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A method for the production of Clostridium haemolyticum spores on solid medium
ff~,t~r~m] t*J Bied*,gic, d Stand4r,&&atien (19811 9 , I t % - 1 1 9
A m e t h o d for t h e p r o d u c t i o n C/ost,qa)'um haemolyticum s p o r e s on solid medium
of
D. R. Ko]be, ~ K. D. : a n d R . M . ?',re"r v.,gt
CN;tr~atiaem ],~.;ol)ti:u~;" spheres were cultivatcx] o n {iver*dlgest {LD) a g a r m e d i u m l~)r us,a ~ a sr:mdardi~ed ch.~Hen~,e m a t e r i a l i*~ a V . S [7>epartmenr o f AgticeJ~utt: [x~renc y assa F o F C . bae*:,edytieum, [~acterins. C u l t u r e s were i n c u b a t e d in an a n a e r o b i c ¢I~atnb<.r. Spores ~'elre w ~ h e d t~t~)m t h e a g a r stirGce a n d s u s t ~ n d e d in a n CxluaI v o l u m e o f g l y c e r o L Tht- s!-mre sus~*nsion w ~ d i s p e n s e d i n t o g l ~ s amfx~ules a n d scored a¢ -- 6 0 ~C. T h e mose p r o b a b l e numk~ze o f v i a b l e SlmZes ( 3 I P M V S L d e ~ e r m i n c d b y t h e d i l u t i o n t ~ g c t e c l m l q u e , xv.t~ | "0 x iO ~ stx~r~0~:O- 5 rot. T h i s p r e p a r a t i o n coneaitwd 6+2 X t0:' g~+inca P N m e d i a n l e t h a l do~cs (L|)a~O ~ : r 0 +5 m | ~ deter+ mine~.t b y t i t r a t i o n . C u l t i v a t i o n ~*fCo Iv¢em~@¢ice¢m sF<~re5 o n a relatix°eiy s m a l l q u a n t i t y o f s o l i d m e d i u m w:~ a c o n v e m e n e methivd o f p r ~ ! u c l n g a u n i I b r m | y v i r u l e n t sgxm: p r e p a r a t i o n .
INTRODUCTION Clos;ria'ium .6aemolytkwm is the cause o f a highly f~tal d i s e ~ e o f cattle, bacillary h e m o g l o b i n u r i a , or ~red~water ~ disease (Records & Vawter, t 9 4 5 : S m i t h , 1975L Usually, cattle are i m m u n i z e d in the areas o f t~le U n i t e d States in w h i c h the disease is e n d e m i c . T h e efficacy o f Co ]memol)t&vim bacterins is t,¢aluated by a vaccinationchallenge e~say prt~zedure in g u i n e a - p i g s descri[~'d in the C o d e o f Federal Regulations, T i t l e 9 , Chapter i, Sut~zhapter E, Part i 1 3 - 9 2 . C h a l l e n g e i n o c u l u m ( 100 LDa~ dose g i v e n intramuscularly) is prepared from C. h, tono&ticmn spores sust-mnded in 50% glycerol, A l t h o u g h it is prepared from w h o l e cell culture, the spore rs to be the only viable component in che final product. Spore simF~nsions in glycerol have been " R e c e i v e d for p u b l i c a c k m 22 J ~ u a ~ - 1980. + U n i c e d States ~ - p a r c m e n ¢ ~ f A g r k u t c u r ¢ , A n i m a l a n d Plan~ H e M t h laslm,ctlon Service. N a t i o n a l V e t e d n a e ; ~ r v i c ~ I a a b ~ m ~ r l e s . Arnes~ Iowa 5 ~ lO, U . S . a . lN)O2- I157.181/0201 t5 + 05 $02AJO*q,) cf | O g | The llaternat~naf ,~_~s~
t15
D~ R. K O L ~ E ,
K
O. C L A U S A N D
R. ~ l
NERVIG
held at a t e m pe r a t ur e o f - 4 0 e C or lower ~or more than nine years w i t h o u t loss o f virulence. Several m e t h o d s have ~ : e n used ~or cu ltiv atin g C. 'Saemo sF~)res in liquid medit, m, These m e t h o d s require~ from t0 to 20 I o f enzymatic digest o f bovine liver which is relati~'ely exg~nsive and time c o n s u m i n g to prepare. SFmre preparations derived from liquid m e d i u m require concentration o f sFa)res in th e cu ltu re by centrifugation. Studies were directed to the d e v e l o p m e n t o f an improved m e t h o d by cultivation of C. haemo6~tiru~e s ~ r e s on agar m e d i u m in an anaerobic character. Clost ()tirum was selected a.~ ix mo d el ~ c a u s e it is by far the most fastidious o f the pathogenic clostridia ( S m ith , 1975). D e v e l o p m e n t o f an e N c i e n t m e t h o d fi~r prc~tuclng C. spores w o u ld also provide a useful m e t h ~ | fbr p r o d u c i n g sF~)res from other pathogenic clostridia. MATERIALS AND METHODS A Male and female D u n k i n - M a r d e y guinea-pigs w e i g h i n g 4 5 { ~ 5 5 0 g, from the closed colony at tt~e Nat tonal A n i m a l Disease Center, Ames, Iowa, were used in dtis study.
B
I straiv Clo~tridium ha~,lo/yticum 7 1 7 0 was used for the p r o d u c t i o n o f s ~ r e s . This strain v¢~
isolated in t 9 5 6 at M o n t a n a State University, Bozeman, M o n t a n a . from the liver o f a cow which died ofbacitlarF hcmog|obinuriao I~ was preserved in 5 0 % glycerol and held at a p p r o x i m a t d y - - 6 0 eC.
Preparation of liars digest agar med}ttm An enzymatic digest of t l vc r w;~ prepared by m i x i n g 500 g o f c h o p ~ d b~wine liver w i t h ! 000 ml o ( d i s r i l l e d water and h e a i n g tn 64 °C in a water bath. O n e g o f p a p a i n ( N V VII1, D i d o Laix~ratories, Detroit, M1 48232), dissolved in 25 m l o f distilled water, was dmroi~ghiy mi×ed wid~ the liver auspension. T h e papain-liver m i x t u r e was maintained at p H 7 q > 7 " 3 w i t h 5 N - N a O t t and incubated ar 6 4 ° C tbr 2 h. T h e mixture was heattxi to a t:~il, c ~ l e d to room remF~erarure, and tl~en centrifuged at I0 O00 × g fi)r 10 rain. T h e sul~-rnatant was clarified by f l t e r i n g t h r o u g h W h a t m a n No. -t filter t~/f~r and adjusted to p H 7"3. T h e t b | | o w i n g constituents were added to I(X)0 m! o f li~wr digest ( e x p r e s s ~ a~ g/D: trFp~icase, ,tO; a ~ r , 20; and yeast exerg<*t, 10. T h e LD m e d m m was l~eated over a low flame u n til dissolved and d i s t r i b u t e d in i 00 ml volumes into e..ch o f t e n 500 ml s,:rew-capfx'd E r l e m n y e r flasks. A protective cover of a l u m i n u m fi~il was shaped over the ~re,,v cap and extended approxin~atdy 8 e m d o w n the nc~.k o f each flask~ Tile fla~ks o f LD m e d i u m ~ e r e autoclaved at 120 °C fbr 25 rain. To insure highly reduced m e d i u m , the aiitoelaved flasks o f hot LD m e d i u m ~70~C) were placed in an animrobic c | , a m l . r (blodel t024 Anaerobic System, Forma Sl ientific, biarier~a, O t i t 5 7 5 0 ) , and held a m i n i m u m o f I8 h i~fore ~:eding w i t h e . hai,±,v,(yttcum. At wmt'<'ratures alz~ave70 o{5 the LI) m e d i u m w o u l d ~}il violently d u r i n g the gas evacuation cycle in the a i r l ~ k o f the anaerobic charntx.r.
Cultit',ltiort The atmosphere in the anaerobic clmml~w w ~ an o×~2gen- free gas mixture <~ntaining 116
PRODUCTIOns/ OF C. tlAEMOLYTICUM SPORES 8 5 % nitrogen, 10% hydrogen and 5% carbon dioxide. A catalyst o f palladium-coated a l u m i n a pellets w ~ used to activate hydrogen for reduction of any oxygen w i t h i n the c h a m ~ r . Tubes o f F ~ p t o n e - y e ~ t e x r ~ c t - g l u c o s e m e d i u m containing 0 - 0 0 0 1 % r e ~ a z u t i n or 0" 0 0 0 2 % m e t h y l e n e blue were used to m o n i t o r the anaerobic c o n d i t i o n of the atmosphere in the chamber. Each flask w ~ seeded with 0 - 2 ml ofC. haemof~tic-um 7170, d i l u t e d 1:5 in distilled water, using sterile 1" 0 ml syringes w i t h 20 gauge n ~ d l e s . The inoculum was spread over the surface o f the agar by t i l t i n g each fl~k at ~ v e r a l different angles. T h e cultures were incubated at 36 °C ,'br 3 ~ 4 8 h, and then incubated at 24 °(2 for an additional 7 2 - 9 6 h.
H r , st of sp, . Microbial g r o w t h w ~ w ~ h e d from the surface o f LD m e d i u m vcith 25 m! of sterile phosphate buffer (0- 05 M-KH2PO4-N0~HPO.,, p H 7" 0). G r o w t h harvested from two to four flr~ks v¢~ p ~ l e d , filtered t h r o u g h several l a y e ~ o f sterile cheesecloth, and held at 4 °C until rested for purity. To test for purity, the sfx~re preparation wo.s cultured on bovine bloe,d agar plates. T h e b I ~ d plates were incubated aerobically and anaerobically at 3 6 ° C for 72 h. Mic~:~bial g r o w t h xv~ not detc~ct~ on the a e ~ b i c a l l y - i n c u b a t e d bl(g~c| plates. Colonies found on the anaerobically-incubated blood plates were identified ~ C. haemolyticum. Forty flasks o f culture were harvested w i t h i n a 30-day ~ r i ~ Each F ~ l e d spore s ~ p e n s i o n was added to an equal v o l u m e o f sterile glycerol and held at room temF~raturn for a m i n i m u m o f 14 days. A final v o l u m e o f 9 0 0 m l ofharvested spores w ~ m i x e d w i t h 9 0 0 ml o f sterile g l y c e r o l T h e s ~ r e - g l y c e r o l preparation xv~ m i x e d t h o r o u g h l y with a m a g n e t i c stirrer each work day. After retesting for purity, 1-6 ml o f sF~reglycerol preparation was :~septically disw_-nsed into each o f 950 sterile 5 m l a m p u l e s using an automatic A m p u l e Filler-Se~er ( K a h l e n ~ r g - G l o b e Co., Sarasota, FL 33578L D u r i n g filling, the s ~ r e - g l y c e r o l p r e p a ~ t i o n was held at room t e m ~ r a t u r e and continually mixc~d w i t h a m a g n e t i c stirrer to m a i n t a i n h o m o g e n e i t y . Samples of the final sealed lot of amg~ules were restcxt fi~r purity. T h e final p ~ d u c t , labeled C!ostridium baemolk[Hct¢m Challenge Culture IRP 234. was held at a remF~rature o f - - 6 0 °C. An}' ~lr~her r e , f e n c e to the s ~ r e - g l y c e l o l p r e p a ~ t i o n shall ~ identified ClostrLdiam haemol~icum Challenge Culture IRP 234 or simply a_s 1RP 234.
Eraluation of C l o s t r i d i u m h a e m o l y t i c u m cu/tu~ IRP 234 E i g h t titrations in guinea-pigs, w e i g h i n g ~t5C~550 g, were conducted d u r i n g a seven-week p~zriod to d e t e r m i n e the LD3o in each 0" 5 ml o f IRP 234~ T h e LD:,0 w ~ bo-Aed on the highest dilution o f s t ~ r e susF~nsion causing 50% o f the animals to die w i t h i n 72 h F~)stin~:ularion. ~ r i a l renfi)ld d i l u t i o n s ( l O - ~ - l O -¢) of IRP 234 were made in freshly prepared 5% CaCI: solution. The d i l u t e d s p o ~ SUSF~nsions were held at ~ o m temw:rature ~ IO rain and then placed in an ice bath. Each o f five guinea-pigs were ine,culatc~J intramuscularly in the hind leg w i t h 0"5 m l o f each d i l u t e d s ~ r e s u s ~ n s i o n . T h e LD:,o value and the 9 5 % Confidence Limits were d e t e r m i n e d by probit analysis (Finney, I97 I). T h e most probable h u m o r o f viable Sl~ore ( M P N V S ) in 0* 5 m l o f IRP 234 vv~ estimated by the d i l u t i o n t u ~ t e c h n i q u e (Harrigan & McCane, 1966). ~ r i a l ten.,*bld dilutions ( I 0 - ~ - I0 ~¢) o f IRP 23,1 were made in O - 8 5 % NaCI ~ l u t i o n , and O" 5 m l o f I17
D~ R. KOLBE, K. D. CLAUS A N D R, M. N E R V I G e a c h d i l u t i o n was a d d e d to e a c h o f five tuLms o f L D b r o t h , e n r i c h ~ w i t h t r y p t i c a s e a n d yeast e x t r a c t . T h e c u l t u r e s w e r e i n c u b a t e d a n a e r o b i c a l l y at 3 6 °C fbr seven d a y s . T h e h u m o r o f rubes ~ r d i l u t i o n c o n t a i n i n g b a c t e r i a l g r o w t h w a s used t o c a l c u l a t e t h e M P N V S b y r e , f e n c e t o p r o b a b i l i t y rabies for t h e e s t i m a t i o n o f b a c t e r i a l n u m b e r s .
DISCUSSION The titrations of C C h a l l e n g e C ~ k u r e I R P 2 3 4 in g u i n e a - p i g s is s u m m a r i z e d in T a b l e 1o T h e r e were 6 1 9 3 0 0 g u i n e a - p i g LD.~ d o s e / 0 - 5 m l o f I R P 2 3 4 (log 5 - 7 9 1 9 w i t h 959~ c o n f i d e n c e L i m i t s o f l o g 5 - 6 I 9 0 a n d l o g 5 " 9 6 5 t). T h e r e w e r e 1 9 8 8 0 0 0 viable s p o m f f 0 " 5 m l o f l R P 2 3 4 ( l o g 6 - 2 9 8 5 ) , as d e t e r m i n e d by t h e m o s t p r o b a b l e n u m b e r m e t h o d . T e s t results are s h o w n in T a b l e 2. T h e r e f o r e , t h e m e d i a n l e t h a l d o s e for 4 5 f > 5 5 0 g g u i n e a - p i g s is a p p r o x i m a t e l y t h r e e slmres. Closlridimn haemolyticmn spores p r o d u c e d ~ d e s c r i b e d in t h e M a r e r i a l s a n d M e t h o d s r e s u l t e d in a u n i f o r m l y v i r u l e n t s~ore c h a l l e n g e p r e p a r a t i o n w h i c h has to d a t e r e m a i n e d stable. T h i s p r e p a r a t i o n w i l t t~m used fiat p o t e n c y a ~ a y s tD, b o t h l i c e n s e d m a n u f a c t u r e r s o f v e t e r i n a w b i o l o g i c s a n d in g o v e r n m e n t l a ~ r a e o o , t e s t i n g p r o g r a m s ° D u r i n g t h e year 1978, m o r e t h a n l ~ u r m i l l i o n doses o f C. !yt&'umb a c t e r i n w e r e p r o d u c e d in t h e U n i t e d Stares by five licensed m a n u f a c t u r e r s . It is c o n c l u d e d t h a t C. !yticum c u l t i v a t e d o n L D a g a r m e d i u m in an a n a e r o b i c T^~L~ 1.
Gulnea-plg
LDao
doses
~r
0"5
ml
o f Clost
icam cha|lenge culture IRP 234
Titration
| 0 ~a
Challenge culture dilutions 10 -~ 10 -a |0 ~
10 ~
I
5"
5
5
3
0
2 3 4 5 6 7
5 5 5 5 5 5
5 5 5 5 5 5
5 4 4 5 5 5
0 0 3 3 1 3
0 0 0 0 0 0
8
5
5
4
2
0
40
40
37
15
0
Total deaths
t Data expressed as num~.r of deaths in each group of five ~ulnea~p/gs~ l ~ g dosa2 ~--3*00~30 --4"00000 --5-00000 --6-00000 -7-00000
P m b i t analysis nding Tested
No+
40 40 37 15 0
40 40 40 40 40
~ ResFnmse IG~)*0 lO0-O
92"5 37-5 00-0
F = I95"6; d r . ~ 3; s|ope = -- !'9430;X ~ ~ 0"69|0; endpoint L ~5 ml; log 5"7919; ~¢i|og 619 3C~; 95% confidence |imie$ 5"619C~5~9651.
118
P R O D U C T I O N OF C, H A E M O L Y T I C U M TABLF. 2.
Tit r~t ion
Most probable n u m ~ . r o f viaMe sF,0res F~.WO" 5 m l o f C/0f icHm challenge C~tltUe I R P 234 Challenge culture dliutions 10 °'* I0 -:a I0 -~
t 0 -v
Log,o ( M P N ) 6~114 6- 740 6~740 6-954 5- 544 5"699 6- 2985
!
5"
4
0
0
2 3 4 5 6
5 5 5 5 5
5 5 5 I 2
2 2 3 0 0
0 0 0 0 0
Mean
SPORES
NIPNVS/O" 5 mL * Data expressed ~ h u m o r of tubes cor~tainlng b:~:~eria| growth in five inocu~ laced tuh~-s of medium. c h a m | ~ r will p r o v i d e a c h a l l e n g e r e , g e n t w i t h o p t i m u m n u m b e r s o f u n i £ o r m l y v i r u l e n t spores. T h i s m e t h o d w i l l also p r o v i d e a c o n s i d e r a b l e s a v i n g o f t i m e a n d m e d i u m in pregntring t h e c h a l l e n g e r e a g e n t . S e v e n a d d i t i o n a l SF<~re p r e p a r a t i o n s have ~ e n p r o d u c e d b y t h e d e s c r i b e d m e t h o d f r o m six s t r a i n s o f C. a n d o n e s t r a i n o f C . sor~'lliL T h e s e s t r a i n s o f c ! o s t r i d i a w e r e c u l t i v a t e d readily o n a m e d i u m c o n t a i n i n g b l o o d a g a r b~.se, yea~st e x t ~ c t , s o l u b l e starch, c y s t e i n e H C l , a n d agar~ Since s ~ r u l a r i o n ~ c u r s m o r e r e a d i l y in t h e s e closrridial SF~cies t h a n Co , m i c r o b i a l g r o w t h f r o m o n l y 10 f l ~ k s o f c u l t u r e w0~ sufficient t o p r e p a r e a p p r o x i m a t e l y 1 0 0 0 a m p o u l e s o f c h a l l e n g e m a t e r i a l . REFERENCES Finney, D. J. ( I 9 7 t). Probit Arml)si~ 3rd edn. London: C ~ b r i d g e University Press. Harrigan, V~;'o F. & McCane, ~l. E~ (1966). ~boratoO" i~letk~..~ m L ltcreomlo~, pp. 3 I % 3 2 3 . London: Academic Press. Records, E~ & V a w r e r L. R. (1945L Bacillaty n e m o g l o b i n u r i a o f cattle and s h ~ p ( R e d Water disea~:). University of Nevada Ex|~rimentM Station (University o f N e v a d a , Reno, N e ~ d a L B 173~ S m i t h , L. |3~ S (1975). Tb,- 1%abogeni~ Am~¢robic Bacteria. 2nd e d m Springfield, Illinois: C h u t e s C. Thomas.
119
A m e t h o d for t h e p r o d u c t i o n C/ost,qa)'um haemolyticum s p o r e s on solid medium
of
D. R. Ko]be, ~ K. D. : a n d R . M . ?',re"r v.,gt
CN;tr~atiaem ],~.;ol)ti:u~;" spheres were cultivatcx] o n {iver*dlgest {LD) a g a r m e d i u m l~)r us,a ~ a sr:mdardi~ed ch.~Hen~,e m a t e r i a l i*~ a V . S [7>epartmenr o f AgticeJ~utt: [x~renc y assa F o F C . bae*:,edytieum, [~acterins. C u l t u r e s were i n c u b a t e d in an a n a e r o b i c ¢I~atnb<.r. Spores ~'elre w ~ h e d t~t~)m t h e a g a r stirGce a n d s u s t ~ n d e d in a n CxluaI v o l u m e o f g l y c e r o L Tht- s!-mre sus~*nsion w ~ d i s p e n s e d i n t o g l ~ s amfx~ules a n d scored a¢ -- 6 0 ~C. T h e mose p r o b a b l e numk~ze o f v i a b l e SlmZes ( 3 I P M V S L d e ~ e r m i n c d b y t h e d i l u t i o n t ~ g c t e c l m l q u e , xv.t~ | "0 x iO ~ stx~r~0~:O- 5 rot. T h i s p r e p a r a t i o n coneaitwd 6+2 X t0:' g~+inca P N m e d i a n l e t h a l do~cs (L|)a~O ~ : r 0 +5 m | ~ deter+ mine~.t b y t i t r a t i o n . C u l t i v a t i o n ~*fCo Iv¢em~@¢ice¢m sF<~re5 o n a relatix°eiy s m a l l q u a n t i t y o f s o l i d m e d i u m w:~ a c o n v e m e n e methivd o f p r ~ ! u c l n g a u n i I b r m | y v i r u l e n t sgxm: p r e p a r a t i o n .
INTRODUCTION Clos;ria'ium .6aemolytkwm is the cause o f a highly f~tal d i s e ~ e o f cattle, bacillary h e m o g l o b i n u r i a , or ~red~water ~ disease (Records & Vawter, t 9 4 5 : S m i t h , 1975L Usually, cattle are i m m u n i z e d in the areas o f t~le U n i t e d States in w h i c h the disease is e n d e m i c . T h e efficacy o f Co ]memol)t&vim bacterins is t,¢aluated by a vaccinationchallenge e~say prt~zedure in g u i n e a - p i g s descri[~'d in the C o d e o f Federal Regulations, T i t l e 9 , Chapter i, Sut~zhapter E, Part i 1 3 - 9 2 . C h a l l e n g e i n o c u l u m ( 100 LDa~ dose g i v e n intramuscularly) is prepared from C. h, tono&ticmn spores sust-mnded in 50% glycerol, A l t h o u g h it is prepared from w h o l e cell culture, the spore rs to be the only viable component in che final product. Spore simF~nsions in glycerol have been " R e c e i v e d for p u b l i c a c k m 22 J ~ u a ~ - 1980. + U n i c e d States ~ - p a r c m e n ¢ ~ f A g r k u t c u r ¢ , A n i m a l a n d Plan~ H e M t h laslm,ctlon Service. N a t i o n a l V e t e d n a e ; ~ r v i c ~ I a a b ~ m ~ r l e s . Arnes~ Iowa 5 ~ lO, U . S . a . lN)O2- I157.181/0201 t5 + 05 $02AJO*q,) cf | O g | The llaternat~naf ,~_~s~
t15
D~ R. K O L ~ E ,
K
O. C L A U S A N D
R. ~ l
NERVIG
held at a t e m pe r a t ur e o f - 4 0 e C or lower ~or more than nine years w i t h o u t loss o f virulence. Several m e t h o d s have ~ : e n used ~or cu ltiv atin g C. 'Saemo sF~)res in liquid medit, m, These m e t h o d s require~ from t0 to 20 I o f enzymatic digest o f bovine liver which is relati~'ely exg~nsive and time c o n s u m i n g to prepare. SFmre preparations derived from liquid m e d i u m require concentration o f sFa)res in th e cu ltu re by centrifugation. Studies were directed to the d e v e l o p m e n t o f an improved m e t h o d by cultivation of C. haemo6~tiru~e s ~ r e s on agar m e d i u m in an anaerobic character. Clost ()tirum was selected a.~ ix mo d el ~ c a u s e it is by far the most fastidious o f the pathogenic clostridia ( S m ith , 1975). D e v e l o p m e n t o f an e N c i e n t m e t h o d fi~r prc~tuclng C. spores w o u ld also provide a useful m e t h ~ | fbr p r o d u c i n g sF~)res from other pathogenic clostridia. MATERIALS AND METHODS A Male and female D u n k i n - M a r d e y guinea-pigs w e i g h i n g 4 5 { ~ 5 5 0 g, from the closed colony at tt~e Nat tonal A n i m a l Disease Center, Ames, Iowa, were used in dtis study.
B
I straiv Clo~tridium ha~,lo/yticum 7 1 7 0 was used for the p r o d u c t i o n o f s ~ r e s . This strain v¢~
isolated in t 9 5 6 at M o n t a n a State University, Bozeman, M o n t a n a . from the liver o f a cow which died ofbacitlarF hcmog|obinuriao I~ was preserved in 5 0 % glycerol and held at a p p r o x i m a t d y - - 6 0 eC.
Preparation of liars digest agar med}ttm An enzymatic digest of t l vc r w;~ prepared by m i x i n g 500 g o f c h o p ~ d b~wine liver w i t h ! 000 ml o ( d i s r i l l e d water and h e a i n g tn 64 °C in a water bath. O n e g o f p a p a i n ( N V VII1, D i d o Laix~ratories, Detroit, M1 48232), dissolved in 25 m l o f distilled water, was dmroi~ghiy mi×ed wid~ the liver auspension. T h e papain-liver m i x t u r e was maintained at p H 7 q > 7 " 3 w i t h 5 N - N a O t t and incubated ar 6 4 ° C tbr 2 h. T h e mixture was heattxi to a t:~il, c ~ l e d to room remF~erarure, and tl~en centrifuged at I0 O00 × g fi)r 10 rain. T h e sul~-rnatant was clarified by f l t e r i n g t h r o u g h W h a t m a n No. -t filter t~/f~r and adjusted to p H 7"3. T h e t b | | o w i n g constituents were added to I(X)0 m! o f li~wr digest ( e x p r e s s ~ a~ g/D: trFp~icase, ,tO; a ~ r , 20; and yeast exerg<*t, 10. T h e LD m e d m m was l~eated over a low flame u n til dissolved and d i s t r i b u t e d in i 00 ml volumes into e..ch o f t e n 500 ml s,:rew-capfx'd E r l e m n y e r flasks. A protective cover of a l u m i n u m fi~il was shaped over the ~re,,v cap and extended approxin~atdy 8 e m d o w n the nc~.k o f each flask~ Tile fla~ks o f LD m e d i u m ~ e r e autoclaved at 120 °C fbr 25 rain. To insure highly reduced m e d i u m , the aiitoelaved flasks o f hot LD m e d i u m ~70~C) were placed in an animrobic c | , a m l . r (blodel t024 Anaerobic System, Forma Sl ientific, biarier~a, O t i t 5 7 5 0 ) , and held a m i n i m u m o f I8 h i~fore ~:eding w i t h e . hai,±,v,(yttcum. At wmt'<'ratures alz~ave70 o{5 the LI) m e d i u m w o u l d ~}il violently d u r i n g the gas evacuation cycle in the a i r l ~ k o f the anaerobic charntx.r.
Cultit',ltiort The atmosphere in the anaerobic clmml~w w ~ an o×~2gen- free gas mixture <~ntaining 116
PRODUCTIOns/ OF C. tlAEMOLYTICUM SPORES 8 5 % nitrogen, 10% hydrogen and 5% carbon dioxide. A catalyst o f palladium-coated a l u m i n a pellets w ~ used to activate hydrogen for reduction of any oxygen w i t h i n the c h a m ~ r . Tubes o f F ~ p t o n e - y e ~ t e x r ~ c t - g l u c o s e m e d i u m containing 0 - 0 0 0 1 % r e ~ a z u t i n or 0" 0 0 0 2 % m e t h y l e n e blue were used to m o n i t o r the anaerobic c o n d i t i o n of the atmosphere in the chamber. Each flask w ~ seeded with 0 - 2 ml ofC. haemof~tic-um 7170, d i l u t e d 1:5 in distilled water, using sterile 1" 0 ml syringes w i t h 20 gauge n ~ d l e s . The inoculum was spread over the surface o f the agar by t i l t i n g each fl~k at ~ v e r a l different angles. T h e cultures were incubated at 36 °C ,'br 3 ~ 4 8 h, and then incubated at 24 °(2 for an additional 7 2 - 9 6 h.
H r , st of sp, . Microbial g r o w t h w ~ w ~ h e d from the surface o f LD m e d i u m vcith 25 m! of sterile phosphate buffer (0- 05 M-KH2PO4-N0~HPO.,, p H 7" 0). G r o w t h harvested from two to four flr~ks v¢~ p ~ l e d , filtered t h r o u g h several l a y e ~ o f sterile cheesecloth, and held at 4 °C until rested for purity. To test for purity, the sfx~re preparation wo.s cultured on bovine bloe,d agar plates. T h e b I ~ d plates were incubated aerobically and anaerobically at 3 6 ° C for 72 h. Mic~:~bial g r o w t h xv~ not detc~ct~ on the a e ~ b i c a l l y - i n c u b a t e d bl(g~c| plates. Colonies found on the anaerobically-incubated blood plates were identified ~ C. haemolyticum. Forty flasks o f culture were harvested w i t h i n a 30-day ~ r i ~ Each F ~ l e d spore s ~ p e n s i o n was added to an equal v o l u m e o f sterile glycerol and held at room temF~raturn for a m i n i m u m o f 14 days. A final v o l u m e o f 9 0 0 m l ofharvested spores w ~ m i x e d w i t h 9 0 0 ml o f sterile g l y c e r o l T h e s ~ r e - g l y c e r o l preparation xv~ m i x e d t h o r o u g h l y with a m a g n e t i c stirrer each work day. After retesting for purity, 1-6 ml o f sF~reglycerol preparation was :~septically disw_-nsed into each o f 950 sterile 5 m l a m p u l e s using an automatic A m p u l e Filler-Se~er ( K a h l e n ~ r g - G l o b e Co., Sarasota, FL 33578L D u r i n g filling, the s ~ r e - g l y c e r o l p r e p a ~ t i o n was held at room t e m ~ r a t u r e and continually mixc~d w i t h a m a g n e t i c stirrer to m a i n t a i n h o m o g e n e i t y . Samples of the final sealed lot of amg~ules were restcxt fi~r purity. T h e final p ~ d u c t , labeled C!ostridium baemolk[Hct¢m Challenge Culture IRP 234. was held at a remF~rature o f - - 6 0 °C. An}' ~lr~her r e , f e n c e to the s ~ r e - g l y c e l o l p r e p a ~ t i o n shall ~ identified ClostrLdiam haemol~icum Challenge Culture IRP 234 or simply a_s 1RP 234.
Eraluation of C l o s t r i d i u m h a e m o l y t i c u m cu/tu~ IRP 234 E i g h t titrations in guinea-pigs, w e i g h i n g ~t5C~550 g, were conducted d u r i n g a seven-week p~zriod to d e t e r m i n e the LD3o in each 0" 5 ml o f IRP 234~ T h e LD:,0 w ~ bo-Aed on the highest dilution o f s t ~ r e susF~nsion causing 50% o f the animals to die w i t h i n 72 h F~)stin~:ularion. ~ r i a l renfi)ld d i l u t i o n s ( l O - ~ - l O -¢) of IRP 234 were made in freshly prepared 5% CaCI: solution. The d i l u t e d s p o ~ SUSF~nsions were held at ~ o m temw:rature ~ IO rain and then placed in an ice bath. Each o f five guinea-pigs were ine,culatc~J intramuscularly in the hind leg w i t h 0"5 m l o f each d i l u t e d s ~ r e s u s ~ n s i o n . T h e LD:,o value and the 9 5 % Confidence Limits were d e t e r m i n e d by probit analysis (Finney, I97 I). T h e most probable h u m o r o f viable Sl~ore ( M P N V S ) in 0* 5 m l o f IRP 234 vv~ estimated by the d i l u t i o n t u ~ t e c h n i q u e (Harrigan & McCane, 1966). ~ r i a l ten.,*bld dilutions ( I 0 - ~ - I0 ~¢) o f IRP 23,1 were made in O - 8 5 % NaCI ~ l u t i o n , and O" 5 m l o f I17
D~ R. KOLBE, K. D. CLAUS A N D R, M. N E R V I G e a c h d i l u t i o n was a d d e d to e a c h o f five tuLms o f L D b r o t h , e n r i c h ~ w i t h t r y p t i c a s e a n d yeast e x t r a c t . T h e c u l t u r e s w e r e i n c u b a t e d a n a e r o b i c a l l y at 3 6 °C fbr seven d a y s . T h e h u m o r o f rubes ~ r d i l u t i o n c o n t a i n i n g b a c t e r i a l g r o w t h w a s used t o c a l c u l a t e t h e M P N V S b y r e , f e n c e t o p r o b a b i l i t y rabies for t h e e s t i m a t i o n o f b a c t e r i a l n u m b e r s .
DISCUSSION The titrations of C C h a l l e n g e C ~ k u r e I R P 2 3 4 in g u i n e a - p i g s is s u m m a r i z e d in T a b l e 1o T h e r e were 6 1 9 3 0 0 g u i n e a - p i g LD.~ d o s e / 0 - 5 m l o f I R P 2 3 4 (log 5 - 7 9 1 9 w i t h 959~ c o n f i d e n c e L i m i t s o f l o g 5 - 6 I 9 0 a n d l o g 5 " 9 6 5 t). T h e r e w e r e 1 9 8 8 0 0 0 viable s p o m f f 0 " 5 m l o f l R P 2 3 4 ( l o g 6 - 2 9 8 5 ) , as d e t e r m i n e d by t h e m o s t p r o b a b l e n u m b e r m e t h o d . T e s t results are s h o w n in T a b l e 2. T h e r e f o r e , t h e m e d i a n l e t h a l d o s e for 4 5 f > 5 5 0 g g u i n e a - p i g s is a p p r o x i m a t e l y t h r e e slmres. Closlridimn haemolyticmn spores p r o d u c e d ~ d e s c r i b e d in t h e M a r e r i a l s a n d M e t h o d s r e s u l t e d in a u n i f o r m l y v i r u l e n t s~ore c h a l l e n g e p r e p a r a t i o n w h i c h has to d a t e r e m a i n e d stable. T h i s p r e p a r a t i o n w i l t t~m used fiat p o t e n c y a ~ a y s tD, b o t h l i c e n s e d m a n u f a c t u r e r s o f v e t e r i n a w b i o l o g i c s a n d in g o v e r n m e n t l a ~ r a e o o , t e s t i n g p r o g r a m s ° D u r i n g t h e year 1978, m o r e t h a n l ~ u r m i l l i o n doses o f C. !yt&'umb a c t e r i n w e r e p r o d u c e d in t h e U n i t e d Stares by five licensed m a n u f a c t u r e r s . It is c o n c l u d e d t h a t C. !yticum c u l t i v a t e d o n L D a g a r m e d i u m in an a n a e r o b i c T^~L~ 1.
Gulnea-plg
LDao
doses
~r
0"5
ml
o f Clost
icam cha|lenge culture IRP 234
Titration
| 0 ~a
Challenge culture dilutions 10 -~ 10 -a |0 ~
10 ~
I
5"
5
5
3
0
2 3 4 5 6 7
5 5 5 5 5 5
5 5 5 5 5 5
5 4 4 5 5 5
0 0 3 3 1 3
0 0 0 0 0 0
8
5
5
4
2
0
40
40
37
15
0
Total deaths
t Data expressed as num~.r of deaths in each group of five ~ulnea~p/gs~ l ~ g dosa2 ~--3*00~30 --4"00000 --5-00000 --6-00000 -7-00000
P m b i t analysis nding Tested
No+
40 40 37 15 0
40 40 40 40 40
~ ResFnmse IG~)*0 lO0-O
92"5 37-5 00-0
F = I95"6; d r . ~ 3; s|ope = -- !'9430;X ~ ~ 0"69|0; endpoint L ~5 ml; log 5"7919; ~¢i|og 619 3C~; 95% confidence |imie$ 5"619C~5~9651.
118
P R O D U C T I O N OF C, H A E M O L Y T I C U M TABLF. 2.
Tit r~t ion
Most probable n u m ~ . r o f viaMe sF,0res F~.WO" 5 m l o f C/0f icHm challenge C~tltUe I R P 234 Challenge culture dliutions 10 °'* I0 -:a I0 -~
t 0 -v
Log,o ( M P N ) 6~114 6- 740 6~740 6-954 5- 544 5"699 6- 2985
!
5"
4
0
0
2 3 4 5 6
5 5 5 5 5
5 5 5 I 2
2 2 3 0 0
0 0 0 0 0
Mean
SPORES
NIPNVS/O" 5 mL * Data expressed ~ h u m o r of tubes cor~tainlng b:~:~eria| growth in five inocu~ laced tuh~-s of medium. c h a m | ~ r will p r o v i d e a c h a l l e n g e r e , g e n t w i t h o p t i m u m n u m b e r s o f u n i £ o r m l y v i r u l e n t spores. T h i s m e t h o d w i l l also p r o v i d e a c o n s i d e r a b l e s a v i n g o f t i m e a n d m e d i u m in pregntring t h e c h a l l e n g e r e a g e n t . S e v e n a d d i t i o n a l SF<~re p r e p a r a t i o n s have ~ e n p r o d u c e d b y t h e d e s c r i b e d m e t h o d f r o m six s t r a i n s o f C. a n d o n e s t r a i n o f C . sor~'lliL T h e s e s t r a i n s o f c ! o s t r i d i a w e r e c u l t i v a t e d readily o n a m e d i u m c o n t a i n i n g b l o o d a g a r b~.se, yea~st e x t ~ c t , s o l u b l e starch, c y s t e i n e H C l , a n d agar~ Since s ~ r u l a r i o n ~ c u r s m o r e r e a d i l y in t h e s e closrridial SF~cies t h a n Co , m i c r o b i a l g r o w t h f r o m o n l y 10 f l ~ k s o f c u l t u r e w0~ sufficient t o p r e p a r e a p p r o x i m a t e l y 1 0 0 0 a m p o u l e s o f c h a l l e n g e m a t e r i a l . REFERENCES Finney, D. J. ( I 9 7 t). Probit Arml)si~ 3rd edn. London: C ~ b r i d g e University Press. Harrigan, V~;'o F. & McCane, ~l. E~ (1966). ~boratoO" i~letk~..~ m L ltcreomlo~, pp. 3 I % 3 2 3 . London: Academic Press. Records, E~ & V a w r e r L. R. (1945L Bacillaty n e m o g l o b i n u r i a o f cattle and s h ~ p ( R e d Water disea~:). University of Nevada Ex|~rimentM Station (University o f N e v a d a , Reno, N e ~ d a L B 173~ S m i t h , L. |3~ S (1975). Tb,- 1%abogeni~ Am~¢robic Bacteria. 2nd e d m Springfield, Illinois: C h u t e s C. Thomas.
119