib6itive for the typical laboratory in all conventional anaerobic isolation techniques. The anaerobic pouch procedure is not only conven,'i%- but also was demonstrated to be a highly efficient device for isQlpt oniof C. botulinum type A and B spores from fresh pork, beef, and ch'ckere'inoculated with at least 100-fold greater numbers of saprophytic putrefac'tiveanaerobic spores. The most obvious conclusion resulting from the'. [rey is that the level of P.A. spore contamination in raw meat is very low e` the'plant level. Furthermore, botulinal contamination rarely occurs. These results assume greater significance when it is considered that the bloody neck area and trimmings were selected as those portions of beef and pork which would most likely have the greatest contamination. These resilts,.,therefore, are in agreement with the low Isvels of P.A. contamination pre~iously reported in the literature (Harriman et al, 1948; Burke, et alF .L958; and Steinkraus and Ayers, 1964). The fact That one botulinal spore lia d

23 etected from among the 19,727 P.A.'s iso
etected from among the 19,727 P.A.'s isolatced emphasized tOe exceedinsly. 9'A, probability of detecting botulinal spores in 6nything out an exterlsiVe survey such as des- cribed herein. The large number of samples permitted the demonstration of statistically significant differences in levels of F... spore containination. However, while it may be of academic interest that beef and pork, for e:kample, had higher leve2ls of P.A. ccntamination than chicken. the differ- enceL. were so smail as to be of nc onmiercial importance. It must be emphasized that the survey was directed specifically toward the enumeration and isolation of mesophilic putrefactive anaerobic spores, particularly those of C. botulinum. Vegetative cells 'of P.A.'s would have been destroyed during the pasteurization of the samples. However. it is also true that vegetative cells of P.A.'s would be readil., destroyed during a commercial thermal or rauiation procets applied to mest'products D (OgeK lo T I(, Hall M. I. 1'Ot$Er AAd k H. Lewisg 0641. "-'uwsnittatibn, ot l 1oas.r diwa pt; rilenge~ in foods .ppl .f t ruboa. to~ 191. !99. Ayres., J C 195's (ks cit~d in iRie.smnn H. 1963. Spfe beat processing of cionned cored *#at# v1th rtg~rO o bacterial spores. ,food TOechnol 1? .19z4q) Ayres,

24 J. C; and k, ' -r xdavs. 195). iiccurat
J. C; and k, ' -r xdavs. 195). iiccurattsc and nrt-urp ~c, bacteria in canned beef Food lehvcv 7: !!-33 Bleael. B, 0,, and R X., Gretnbtrg, 196, 1I1uch method for the isolation and onumsrption of CiO1Lr1i4Js .ppl. ?icrobkh1 13 281-285, Burke, B V tK.. R, Stainkri'us, and 1. L. .4yres. 1950. Methods tor determining the incidence of puttrefe.ctive anaerobic sports in meat products Food Technol. 4 21-?5 Crisloy., F. n, ,end (;. C briz 0)61 'o*b. jbterv~tions of the offact of ftitrates of severp) rtpre~stntatLiVe tonclcitaflt bacteris on Clostridium batultno tvp# A. Qn " irirobiol. I- 693-61q, of putreb;ctive clostruidiu spores Ln heat procoused meat products Bact Pra, 0 Harril'.ra L s.V .~ ~~ hunn R. HRester Ihf incidence of putr~fvv'tve anatrcibts in pork soc .III. Sac. rpsper. Peoria, Illinois flcv.,b~r 11 1944A Schack- W. R ,R & Gratabtrg C, SiBkdgLtc and I P 'Uillik-r 1958 Thermal prc.~*ssing charit: Lr-n*Lc of canned fton-ci'mmiu~~ted swats Tor'd Res 2, i "12 1 SIII Lker .H it e tenberg K 5 ' :hack 1958 YJ fect of individual cazr~ng 1ngrtdtonts on th* .ito stability of canaed COMI"Uted meats fo'od 7C.hnr.i 1 Z 551 551 n iknkr A iC r' ..6 i9t, Incidence, at putrFictaeiv Afl*eroot. srores in *w#Ate )* aoo" i 2 87.93 Wbeiton r and '; h, Pratt 1q#) ":

25 4IOpAra~tve studios on media ior cauntli
4IOpAra~tve studios on media ior cauntliw'g ravrobic bctirahski spore* :1 J. food .Ski 26 261-268. Iiheston q, f Pratr .n4 J P' J.- kon, 1959. rosparative studios op %,i for cauDCiD3 -vearobic b-ztvriul spores TOO'd lies. 114 110-' Table 1. Reduction of standard plate count microflora in raw meats at various pasteurization temperatures Mean Temp Time Product Log Product TYg log _c min sample .Aeduction sample reduction reduction 50 0 Pork A -Bee.*&' C o 15 0.5 0.1 0* .0 30 1.2 0.5 0.85 45 0.9 0.9 0.90 60 0.5 1.2 0.85 55 0 Pork B -Chicken A -- 15 0.3 0.3 0o0 30 2.5 1.1 N.bO 45 2.3 1.0 1.65 60 2.7 1.4 2.05 60 0 Beef A -Chicken B .o 15 4. 3.7 4.10 30 4 4.3 45 3.4 4.2 NO 60 4.7 6.3 5.50 65 0 Beet B -Chicken C -- 15 7.3 4.7 6.00 30 7.9 5.7 6.8o 45 7.5 5.7 6.60 60 8.2 5.3 6.75 Table 2. Enterococcus survival in uninoculated meats at 55 and 60 C Time Log reduction enerococci min. boc Beef 0 -- 15 -12-0- 30 0 Z' 2 45 0 wP2 60 2 ;2 Chicken 0 - 15 1 1 30 1 2 45 2 3 60 4 Pork 0 - 15 30 o 1 45 .2 60 3 I 12 -- Tabie 3. Putrefactive tnaerobic Apore survival in uninoculated Meats Temperature (OC) Time Minuteas 5O 60 70 Pork 0 P P P P P 15 P P P P P 30 P P P A P 45 P P P A A 60 P P P A A Beef 0 P P P P A 15 P P P P A 30 P P P P A 45 P P P P A 60 P A A A A Chicken 0 P P P

26 P A 15 P P P P A 30 P P P A 45 P P P P
P A 15 P P P P A 30 P P P A 45 P P P P A 60 P A P P A P -Present A -Absent Table 4. Detection of P.A. 3679 spores by the Peptone Colloid M method P.A. spore Calculated Actually detected inoculHM! spore / .spore/ , 0 (indigenous) 0.37 0.1 0.47 0.82 1.0 1.37 1.32 10 10.37 10.8 100 100 76 * Geometric mean of 6 experiments in meat 1 Table 5. Detection of C. botulinug 62A spores in meats inoculated wiT P.A. 3679 spores by the Feptone Colloid MPN metaod. Inoculum NPN total MPN confirmed P.A.3b79 C.botulinum b2A P.A.'s/g C.botuQinua/g O(indigenous) 0 0.62 0.3 0.1 0.1 0.98 0.69 1.0 1.0 2.3 1.8 10 10 14 7.6 100 100 54 21 SGeometric mean of 6 experiments in meat Table 6. Comparison of pouch and NPN recovery methods for detection of indigenous P.A. spores in raw meats. P.A. spores/g Nethod Geometric sstem Method Beef Pork Chicken Mean Pouch Angelotti Agar 5.00 1.66 3.00 3.03 Pouch Brewer Agar 1.36 0.41 2.73 1.23 Pouch Peptone Colloid Agar 3.95 0.41 3.33 1.73 MPN Peptone Colloid Broth 0.53 0.36 4.30 0.93 I Table 7. Comparison of pouch and MPN recovery methods for detection of C. botulinum spores in raw meats.a Beefb Porkb Spores confirmed % Spores confirmed % Method ./ botulinal/g botulinal j botulinal botulinal/g Pouch: Angelotti 3.6 2.2 61 6.7 .i 42 Pouch: Brewer

27 6 5 2.6 40 0.43 -- Pouch: P.C. agar 5.1
6 5 2.6 40 0.43 -- Pouch: P.C. agar 5.1 2.0 39 1.4 0.38 27 MPN: P.C. broth 2.3 0.58 25 3.5 2.3 66 C Chickeno Summation Pouch: Angelotti 7.9 2., 34 5.8 2.7 47 Pouch: Brewer 7.5 2.4 32 3.0 0.97 32 Pouch: P.C. agar 5.8 2.0 35 3.5 1.2 33 MPN: P.C. broth 3.0 0.91 30 2.9 1.3 45 a = Inocu.um cc.tained P.A. 3679, C. botulinum 33A, and C. botulinum 113B b w Geometric mean of 3 experiments c -Geometric mean of 2 experiments Table 8. Recovery of C. botulinum from chicken by pouch method* P.A. spores C. botulinum spores q C.botulinum Total Per g Total Per g % Test #train Picked Chicken Confirmed thicken Aetected 1 41B 264 110.0 2 0.83 0.76 2 41B 168 70.0 3 1.25 1.79 3 41B 214 89.2 0 0 0 4 33A 246 102.5 2 0.83 o081 5 33A 213 88.7 2 0.83 0.93 6 33A 192 80.0 3 1.25 1.56 Mean 216 90.1 2 0.83 0.92 *Inoculum contained P.A. 3679 and C. botulinum spores at 99:1 ratio. Table 9. Recovery of C. botulinum from pork by pouch method* P.A. Spores C. botulinum spores Test C.botulinum Total Per g 1Total Per g % -train iIcked t|ork eonfirmed ,jbbrk detected 1 33A 145 60 5 2.08 3.47 2 33A 452 188 23 9.62 5.12 3 33A 288 120 14 5.83 4.86 4 41B 218 95 9 3.75 3.94 5 41B 316 132 16 6.67 5.05 6 41B 411 171 4 1.67 0.91 Mean 305 128 11.5 4.94 3.86 *Inoculum contained P.A. 3679 a'.i C. botul

28 inum spores at 99:1 ratio. S~19 Table 10
inum spores at 99:1 ratio. S~19 Table 10. Recovery of C. botulinum from beef by pouch method.* P.A. spores -C.botulinum slores C.botulinum Total Per g Total -Per g % Test strain Picked A7eef Confirrj:d Ueef detected I 33A 150 63 3 1.25 2.0 2 33A 83 35 5 2.09 6.o 3 33A 160 67 1 0.42 0.67 4 41n 112 47 3 1.25 2.7 5 41B 264 110 6 2.50 2.3 u 41B 295 123 2 0.83 0.76 Mean 178 74 3.3 1.38 1.87 *Inoculum contained P.A. 3679 and C. botulinum spores at 99:1 ratio. . Table 11. Summation of C. botulinum detection results by pouch method* Number of P.A. Confirmed % C.botulinum Meat wxperiments spores/g C. botulinum spores/g spores detected Chicken 6 90 0.8d 0.92 Pork 6 128 4.94 3.86 Beef 6 74 1.38 1.87 .97 2.38 2.45 *Inoculum contained P,A. 3679 and C. botulinum spores at 99:1 ratio. Table 12. Incidence of mesophilic putrefactive anaerobic spores, including C. botulinum, in raw beef, pork and chicken No. of Total no. P.A.'s Mean No. botulinal s amples isolated P.A. 's/g isolates Beef, bloody neck area 298 2929 3.277 0 Beef trimmings 326 2742 2.803 0 Pork, bloody neck area 319 3655 3.820 0 Pork trimmings 337 2308 2.317 0 Chicken, anterior 373 2673 2.390 0 Chicken, posterior 379 3071 2.700 1 Chicken, giblets 326 2349 2.403 0 TOTALS 2,358 19,727 2.816 1 A Table 13. Distri

29 bution of raw beef, pork and chicken sam
bution of raw beef, pork and chicken samples according to level of contamination with putrefactive anaerobes. No. gamples at each P.A. Concentration Beef Pork Chicken Total iio, samples at BI. r,.. An- Pos- aach P.A. P.A.,s!g deck Nrmgs. ieck Irmgs. terior ternor Giblets conc. CO.33 15 41 16 32 27 31 26 188 0.33 20 32 19 51 40 35 37 236 0.66 18 15 26 20 39 30 38 186 1.00 16 16 17 24 15 23 16 127 1.33 13 29 20 23 20 21 26 153 1.66 24 29 18 23 15 25 29 163 2.00 33 42 34 35 -1 45 31 272 2.33 33 23 28 34 41 31 20 209 2.66 15 19 19 30 31 30 14 159 3.00 18 16 24 11 16 16 17 117 3.33 14 8 13 10 21 12 9 88 q.66 9 2 7 6 8 12 9 57 4.00 9 4 10 4 3 8 8 47 4.33 9 3 7 5 5 9 7 45 4.66 8 2 9 3 2 6 3 34 5.00 1 3 6 4 8 i 4 5.23 3 5 5 1 7 1" 9 29 5.66 3 3 3 2 1 15 6.00 2 2 4 1 5 24 6.33 3 2 1 2 2 1 14 6.66 2 3 3 1 Table 13 (nontinved) No. samples at each P.A. Concentration Beef Pork Chicken Total !1o. ,samples at BI. BI. An- Pos- each P.A. P.A.'s/g neck Tm.a. ieck T-rmgs. terior terior Giblets 0onc. 7.00 2 2 4 2 4 1 15 7.33 2 2 2 5 2 13 7.66 1 1 2 3 1 1 9 8.00 1 2 3 1 2 9 8.33 3 3 1 2 1 2 12 8.66 2 2 1 5 10 9.00 1 1 1 2 5 9.33 1 1 2 9.66 1 1 2 10.00 2 2 1 1 1 7 10.33 2 1 1 1 5 10.66 1 2 11.00 3 2 2 1 8 11.33 1 1 1 3 11.66 1 1 2 12.00 1 1 2 12.I3 2 1 1 14 12.66 1 1 1 13.00 1

30 1 1 16 1 -7 14.00 1 1 1 f .,,Table 13 (
1 1 16 1 -7 14.00 1 1 1 f .,,Table 13 (continued) Beef Pork Chicken Tota l no. famples at BI. BI. An- Pos- jach P.A. .reck ?rrgs. heck '.Trms. ternor terior Giblets C'onc. 14.33 1 2 1 1 5 14.66 15.00 1 1 1 1 4 15.33 1 1 15.66 16.00 1 1 16.33 16.66 1 1 2 17.00 1 17.33 17.66 1 1 18.oo 1 1 2 18.33 18.66 1 1 19.00 1 1 19.33 1 19.66 1 2 20.00 1 2 20.33 20.66 1 21.00 1 2 21.33 1 1 21.66 1 1 8 4 a' I Table 13 (continued) Beef Pork Chicken Tctal no. samples at BI. BI. An- Pos- 5ach P.A. SPneck '_mgs. _eck Tgs. terior terior Siblets conc. 22.00 22.33 22.66 1 1 23.00 25.66 1 2 31.33 1 1 32.00 1 1 35.66 1 1 21.00 1 1 51.66 1 67.66 1 1 69.oo 1 1 71.33 1 1 115.00 1 1 Total nio. samples 298 32C 319 337 373 3 3 3mples 4'3.1' ) 3I.`)0 24.J5 37.69 32.44 31,40 f6,.0 31.26 1 P.A./gm 31.26 or less -t 'amplen 68.79 80.?7 69 `8 S3.3O 79. 9 75.73 78 �3 7n.76 ' 3P.A.'s/gnr or less "C. botuflnum type C spore isolited TABKE 14 Incidence of P'utrvfactive inaerobea In faw beef, Pork, and Ohioken No. 4amples Total P.A.'s Mean P.A.,'s Beef 624 5671 3.03 Pork 656 5963 3.03 Chicken 1078 8093 2.50 27 TABLE 15 Distribution of larnples and Ooncentrations of Putrefactive Anaerobes According to 6easons No, of Total rto. PA.ts Mean No. botulinal lamples -tsolated PAo's/k isolates S

31 prin~g 585 2 45a9 1.422 0 Summer 624 4I3
prin~g 585 2 45a9 1.422 0 Summer 624 4I382 2.338 0 Aut~umn 563 7015 4,,l149 0 Winter 586 5831 3.314 1 q 28 llnrlnnaatfild Security Classification DOCUMENT CONTROL DATA -R&D (Security claesailcathon of tifll, body of abetrecl and indexing annotation must be entered when the overall report it clelaaefed" 7 OIGINATIN G ACTIVITY (Corporate author) 2. RCPORT IECURtITY C LAISIFICATION Swift & Company, Research Labs. Unclassified Packers & Exchange Avenue 21 GROUP Chicago, Illinois 60609 3 n9PORT TITLE INCIDENCE OF CLOSTRIDIUM BOTULINUM IN RAW MATS 4 DESCRIPTIVE NO'ES (Type of report and Inclusive date&) Final Period 22 July 63 -1 Dec 65 5 AUTHOR'S) 4Last name, first name, Initial) Tompkin, R. B. * REPORT DATE 7. TOTAL NO OF PAGFV 7b NO OF REFS June 1966 28 13 08. CONTRACT OR GRANT NO. 90 ORIGINATOR'S REPORT NUMDER(S) DA19-129-AMC-161 (N) b. PROJECT NO. 1-K-0-12501-A-033 C Sb OTHIER REPORT NO(S) (Any other numbers that may or eewaened 66-44-fi) FD-50 t0 AVA ILABILITY/LIMITATION NOTICES Distribution of this report is unlimited. Release to CFSTI is authorized. II SUPPLEMENTARY NOTES 12 SPONSORING MILITARY ACTIVITY U.S. Army Natick Laboratories Natick, Massachusetts 13 ABSTRACT Two thousand three hundred fifty eight (2,358) raw meat samples were analyzed for putref

32 active and botuinal spores. Of 19,727 P.
active and botuinal spores. Of 19,727 P.A. isolates, only one was botulinal (Type C). 777% of the samples had 3 P.A.';!g or less. The overall mean was 2.82 P.A.'s/g. DD A'IP 1473 Uncla.sified Security Classification Uinclassi fied KCY WOODS LIKA-IK I ROE Y RO9E W "OLE a leolatior, 8. 8 Clostridiust Botulinuim 102 1,2 Meat15 Raw00 Coun ting 0 8 Ratios8 Anaerobic spores 1 Putrefactive 19 1 INSTIIJCTIONII I. ORIGINATIN4G ACTIVITY: Enter the ntime anid address 10. AVAILABILITY/LIMITATIONNTCSCEtraytm Of the contractor, subcontractor. grant@*, Department of De. itatlons on further dissemination of the report. other than those fense act ivity or other organization (corporate author) Issuing imoeib euiy classification, using standard statements the toot Impse bya. ui 2a. REPORT SECUN4TY CLASSIFICATION: Enter the over- (1) "OQualified requesters may obtain copies of this oll-security classificat ion of the report. Indicate whether rpr rmDQ "Resiricted Data" Is Included, Marking..1 to be In aecord.rertfoDD. once with appropriate security regulations. (2) "F1oreign announcement and dissemination of this 2b. GROUP: Automgtlc downgrading Is specified In DoD Di. report by DDC is not authorized." rective 5200. 10 and Armed Forces Industrial Manual. Enter (3) tIO. &. Gov

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