Isolation and properties of a phage lytic for non-smooth Brucella organisms

AA

. ~

o.f

1979 ~, 349-360

pro~ for n

M.y.

A pb~o~ l ~ c for

m

of a

B.J~l~

c ~"

*

t

ce:~ o f ~ ~ ~.ated'from a o f ~a,~e wi~ ro~h ~ ~ ~e p ~ of N-me~hyl-r,~%r~ttoI~ did r ~ t | ~ ~ t h ~ s of ~ . ~ s t r ~ j ~ n o r ~hose o f o ~ bt.q the c o ~ of ~ ~ H ~ m rou~ a small

~e ~ ~IO ~ h

c~l =rod I ~

phage. P.~ ~ J f Fr~ge R ~ ~ both ~

fotmd m plaqu

piu~e m im D N A and

prem~ ~

~-labil¢

INTRODUCTION

g I)~

lys~ ~

&

*

~ e in t h e ~d 1963; ~ , 1975). H o d are ~ an ~ ~orb ~ l ~ of az~ in the ~ 1957; ,1963; I~; Jones, ~ & ,1972; Morr;~ & 1973; , & b; & ~1=~, 1976). T ' n ~ ~e ~ muted are

f~

~ &

16

c2~on of the bru~L~t o , r e p l l c a t e in .and coloni~ & ,1968; I~; ha-,~ ~ d ~ ~v

1979.

2,49

M. J. CORBEL

As the p of ion is not only w i ~ Ioss of ~ s i f i v l t y to lw i s by p h a ~ bu t a ~ with mo~fieation or Io ~ of the s m c ~ h s u f f a ~ an6gens e of the g e n t , non~-a ~ d i ~ c u l t to identify by tion~ meth~ & ,1975; M & C o r ~ , 1976). For this an of have been for d e ~ i t i v e id~fification o f i~ ~ of the ~ u s & , l ~ ] a , b; & MCul ,19~; lo~m, Dr'an & n~re~q 19721 s, 1973). none of t h ~ t e e h n l q u ~ is m d i l y applicable to the ~ u t i n e d i a ~ o s t i c labo and there is d ~ r l y a ~'qJa t for s i m p l ~ taxonomic urn. extension o f the p h a ~ g scheme ~ include x~xmld largely .fulfil this requh-ement, at were trade to m ~ i f y the host ~ n g e of existing phages. ~tre of a re of of th~-e to a e , f o | l c r ~ by incubation m t h e ~ of non-smoo~h brueella ce]~, r e s u l t ~ in the p r o d u ~ i o n of a phage which o n preli examination ~,¢s ob to f o ~ p l a q u ~ on a s~.~in of Br. (Corbel, I I n the pre.~ent the p r o ~ . r t i ~ of th_is k~late, d ~ s i ~ a t e d phage R, ]m*ee b ~ n su to detailed nation.

MATERIALS

AND METHODS

~e s~alp~ and other o ~ a n i s m s used, ~ t h G~e ~ p ~ o n of tht~ ser o ~ t ~ , ~ r c ~ o m the ~ | t u ~ coll~=ion kept at this r~: T h e ~ u ~ strain u~ed in t h ~ ~ d from a subof the isolate d by & (1930- ~ C ~ i o J ~ ~ m p l e u~ed had b ~ kept Lu ~ e ] ~ n d i t i o n at 4 °C sLn~ 1952. ~ i a n t s of o t h ~ were o b ~ by ~ ' . ~ i n g the orgamis,-ns in static AAbimi b r , ~ a broth at 37 ~C for 7 - I 0 days, by tv.t~ onto ~r~am d e x t r ~ e ~mar. Colonies the ~ha e ~ of rou# in refl ry, 1933) were ] t u ~ and for autoagglut ~¢ in ~ 1 5 and 0-I a~ (Braun & B~on , 1947)and for C~e~ r e a s o n ~ an re~de mon fie for the A, M and R of t h e genus ng to ~ t o n , J o n ~ & Pietz (1975 L The ~ r . , s were those u ~ in p r~-io u s .s tu d i~ , 1975; & "~n~, I . ~nd w ~ kh'~dly by D r Y. V¢orld H ~ ' ~ O ~ a ~ t i o n , G ~ e ~ , S w i t ~ l a n d . .All -~-e on ~" cally at 3 7 °C. o~i~.s

were g ~

on t _ ~ f i ~ s o y

agar i n o a ~ t e d ~ r o b i ~ ¢

at 37 eC.

P~ge ~

T h e o r i # ~ o f the T b ~ ~), ~dge ~ d previo~y ~Io~ ~ aL, 1973; & Cor~, 1 D ~ ~|a~ ~ma bat~,of ~ pb~

a al., ~ at ¢ ~

3~

~

~-~ ~

by ~ o n ~ . h toluene m

045 ~ ~ity.

strains ~ve~ ~

ran~ ~

d~

~d

P H A G E L Y T I C FOR N O N - . S M O O T H B R

LA

of p-h~e T h e m ~ o d h ~ ~ n d e s ~ b e d in det~l in a p ~ i m i n a r y repo~ (Corbd, 1977a). B a mixture of ridge, MC/75 and D ~ m in d sJth Bn a~t~ c.lls m the ~ 0 f N - m e t h y l - N ' - n i t ~ - N - n i ~ s ~ g ~ a a n i d i n e(tR.~ph N. EmanueI Ltd, L~ndon)~ ~ e ~nariant, ated R, was cloned from p l a q u ~ which a p p ~ e d on c u l t u m of rough cells recovered f r m tMs ~faterial from ~ngle # a q u ~ v ~ diluted in Albimi bracella broth and used m inoculate fresh euloares o f B r . ~ed to f o ~ confluent layers on ~ a m d ~ r . C l o n ~ of phage were from individual plaques which d e v e l o # on t h ~ cultures and the cloning p ~ s ~ugh six ~rlal transfers on ~ r . ~ A ~ r k i n g phage S ~ ~g~s prepared by i ating large n t i m ~ of ~ r t i m d a~ ~ -~th Br. ~ ~th s~ent cloned materi~ to prcMu~ con~ fluent lysis. A f t ~ 48 h in n at 37 ~C, the # a g e xs-~sh by ~-ashing the agar la2~xn-s with se.~am de~t~se broth. This ~ . l d e b ~ t e v ~ d by ~ n t r i f u ~ d o n at I0~×g for 15 main, followed by ion 0-8~ rane f i l t ~ (Oxoid, I~3ndon) and s t o ~ at. ~4 *C u-ntil ~ r e d .

T h e p h a ~ ~rim'~t, d e s i ~ a t c d R in view of i ~ a h i l i ~ to l)me Br. cultures, ~ d by- pIa~Jng scrim dilutions in Albimi b ~ l h broth onto s~rum d aga r inoculated with B,-. 45]20 to produce ~ n f l u e n t ~ The were at i n t e r ~ s of 24 h ~ d # a q u e counts Made alter 72 h inc,~~fi0n. Rou~ne t ~ t dilution ( R T D ) ~ d ~ n e d ~ the lo~x~t c o n c e n ~ t l o n pr*-duemg confluent 1.-~ o f B r . 45/20 c u t t u m under the conditions described.

of hoar r ~ e ~t-~ w ~ done ~ e n to 1~1orrlsct aL (1973). Phage rationsw e ~ ~d at , le~ R T D and I ~ I~:TD. ~ e e.o.p, o f t h c on di t h ~ strains was determined by inco ing u ~ t v o l u m ~ of ~ , a l dilutions of phage su into agar with the tire ba~J~la s ~ n . P i q u e were trade after 72 h i n ~ b a t i o n at 37 °C. ~ e abili~.of the to replicate in bru~IM ~ n s in liquM m ~ i u m -.~,s ~ by ~ e tire by ~$orris & C o r ~ | (19~'3). ~eld-s were h'x d u # i ~ t e on l a ~ . s of Br. 4 5 [ ~ and Br. m~h'~ 49. SingI~step m were done on Br: 45/~3, B r . S t U n I9, Br. cm,~ R?d 6166~ B r . ~ 6 and on r o u g h and ~ o o t h c ~ l o n ~ f o ~ of Br. Rev I, Br. 5K33 and Br. ~ Thomsen. ~ u e u ~ used x ~ that d ~ ~ by & (19773) that the p h a ~ yield ~ on confluent of B r in e a ~ t.

were done usi~ heat-~lled ~ o f Br. B3196 ~i S), Br. ~ R~I 1~. tnt.rk

Br.

, Br. ~

(

Br. R e v l, Br. Br. ~ 1330,

and

$99, Br. 63/9, Br. n,~co//,

~d 351

M . J. C O R B E L

10 dry weight. T o 0-9 ml volumes of ~ e s e suspensior~ 0.1 ml volumes of t~hage l y ~ t e c o n f i n i n g about 10~ p.Lu./mI were added and the r ~ incu at 37 °C for I h. Re te volumc-s of 0-I m~ of t h e r ~ were t i t ~ e d for p l a q u e * f o x i n g a ~ ¢ i ~ on confluem lawns of Br. 45/20. A ~ procedu~ ~ . ~ ~ with live cells of strains except that after the adsorption period the rium s u s ~ s l o n s vce~ centrifuged at 5 C ~ × g/20 min, the supematan~ passed through 0.45 ~ filte~ and then d for p l a q u e * f o x i n g a on Br. abort~ . ination of l~ l Bu density m~,,su~men~ were made b y i s o p y ~ i ¢ ~ n t ~ ion of phage in continuous I suc~e ~d w / v CsSO 4 ~ d l e n ~ . Centdfugation - . ~ ed for 12 h at 1 ~ 000 × g and 8 °C. Fractions were for phage activity and t h d r s ~ f i c ~ d e t e ~ n e d b y m ~ n s of a mi ometer. T h e nucleic acid ~ tion of the ~ determined on prepa~tiov~ purified from crude Iysates by incuba~on for I h at 37 °C ~ t h RNase (1C~3 Kunitz unim ml -l) ~ d DNase ( 5 ~ K u n i ~ u n i ~ ml -I) followed by i ic centrifu~tion L,1 a CsSO a ~ d l e n t . Phage re~vered from the ~ d i e n t was di~ysed a ~ n s t and pelleted by centri at 1 C a 3 ~ x g for 1 h. x~,~ d e t e ~ i n ~ by the difhenylamineacetal e method ( G i l ~ & M ) ~ , 1965) and RNA by the B i ~ orchaol method (Mejbaum, 1939). ~rte stability of the phage to h ~ t and to various ~ and chemical en~ determ2ned using the c o n d i t i o ~ employed in previous s t u d i ~ ( M o r n s et a l , 1973; Corbel & as, 197~) t where ind to the ~ n t r ~ - y .

ination of n~t,~tively stained s ~ m e n s was done ~ d ~ d et aL, 1 ~ 3 ) . ~ t h c ~ d e lysates and phage purified ~ for c h e m i ~ l

pr~dously ( M o ~ ~-e~ n~.

tests

neuL~ p

~

t ~ t s were done a ~ i n g to the method of Adams (1959) usLng bed b y M o r ~ s eZ aL (I973).

RESULTS H ~ r a ~ e ~ e.o~. Phage R p ons stand at R T D did not p r ~ u c e or te on or c u h u ~ o f Br. b I, 2 or 3 or Bt. ~-/s I, 2, 3 or 4. Simfi~ly, no l ~ s or we~ p~u~ on any of the of the naturally n~a-smooth s ~ , Br. ~ ~ d Br. ~ , s . I the ¢on~trafion to I ~ R ~ ~ d not tb.ese r e s u l ~ but at ~n ~ of I ~ or ~ n e s of ly-~s on ~ m e Br. ~ ~ d Br. c,~. w~ ~ in ~ e ~ but often the ~ the a , ~ was ~ o r c with tested on ~ t ~ of Br. ~ 1, 2, 3, 4, 5. 6 , 7 or 9, , no p were by at R T D . those stan ~2

PHAGE L Y T I C FOR N O N - S M O O T H BRUCELL.4 at concenL~dons of I ~ and lif t RTD, ~ e t e p l a q u ~ or z o n ~ of semi-confluent lysis at the test Sites ~ t e r 2-3 days in on 1), O n C~a~t',l~ of Br. and ~ l other nonstr~ of Br. ¢xa,~ned, includb~g all which au urinated in ac~fla~ne solution, the phage R p ons at produced ~ n e s of ~ n f l u e n t lysls after 24-48 h incubation. At con~nt~riov~ the phage R produced d i s ~ t e p l a q u ~ which w e ~ visible ~ t e r 24 h but did not d ~ally until after h in . At phage ~ n ~ n t r ~ tions b~gher ~ a n R T D z.on~ o f I ~ apparent ~ t h i n 24 h I). T h e # a g e s obtained from single plaques p ~ u ~ by ph~-~ R on smooth B r . S19 cul~Jres were d e $ ~ a t e d R]S variant. Some isolates f o x e d p l a q u ~ on all smooth Br. Br. and Br. ~ o d l t u ~ tested but dM not lyse B r . nor any n b r u ~ l l a strains ~ m i n e d . Other R/S - , ~ r i ~ were l ~ i c only for s Br. ~ns. T h e e:o.p, o f R on ~ u g h Br. strains ~,,s in the order of I ~ times greater than o n o3mplet~¢ of this s ~ i ~ . T h e e.o.p, on s t u n s - a h i ~ w e ~ parti~ly a t ~ to ~ e i n t e r m ~ i a t e ~ rough p_ha~ ~ s s i m i l ~ to tbmt observed on strafers in t h e ~ m # e t e l y ~ u g h or m u ~ i d state. One ~.il~re o f B r . aborfus $99 which was in the i ediate # ~ w-as s iMe to lysls by both # a g e R and the # a g e s a~ive upon smooth

No of ~ # i c a t j o n of # a g e R w ~ o b t ~ n e d when ~ n t i n u o u s l y broth c u l ~ r e s o f Br. st~.qs Rev 1 ~ d B115, Br. ~ T h o m ~ n , Br. n e o ~ . a e 5K33, Br. ~ B r . canls R£Wi 6/66 or ..**~riantsof Br. ~ t o ~ ~ d Br. ,uls were for p tion. T h e resul~ were similar for ~ l strains when Br. aborlus 45/20 ~ ~ d ~ the indicator, propagation ~ ate d in smooth B r . ~ t u s S19 cult,ires under the ~ e ~ n d i t i o n s , an i n ~ . . s e in the p.f.u, ration in the order o f I ~ tL~nes the input was when the yield ~ tiLrat~ on Br. S|9 cultures. N o overaJl i in yieM ~ when the titrations were ~ r f o ~ e d on Br. cuhures, r. Br. ~ used ~ the p ng strain, ~ in in p~fm. ~nt Lq the order of I ~ times th~ phage input was o b t a i n ~ ~-Emn ~ e yield was on Br. ~ ~ e indicator strain. V~rhen the titrations were ~ r f o r m e d on S Br. S19 oaltures, the I i in p.f.u, ra~ons ~ I ~ mad I ~ tlme~ the input ~ on this strain. ~nese ~ r e c-onfi~-d by Si ~ ~ on Br. ~ t u s 45/20 ¢lalt~a~ p h ~ R had a latent ~ r i ~ of a p p ~ x i m ~ l y 2"5 h ~ d a burst ~ ~ n g from ~ to 38 over 6 ~ p e f i m e n m . T h e burst size for the R/S vh_rian~ a~ive on smooth at. S19 co,aid not ~ detetm-fined p ~ l y b e c a ~ of the low ~ n ~ n t r a t i o n o f these in the inpuL ~ o r e , the e-ciden~ ra that a propo~ion of R/S ~,~ tly p ~ u ~ R was d ~ Br. 45120 or~h~ b ~ c e l l a oalt-¢res. T h ~ the RiS could ~ o t ~ i m L q a t e d ~ or of the p h ~ R ~ stock ~ Br. ~lls.

~ s d~o~ polnt znm~s of

~

R on Br. a n d other rough a t . slowly. T h - ~ a:-ter 24 h incu they were visible ~ pin ~ ~ a ~ o i d zon~ o f After 48 h

3~

9~

• t

'S 7, R 'S

6' R

'8

S' R

'8

4' R

'S

3'R

'S

2' R

1' R 'S

"S

By. ahoy,tin

2 2 4 3 4

2 2

2 2 2 2 5 4

3 2

18

Number of stn ins h ~ coionM

0

0

0

2 0 4

0 0

0

2 0 0

2 0

0

0 5

2

0

0 0 0 0

M

0 2 0 3 ¢

0 2

4

2 0

0

2

!7 0 2 0

R

0

0

2 0 ¢

2 0

0

0 S

2

0

1 3 0 2

RTD

0

0

2 0 ¢

2 0

0

0 5

2

0

t 3 0 2

10t RTD

Tb

......

0

0

2 0 ¢

2 0

0

0 5

2

0

1 3 0 2

1D

0

0

2 0 4

2 0

0

0 5

2

0

1 3 0 2

t0 t RTD

Wb

0 2 0 3 ¢

0 2

4.

2 0

0

2

6 0 2 0

RTD

..:........J.........................

rr::: -

Number lysM by phag~ at RTD and 10t RTD

,~-~--.................

::

No i ~n~ of Br. a ~ s biotype8 we~ a~ltble. ' 0.1% w[¢aqueo~, S ,, Stash, R a Non.smith, in¢Iudir~fnte~iate, inlaid ~d ~u~.

0 2 0 3 ¢

0 2

0

4

0 0 0

0 2 0

0

1 3 0 2

A

withantt~" m n~pevfic for antigens

2

18 0 2 0

amflavtne

NumMr Lgglutinating

Number

TAW 1, The s~ptibUi~ of sm~oothgad non.smooth st~ins of Be, ~ r t u t to l:,'sm'by phage

!8 3 2 2 2 2 2 5 ¢ 2 2 2 2 ¢ 3 ¢

10tRTD

R

0

*t

P H A G E L Y T I C FOR N O N - S M O O T H B R U C E L L ~ ~ b a t i o n the p l a q u ~ ofa d~ a turbid pe~pheraI zone 3 ~ ~ in di d~er of ~ e ~ zone Mi pie n of ~ sho~

c e n t ~ zone 1-2 m_m h'~ (Plate 1). On more p ~ in size b a t ~ e ~ tu that it ~ n n~e~us

surrounded by ineuba~on t ~ re~ . of

T h e p h q a ~ p ~ u ~ - - d by R on Br. were m u ~ s , ~ l e r ~ n t h o s e whic~ developed on ~ e cultures. T h u s a:~er 72 h incu they turbid ~ n ~ of I ~ r t i ~ eleafing 0.5-1 ~ in di A~ in Be. S19 the p l a q u ~ much reaching ~ m,m in ~'ter ~ h Ln~ba~on 2).

R ~ d n ~ a ~ r b a p p ~ a b l y to l l ~ cells of any of the o ~ i s w ~ tested, induing and s m ~ i Tb~ ~ in ~ n t ~ t to results obtahned ~ ~ R/~ t (Table 2). % ~ e n the w ~ ~ r f o r m ~ wiLh live b a c t ~ c~h, R bed to Br. and to a l ~ e r ~ t , t o c~l~ of other bruceHa strains. T h e r e was ~ si~ificant ~ smooth Br. 819 and smooth Br. 5K33 cells, on to o t h ~ ~ p . and to ~ x , o n o . ~ l l y was ~ e lim3m of ~ ~r.

On i~pycnic ~ b ~ d band ~ g

ion in s u r e gradients p1-@~ R p r e ~ t i o r ~ formed a single L'~ buo)~nt dePUty from I - 1 ~ g m| ~z to 1":2430 g mI -I ~ d with a

T ^ e L Z 2. A d ~ ~

of p b ~ e ~ R,and RI~ to B m c d l a ¢elh

P~por~of

p~s ~n~g

aft~ a~rpfi~

R S ~ ' ~ ~ - d for Br. $99

b

~

~

PalS V ~

oeHs

Live ceils

Kill~ ~

38

61

67 6s

1 55

4 s ~ o (,o~h)

5 B31% B,. ~ RM

~e

of p h a ~

12

6]66

B,. R~" I BI 1S ( bi~ 2 ~¢pe 3 ~.er Br. 5~L33 Br,, ~ 63/290 Br. ~ I 1330

85 9s

l'qD 70

~ I~

2 89

2-7 101

97 75

1~ 96

91 IWD

75-5 103

~D 33 69

93 ~ 105

N D ~$ 95

105

~

93

I

ND

1~

99 1"2

355

M. J. C O R B E L m~-~ b u o y a n t density o f 1-1875 g m l - L P h a ~ p a r t i d ~ o f R and R / S h ~ range w e r e in C~s b a n d . On ic c~ntrlfugation in CsSO~ ~ d i e ~ , ~ phage R fio~ d into ~'o of d t density. ~ e m a j o r porzion of t h e phage ;xas p r ~ n t Ghe density band, ~ r m p o n d l n g to a nt d ~ i t y o f 1 - 3 8 ~ g ml -~. T h i s ~ion p ~ d u c e d # a q u ~ on both rough ~ d Br. c u l t u r e . . ~ e low ~ t y fraction, ding to a b~loyant of ! g mt -1, p ~ u ~ d # a q t t ~ only on c u ~ m of Br. ~ e ph~a~ p m i d ~ p r ~ n t in hi~n a n d low d ~ fra~ions we~ mo~nolo#Mly similm% ~de infl-a. I n t e ~ s o f total particle ~ n ~ t r a f i o n . the low d e n s i ~ ~ s =ppzx:*-~mately 1 ~ ~ m ~ I ~ e ~ e n t m p r o d u ~ n g p l a q u ~ o n Br. than t h e high d e , ~ t y , t i ~ that it m a y have ~ n ~ n e d a high on o f i n m i v e uni~ ~ e only nucl~c a d d in p u r i f i ~ R preparations ~ a s D N A . T h e ~ a b i l i t y o f p h a ~ R ~ d an R / S t t o ~mrious ~ and ~ c a l ~ n ~ is s u m m a r i z ~ in 3. I n t h d r ~ s t a n e e to diethyl ¢ t h ~ and toluene and t h d r bility to ~ 1 o . ce a n d cationic d they w~rc c o m e ~ t h e brucellalyric for T ^ n L E 3.

of R m'~d its RIS ~ a n t ¢hemicml i n a ~ agents

~g

~t

R .

_

.

~

...............

1~

D i e ~ y l e*-~er Toluene

I01 98

9

~ 105 5S

Lipase DN~

55 93 89 65 C'~5 92 4"5

91 59 0-1 95 3"5

45F~ ~

for P ~ ~ ~ - h

of p l - ~ ~ RJS ~ Phtqt.m ~ . t s for R w ~ d ~ ~ ~ ~ . ~ . ~ L t S19.

o f crude 3~

98 101 39

Fi~

0~2-M-Dith 37 * e l i h 04~.. M*}~aIO,; p H 4-5 15 °C ~Na 60 "C/1 h

n=

_

1(~3

15-5

te

of ~ ~

h

~.L B: ~

~R

~d

R~

o

C~I5~ N a ~

I~./o ~ m m d 1% T d t o n X - l @

~

~ent

~a

I

m

f~

65

Br.

75 ~ d ~

in

~d

P~tc | . P|~qu~ produced b-¢ p b ~

P~© 2 . P~u~'~ p st 37 C.

~

R on ~ u ~ h Br. ~ o r t . s 45~20 after ~ h i n C ~ t i ~

by pbJge v a ~ T RIS ~ ~ h

Br,

$I9 after 4 8 h

~cu~rm

L ~ ~

~6T

Plate 3. N e ~ t i v e | y s t a i n ~ low d ~ i t y fraction o f p ~ g e R r~,~oeered from a C s S O , g ~ d i ~ t after ic c©ntfifu~tic,\r~ "FI~e p}~© p~nlcle$ arc l n ~ t i y intact and ha~'e h ~ g o r ' . a ! h ~ d s with short tubular |~ils. Stained w i t h I o, ~o ~ t a s s i ~ m p h ~ p h o t u n ~ t a t e . ~ t i o n × 40 0"~.

Plate 4. r~ pho,

}iirh d ~ - i t y of R ~ the ~ m ¢ ~ O 4 ~ in Plate 3. T h e n e ~ t i ~ l y * ~ pF~ |y i ~ i t h t h e e o f ~¢ ~ d e i t y . S ~ ~ t . h 1 ~g e..Ma~flcatlon × ~ ~.

d~m-

PIIAGE LYTIC

FOR NON-SMOOTH BRUCELI..4

~ b u h r tails 20 and 30 nm in length. T h e r e , ~ s no e~dence of a b ~ e plate hut s~a~a~ ve of sho~ tail fibres were seen in some preparations. Idemical p ~ i e | ~ were seen in phage R p purified by ~ntHfugation in ~ S O ~ d e n s i ~ ~ d i e n ~ . T h e only differen~ seen ~ t - w ~ n ~ae low density and high d part~ ted on the ~ d i e n t ~ s that ~ e tended to have heads slightly h r g e r in diameter than those of the 1alter ( P l a t ~ 3 and 4). T h e proportion of phage p a ~ i e l ~ g~as similar in both high and low density fractions and evidently did not acc~0unt for the differ¢~aee in ~dimentation behaviour o f the two f~ctions. |nation of purified prepa~tions of an R/S x~a_~ant showed p a ~ i c l ~ m o ~ h o l o g i ~ l y indistinguishable from those seen in rough Be. at~,rt~ l~sates. l propertles

Neut.1 tes~ c o n d u c t ~ on phage R with homologous m and antise~ to the t h r ~ phage strains ~ d in its initial selection pro~aueedp a r a d o x i ~ r ~ u l ~ . T h u s the homMogous andserxim to phage R produced a lower K x'atue again~ this strain than ag~nst the Weybridge or phages, xmlu~ may not have b ~ n truly compa~bte, however, as the i n d i c t o r s t ~ i n u ~ d for phage R xs~s different from that used for the other phages. A more valid rison xv~ obtained in neutralization t ~ vAth an RIS variant (Table 4). This phage produced K -.~lu~ with a n t i , rum to dge phage ~'hieh were identical ~ L h those given x~th the homologous antigen, ".~qthin the limits of mentM error. Some d ~ e e of erogs-neut~li=tion also occurred with the a n ~ s e ~ to the other phages but the "¢~u~ obtained v.'e~ subst~ntiMly lower than those given by the homologous st~ins.

TABLE 4 . The n e u ~ d o n

o f p ~ a g ~ R and Ri~ ~ d their parent st~ins by h o m ~ o g o m ~ d heterologous ~ t i s e ~

Phage st~in R

R/S %Veyb~dg¢ MCP5 D

K v ~ u ~ o f ~ 6 s e ~ to phage sh-ains XVeybfid~ MC~$ D R 45-0 75-7

3.2

2-8

4,2 6"9 46~

22*8

4-8

7I-1

37-0 39"0 ~'9 13"i 61"1

16"6 8"7

I8.g" 29"6 0

All antise~ ~ere s ~ r ~ with cells of Br. ab-:wtusstt-a&~s 19 ~ d 45120 ~ f o ~ t~tlng. P h ~ e R ~ s tie.ted 'on ~ h ' Be, ~ r t ~ 45J20, the other p ~ on smooth Be. ~ r u s S19.

DISCUSSION na'don of the host ~ n g e of phage R ~ o w e d flint it ~-s~ quite distinct from ~ | bruc~Jla hiCheno d . Thus nu phage isolates h~:ce been reported to be 1)~¢ for Br. ~ d other t2,~dr activity h ~ ~ n c o n f i n ~ to in ~ e smooth or s m o o ~ late & To! I96~; et ~ , 1973; Corbel & ~ o m a s , I b; & Elberg, 1976). ~ ' ~ e lyric action for ~ltu~ by phage R p at high ~ n ~'as e v i d ~ t l y table to ~ e p of R/S in the # a g e 357

M. J. C O R B E L stock. Thtts the produ~ion of plaques on smooth strains couM not be eliminated by re cloning, nor by extensive absorption of the phage seed -~th smooth brucclla celh prior to p ion in the rough st~in,, T h e sm~th-celt-sF~cifie phage m u t a n ~ we~ t m a rdadvely constant propo~ion to the phage panicles a~ive on the rough org~n~n~ and must b~ve been produced concomitamly during the course of replication in the Br. abort~ 4 5 / ~ p ting strain, t ~ t a n d i n g the p r ~ - e n ~ of R/S mutants, it - , ~ evident Chat the of # a g e R p a r t i d ~ were not le o f ly~ing or a d ~ r b i n g to s m i t h brucella celts. This was co d by theea~orption tes~ which showed d ~ l y that the f o r phage R were different from those i n v o l s ~ in ~ e attachment of phages to smooth bmoella cells. ~ e latter have ~ n identified ~ heat-stable s t r u ~ r e s associated with the iipo-poly~cchaHde-protein ~ l u t i n o g e n corn#e× (~dorris a aL, 1973; CoFl:ml& ~ o m a s , 1976a, b; Corbel, I . i n contrast, die receptor for # a g e R ~ - s heat-labile and present on living non h b r u ~ l l a ~lls. T h ~ difference n the receptor sites involved in the attaiehment of # a g e R and the s m o o t h - s : ~ f i e to their h ~ t cells ~ s p r o b ~ y reflexed in the kineti~ of the neutrMization reactions with specific antise~. " ~ i s could ~x#ain ~ e apparent of ~ t i s e r u m to # a g e R producing higher K valu~ with iero~ss-rea~ing th~n ~ t h the homologous antigen. In s # t e of the differences in host ~ n g e and adsorption p r o p e r l y , phage R ~ m M e d the smooth fie brueella-phages in other . "~qus i ~ D N A ~ n t e n t , mor#ol~, stability to # y s i c a l and c h e m i ~ inacti~tlng agen~ mad ~ r o l o g i ~ ficity identified it as a mc~mber of ~ae ~ m e phage family ~ the Tbilisi and ridge g r o u ~ . Detailed examination of its p i ~ indicated a closer resemblan~ to Vv'e#fidge phage than to the other b~cella p h a ~ e = m l n e d . This s u ~ e s t ~ that phage R r ~ y have t~:~n derived from Vqeybfidge phage =tI,mr than the other u~ed in the ori#nal mixtu~. T h e ehara t i ~ of the s -s~cifie R/S m u t a n ~ p r o d u ~ during repli~tion in ~ u g h ¢x,ltur~ lent further su to t h ~ conclusion. "Fi~us, in their host range, ~ r o l ~ c a l s ~ f i d t y and or/her es, t - h ~ resembled ~,%.:~'b~d~ phage, although distinguished by their high c , h l se~.sitivlty. T h e p r ~ n ~ of ~ r t i ~ of two disqin~ i ~ in the phage R on r~q~res ~planatlon. T h e two sub-populations did not c o r r e s ~ n d to the h o s t - r a n ~ ~ti~ , nor did Lhey ~ nd to complete particles and t h ~ ~,~-id-ioutnu~ei¢ a d d cores is'ely. H in view of the rela low infecti~'ity : particle ~ d o of the 1~ d fra~ion it probable that this con a hi# on of inactive p a ~ i d ~ x~th a ~ u ~ nudelc acid content. q~h© p ~ v e nataare of the ~anges' in-~x~lved in the production of # a g e R from i ~ p ~ b a b l e par~qt strain V V e # ~ d ~ xs~_snot established but e~dently invo|ved m ~ i f i c a ~ o n of the # ~ r e genome, probably under the influen~ of ~-meth~l-N'-nitro-N-nRrc~o~ne. Tb~ on ~ r ¢ f l ~ in a c h m ~ in the nature of ~ e Sit~ to W h i c h the phage a~ae/-~o~ on the h ~ t cell, but in~x~Ivedonly a slight a h ~ t i o n Ln the s e r o l ~ c a l S # f i ~ t y . This ~ a t further g ~ e t i c m0difi~fion off existing bruce~amay ~ F - ~ i b l e and that u I d m a t e l y t~zay be applicable to ~ l of the ~ n u s

The audhor h irtdebtt~d to M r E. L T F ~ n ~ for e x ~ t R. M . ~ f ~ the ~ d ~lr R. S a ~

358

~n~, for ~

to ~,~r .

PHAGE LYTIC FOR NON-SMOOTH

BRUC~LLM

REFERENCES Adams, ~I. H . (1959). In B a t ~'~cw York: lnters~en~. ~ton, G. G., John, L. M . & ~ e ~ , D. E. (1975). Labo~tory tecb~r,ique~ in bruc~cHosis,2nd ed, ttealth Non ~ o n ~ r a p h 2~o. 55. Brad, l, XV. & B o n ~ e I l , A. (19¢7). ]ndepemdent variation o f ~ a r a c t e f i s f i ~ in Br. abortus v a f i a n ~ and their detecfion~ A m ~ ¢ a n ~ M of ~, a~esearch 8, 386--390. , Ri. J. (1975). ~ e se ~ rdationsb~ip spp.,} enrerocolitiea ~ r o t y p e IX ~ d 2.~almonella s e r o t ~ o f K a ~ a n n - X . V h i t e group N . Journal o f e 75, 1SI-171. , M . J . (1977a). P r o d u ~ o n of ¢ l)'~ic for n o n - s m i t h strains. A~li 19, 99-108. Cor'~l, i . J. (1977b). on ~ d e l ~ c t e n ' z a ~ o n o f a phage r~ceptor from n e o t ~ a e 5K33. AnnMi 19, ]31--142, Corbel, i . J. & ~iorga~'% ~V. J. B. (1975). for m i n i m ~ standards for d ~ # o n s of n ~ a S ~ d ~ and b o f the genus . Inter2mtlor.~l ffournal o f SystemattLe Corbel, M . J. & Phfllip, J. L It. (1972). T h e relationship o f Bruceila obortus agglutinogenic m-~ to the r sites for ~ i l ~ i p h a ~ . in r~ 2 Sde~ce 13, 91-92. M . J. & ~aorrtas, E. L . (1976a). cs o f s~me new b r u c e ' - p h a g e i ~ t a t ~ ; for I y s o ~ n y xs~thin the ~ u s . meats in 8ta tlon 31, M . J. & q~homas, E. L. (1976~). D e e p , o n o f a n e w phage l ~ c for several sped~. ~wa~t of ~'on 4, 195-201. D o u g l ~ , J. T. & ~ k . r g , S. S. (1976). Isolation o f meHtensis p h ~ e o f b r a d biots~e

~rl~J,

G i l t , K. XV. & Myers, A. ( 1 ~ 5 ) , An improved di amine m e t b ~ for t h e estimation of d d e i c a d d . ~qamre, 206, 93. H~, B. S. (1933). D i s s c ~ o n in the gent, s . of Diseoses 52, 3 Hoy~er, B. H . & M c C , N . B. (I965). P o t ~ u d ~ t i d e ~ ~ brueella deoxyf i ~ u d e i c ~ds. of ~5, ~@t ~48. Jones, L . M., M e ~ , G . & , J. B. (1968). typing ~ a c t l o n s on speci~. 16, 1179-I190. ~ I c E - w ~ , A. D . & Rob-'r~, R. S. (1936). e contagious ~ M n i o n . T h e use o f guineapigs ~ i m m u n i ~ f i ~ s t ~ . I qf C ~ p a r a t ~ : ¢ Pa and ~atics 49, 9~7-I17. 1~ m, "~V. (1939). C~e~r ~ e I b ~ f i m r r t ~ g ~ e i n e r P e n g ~ , ~sb~.-sondere in D e r i c a t ~ der Ad j~r Cheraie ~ q S , I I7-120. ~-fe~er, M . E. & , H. S. ( l ~ l a ) . ~|etaboHc o f the g ~ . I. S e v a l ~ t / o n o f the ra~ by t ~ e I o f each s ~ ~ be idiotiC, o f Bo¢ ~ . 357-395. ~Ieyer, M. E. & Ca~mron, H. S. ( l ~ I b } . N.Ietabolic ~ a m c t e r l z a d o n o f the g ~ Brucella. IL. G ~ d v e me~Mlie ~ s o f the boa biot3T~, ffournal o f 82, 3 VV. J. B. (1963). ~ e on o f oaltu--r~ for l ~ i s b y ph~e,.,Voutnal of ~ , 437-445. •% I O ~ n , ~V. J. B. & ~ r b e l , ~i. J. (1976). R ~ t i ~ s for ~ e d e s ~ p t i o n of s p e c i ~ and b o f the g e n ~ . ta in Standardization 31~ 27-37. J. A . (1973). ~ e ~ of pol ~ i d e gel el reals in taxonomy of . ~Io~, ~1o~ for

J. A. & ~ r b e L ~ i . J. (1973). ~ o f a n ~ phage I ~ i c for ~. of 21, 5 3 ~ 5 4 4 . J.A., , M . J. & PF,illip, J. I. H . {1973) o f three p b ~ e s l ~ c . oj: 20, .

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J, C O R B E L

Miinter, V4. (t963) ~ r ~ tm.hm eh'~i~r B ~ c e l l a . . lattf~a, 16~e, u~ (1 Abt./'Orig.) 190, 348-352. 0 N . N . (1957). l~sprt~tr~-I brtlt~l ofaga ~ e d i k ~ ' t u r bt~ar~ i ~ posI In I I , pp. 8 ~ I 0 2 . M o s c o w : . A ~ d m i y a ~{edi~ins~Mkh SSSR. Os , ~ . N. & , T . A. (I968). ~ o f the a ~ r p t i o n o f b~ to ~Ats o f B r . and ~uis. t lcwii, E_pi&."m i Im 4k5, 79-83. (In R t t ~ , ) Stablefor*d~, A. XV. & J o n e s , L. M. (1963). R e ~ r t o f ~ e S on o f the genus . lnt ~ Ia~:ure a n d T a x o n o m y 13, 145-148. ~ , n G ~ . E. L . ~ ~ r M , / ~ I . J. (I 977). Isol~~atlort o f a l ~ e for s e v ~ s ~ , following p ~ o f Tbilisi in the p r ~ e n ~ o f tr'~ C. A of ~ , 259-261. Ve~hilova, P. A.~ Drano~'~kaya, E. A. & a.-~v, V. ~ t , (1972). ityel'nyy oprFed i ~ pfinadl fi bakteriy k r~Mu . t i Im 49, 98-101.

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