LISTERIA MONOCYTOGENES;
A REVIEW AND COLONIAL DISSOCIATION
By
M itc h e ll L* Gray
A THESIS
Subm itted to the School o f G raduate S tu d ie s o f M ichigan
S ta te C ollege o f A g ric u ltu re and A pplied S cien ce
in p a r t i a l f u l f i l l m e n t o f the re q u ire m e n ts
f o r th e degree o f
DOCTOR OP PHILOSOPHY
Departm ent o f B a c te rio lo g y and P u b lic H e alth
1954
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A f f e c tio n a te ly d e d ic a te d
to
N ell
ACKNOWLEDGEMENT
The t h e s i s perhaps i s th e most c o n crete evidence o f th e e a rn in g
o f a g ra d u a te d e g re e .
I t i s som ething one can p ic k up and hold and
even o c c a s io n a lly scan i t s p a g es.
and no em otion.
But i t i s a cold th in g o f hard f a c t s
Only th e a u th o r and th e few c lo s e ly a s s o c ia te d w ith
him see among th e c o ld f a c t s the high p in a c le s o f e l a t i o n and the d e ep ,
wide v a lle y s o f d isa p p o in tm e n t, and th e even deeper canyons o f d e s p a ir .
Many tim es th e re seemed no escape up th e ste e p and rocky canyon w a ll.
In th e s ile n c e and lo n e lin e s s o f discouragem ent one h eard o n ly the echo
in g ad m o nitio n o f Solomon —- " o f making many books th e re i s no end and
much stu d y i s a w e arin ess o f the fle s h "
---- o r th e cry o f Job ---- "would
t h a t my a d v e rsa ry had w r i t te n a book" ,
But f o r tu n a te ly th e re was a ls o
a lo u d e r , more com pelling c ry - not an echo - a command
dominion over th e e a r th and subdue it" .
"have
With an id e a l no w a ll i s too
s te e p , no ro ck too sh arp and one can alw ays rea ch the p l a t e a u .
p la te a u i s a h o t d e s e r t and s u r e ly no sto p p in g p la c e .
high m ountain top on the h o rizo n s t i l l f a r away.
But the
The g o al i s the
There i s more c lim b in g ,
more s l i p p in g , more s l i d i n g , a n o th e r b r u i s e j b u t a n o th e r view o f the
m ountain to p , and t h i s tim e a view perhaps obscured by th ic k storm clo u d s
o f a p p re h e n sio n .
But i t i s c lo s e r —- one more stream to f o r d , one
more v a lle y to c ro s s and th e n f i n a l l y - the to p .
And f a r below s tr e tc h e s
a l l th e d isa p p o in tm e n ts, a l l th e hopes, a l l the accom plishm ents, a l l
blended in a hazy film o f d is ta n c e .
But even th e m ountain to p i s cold
and windy and one can n ev er lin g e r lo n g , b u t must go back to p la te a u s
and v a lle y s and canyons.
There a re more m ountains to clim b and one must
clim b many i f he i s to have dominion over th e e a r th and subdue i t .
o f t h i s the a u th o r se e s between th e cold f a c t s on th e p r in te d p ag e.
A ll
To th e many who h elp ed me to rea ch the f i r s t m ountain top I want
to sa y " th a n k s " .
One couldn* t q u ic k ly f o r g e t the many k in d words o f
encouragem ent o f D r. H. J . S t a f s e th , m ajor p ro fe s s o r o f the guidance
com m ittee.
H is s o f t spoken v o ice alw ays conveyed s i n c e r i t y and was
n o t e a s i l y ig n o red o r f o r g o t te n .
D r. F rank Thorp, J r . because o f h is
more in tim a te a s s o c i a t io n w ith th e problem was in a b e t t e r p o s itio n to
a i d , and h is broad knowledge and p a s t accom plishm ents were a c o n tin u a l
source o f i n s p i r a t i o n .
Perhaps the in te n s e com pelling in flu e n c e o f
o th e r y e a rs has dimmed a b i t , b u t i t s memory s t i l l compels th o se who
were f o r t u n a t e t enough to e x p e rie n c e i t .
D r. C. A. Hoppert and D r. C.
F . Huffman by t h e i r accom plishm ents in t h e i r chosen f i e l d s p ro v id ed a
c o n tin u a l source o f i n s p i r a t i o n which was k e en ly f e l t in p e rs o n a l con
ta c ts .
There a re many o th e r s who deserve th a n k s t
the te c h n ic ia n s , Miss
S y lv ia Laine and Miss JoAnn S h riv e r who h elp ed w ith th e many blood counts
and fe rm e n ta tio n s tu d ie s ; M rs. A lu b o w ic z.refe ren c e l i b r a r i a n , f o r h e r
a s s is ta n c e in s e c u rin g th e many i n t e r - l i b r a r y lo an s? D r. R. A. R u n n e lls,
head o f th e D epartm ent o f Animal P a th o lo g y ; a l l my c o lle a g u e s in the
departm ent and th e M ichigan A g r ic u ltu r a l Experim ent S ta tio n who g ran te d
a leav e o f absence in o rd e r t h a t the work could be accom plished in a
s h o r te r tim e .
I w i l l alw ays be g r a t e f u l to N e ll f o r the many hours
she sp en t ty p in g the m a n u s c rip t.
And i t would be u n ju s t no t to m ention
th e th r e e dandy boys who n ever a u d ib ly p r o te s te d th e many hours they
l o s t w ith Dad.
One never can f o r g e t th e t i n y , spontaneous v o ice o f the
l i t t l e boy w ith hands fo ld e d a t th e d in n er ta b le - b le s s th e food ---and h e lp daddy w ith h i s t h e s is
M itc h e ll L . Gray
c a n d id ate f o r the degree o f
D octor o f P hilosophy
F in a l ex am in a tio n :
D is s e rta tio n :
1:00 p .m ., J u ly 13, 1954* Room 34^* G iltn e r H a ll
L i s t e r i a m onocytogenes:
A- Review and C o lo n ia l D is s o c ia tio n
O u tlin e o f S tu d ie s
M ajor s u b je c t:
Minor s u b j e c t s :
B a c te rio lo g y
Animal F athology and D airy N u tr itio n
B io g ra p h ic a l Item s
Born:
F eb ru ary 12, 1918* Grand R a p id s, M ichigan
U ndergraduate S tu d ie s : C a lv in C o lle g e , Grand R ap id s, M ichigan,
1937-1941. A .B ., 1941
G raduate S tu d ie s : M ichigan S ta te C o lle g e , E a st L an sin g , M ichigan
1945- 1954. M.S. i n B a c te rio lo g y , 1949
E x p erien c e: B a c te r io lo g is t a t M ichigan Departm ent o f H e a lth , Bureau
o f L a b o r a to r ie s , L an sin g , M ichigan, 1941-1943* F in a l in s p e c tio n
o f H am ilton S tandard V a ria b le P itc h H y d rau lic P r o p e l le r s , Nash
K e lv in a to r C o rp o ra tio n , L an sin g , M ichigan, 1943-1945; A s s is ta n t
in R esearch ( I n s t r u c t o r ) Department o f Animal P a th o lo g y ,
M ichigan A g r ic u ltu r a l Experim ent S t a t i o n , E a s t L an sin g , Mich
ig a n , 1945-1954.
Member o f S o c ie ty o f th e Signa X i; S o c iety o f American B a c te r io lo g is t
M ichigan B ranch, S o c ie ty o f American B a c t e r i o l o g is ts .
Table o f C ontents
Page
I n tr o d u c tio n
PART I .
I
REVIEW
Review
1
Table sum m arizing
i s o l a t i o n s from sheep
28
Table sum m arizing
i s o l a t i o n s from g o a ts
30
T able sum m arizing
i s o l a t i o n s from cows
31
Table sum m arizing
i s o l a t i o n s from a b o rtio n
33
Table sum m arizing
i s o l a t i o n s from chickens
35
Table sum m arizing
i s o l a t i o n s from man
3&
B ib lio g ra p h y
PART I I .
41
COLONIAL DISSOCIATION
In tro d u c tio n
55
M a te ria ls and Methods
57
R e s u lts
64
I l l u s t r a t i o n s o f c o lo n ie s
67
D isc u ssio n
98
D is s o c ia tio n Scheme
1C6
Summary
115
B ib lio g ra p h y
117
I n tr o d u c tio n
Almost th r e e decades have p assed s in c e th e f i r s t d e s c r ip tio n o f
th e b acteriu m known a s L i s t e r i a m onocytogenes.
The p a s s in g o f tw enty
e ig h t y e a rs has added l i t t l e to o u r knowledge o f e i t h e r the b acteriu m o r
to th e h o st o f d is e a s e s w ith which i t has been a s s o c ia te d .
i s th o u g h t to be o f l i t t l e o r no economic im p o rtan ce.
To many i t
I t i s o f te n r e l e g a t
ed to th e c la s s o f la b o ra to ry c u r i o s i t i e s , a b a cteriu m t h a t can produce
a h ig h c i r c u l a t i n g m onocytosis in th e p e rip h e r a l blood o r a marked p u ru
l e n t c o n ju n c ti v i ti s in a s u s c e p tib le la b o ra to ry a n im a l.
But to th o se
who have s tu d ie d i t c lo s e ly i t i s a v ic io u s k i l l e r , and the few who s u r
v iv e i t s a tt a c k a re u s u a lly l e f t w ith perm anent p h y s ic a l o r m en tal d e
fe c ts .
The c o n s ta n tly m ounting evidence su g g e st t h a t t h i s b a cteriu m i s
a c t u a l l y w idely d i s t r i b u t e d in n a tu re and th a t a l l warm blooded anim als
and man a re s u s c e p tib le h o s ts ; in d e e d , a l l may be c a r r i e r s .
S t i l l un
known a re th e f a c t o r s which d i c t a t e t h a t in c e r t a i n s p e c ie s in f e c ti o n r e
s u l t s in e n c e p h a l i t i s , w hile in o th e r s in f e c tio n i s m a n ife ste d by s e p t i
cem ia, o r a b o r tio n , c o n ju n c ti v i ti s o r some o th e r d i s o r d e r .
S e v e ra l f a c t o r s c o n trib u te to th e la c k o f knowledge o f the genus
L is te ria .
These a re an unew areness o f the b acteriu m which o f te n r e s u l t s
in i t s b e in g d isc a rd e d a s a c o n tam in atin g d ip h th e ro id ; the d i f f i c u l t y o f
i s o l a t i n g i t from c e r t a i n anim al t i s s u e s w ith th e consequence t h a t th e
c u ltu r e i s re p o rte d a s n e g a tiv e ; th e m isco n cep tio n t h a t in f e c ti o n w ith
L . monocytogenes i s r a r e ; the i n a b i l i t y to reproduce in th e la b o ra to ry
some o f th e d is e a s e s w ith which i t has been a s s o c ia te d and th e f a i l u r e
to d eterm ine i t s n a tu r a l r e s e r v o i r .
II
I t was f e l t t h a t a through rev iew o f the w o r ld 's l i t e r a t u r e d e a l
in g w ith i n f e c ti o n s w ith L . monocytogenes m ight r e v e a l tre n d s o r c lu es
t h a t would a id in th e s o lu tio n o f the many unsolved problem s su rro u n d
in g th e epidem iology and p a th o g e n e sis o f th e many forms of l i s t e r i o s i s .
I t was a ls o f e l t t h a t a stu d y o f th e c o lo n ia l d is s o c ia tio n o f L. mono
cytogenes m ight le a d to the re c o g n itio n o f a d d itio n a l c o lo n ia l form s;
form s t h a t c o n c e iv a b ly m ight be found in n a tu re o r in in a p p a re n t in f e c
t i o n s , and th u s a id in th e se a rc h to e s t a b l i s h b o th the n a tu r a l r e s e r v o i r
and th e mode o f tra n s m iss io n from h o st to h o s t.
th e r e s u l t s u n fo ld on the fo llo w in g p a g e s.
These were th e g o a ls —-
PART I
REVIEW
REVIEW
L i s t e r i o s i s i s one o f th e most r e c e n tly d e sc rib e d and l e a s t u n d er
sto o d o f a l l th e b a c t e r i a l in f e c tio n s o f man, h is dom estic a n im a ls, and
th e w ild l i f e which sh a re h is c i t i e s and fa rm s.
H u lp h ers, a Swedish work
e r , a p p e a rs to be th e f i r s t to re c o rd an en co u n ter w ith th e c a u s a tiv e
a g e n t, L i s t e r i a m onocytogenes.
In 1911 he i s o l a t e d a b acteriu m from th e
n e c r o tic l i v e r o f a r a b b i t , and h is d e s c r ip tio n o f th e org an ism , p u b lis h
ed in Svensk V e t e r i n a r t i d s s k r i f t . co rresp o n d s v e ry c lo s e ly to t h a t o f
L. m onocytogenes.
Wo doubt th e sm all epidem ic o f m e n in g itis re p o rte d by
A tk in so n in 1917 a ls o was due to t h i s b a c te riu m .
The case re p o rte d by
Dumont and C otoni in 1921 was confirm ed by C otoni in 191*2 a s due to L.
m onocytogenes.
These r e p o r ts in d ic a te t h a t in f e c tio n s w ith members o f
t h i s genus were known p r i o r to i t s f i r s t d e s c r ip tio n in 1926 by M urray,
Webb and Swann.
They i s o l a t e d th e b acteriu m from th e l i v e r o f r a b b i t s
and g u in ea p ig s and named i t B acterium m onocytogenes.
The fo llo w in g y e a r
P i r i e ( I 927) re p o rte d i s o l a t i o n from th e l i v e r o f th e A fric a n jumping
mouse (T a te ra lo b e n g u la e ) o f a b acteriu m which he named L i s t e r e l l a he pa to ly tic a .
The f i r s t rec o g n iz e d a s s o c ia tio n o f t h i s b a cteriu m w ith i n f e c ti o n
in man was in 1929 when N yfeld i n Denmark i s o l a t e d i t from th e blood o f
p a t i e n t s w ith in f e c tio u s m o n o n u cleo sis.
Matthews (1928) re p o r te d an o u tb re a k o f e n c e p h a litis o f unknown
e tio lo g y i n c a t t l e which v e ry l i k e l y was l i s t e r i o s i s .
But th e f i r s t r e
p o rte d i s o l a t i o n o f L. monocytogenes from a dom estic anim al was by G ill
(1931) i n New Z ealand.
He is o l a t e d th e m icrobe from th e b r a in o f sheep
and e s ta b lis h e d th e r e l a t i o n o f t h i s m icroorganism to th e e tio lo g y o f
-
2
-
" c i r c l i n g d is e a s e " * a name s t i l l o f te n a p p lie d e rro n e o u sly to l i s t e r i o s i s *
P le tn o v a and S tik so v e (1950) c laim i t s i s o l a t i o n from p ig s in th e S o v ie t
Union a s e a r ly a s 1924*
In r e c e n t y e a rs i n f e c tio n s w ith t h i s b acteriu m have become so wide
s p re a d t h a t today i t i s co n sid ere d to be the e le v e n th in economic im portance
among th e d is e a s e s o f dom estic a n im a ls.
I t s p ro p e r p la c e in human m edicine
has n ot y e t been d eterm in ed , b u t w ith an in c re a s e d aw areness o f the
d ise a se * and th e m ounting number o f case r e p o r t s , i t may e v e n tu a lly be
o f f a r g r e a te r s ig n if ic a n c e th a n p re v io u s ly su sp ected *
The r o l e o f w ild
l i f e in th e sp re ad o f th e i n f e c ti o n i s s t i l l to be d e te rm in e d , b u t judg
in g from th e numerous sp e c ie s from which i t has been i s o l a t e d , i t would
a p p e a r t h a t i t may have an im p o rtan t p a r t .
I f man i s to p r o te c t h im s e lf ,
and a ls o h is do m estic anim als and w ild l i f e from t h i s i n f e c tio n which
v e ry o f te n k i l l s o r le a v e s i t s v ic tim s p h y s ic a l o r m en tal c r i p p l e s , i t
i s im p e rativ e t h a t a com plete stu d y be made to determ ine e f f e c t i v e methods
o f d ia g n o s is , mode o f tra n s m is s io n , m ethods o f tre a tm e n t and a system
o f c o n tro l*
U n til 1940 th e r e was c o n s id e ra b le co n fu sio n in th e nom enclature o f
t h i s b actd riu m due to th e in te rc h a n g e o f the g e n e ric names L i s t e r e l l a
and L i s t e r i a *
P i r i e ( 1927 ) chose L i s t e r e l l a a s th e g e n e ric name i n honor
o f Lord L is te r * th e w ell known p io n e e r in th e f i e l d o f b a c te r io lo g y .
However, t h i s name had a lre a d y been a p p lie d to a group o f slim e molds
(M ycetozoaire) by Jahn ( 1906) .
B acterium a s a p p lie d by Murray e_t a l*
( 1927) was u n d e s ira b le because th e m icrobe d id not p o ss e s s th e c h a r a c te r
i s t i c s o f t h i s g en u s.
In 193& M esnil proposed t h a t in a l l d u p lic a tio n
o f g e n e ric names, o n ly th e one f i r s t a p p lie d should be c o n sid ered v a l i d .
-
3
-
T h is r e s o lu tio n was ad ap ted by th e comm ittee on nom enclature o f the
T h ird I n t e r n a ti o n a l Congress f o r m ic ro b io lo g y , New York, 1939* (P re v o t,
1944)*
T his in v a lid a te d th e g e n e ric name proposed by P i r i e and in 1940
he proposed th e name L i s t e r i a , which was adopted in th e s ix th e d i t i o n o f
Bergev* s Manual o f D e term in a tiv e B a c te rio lo g y (1 9 4 8 ), and i s now the
o f f i c i a l g e n e ric name.
W ilson and M iles (1948) i n ik e t h i r d e d it i o n o f
Topley and W ilson1s P r in c ip le s o f B a c te rio lo g y and Im munity, employed th e
g e n e ric name E r y s ip e lo th r ix .
T his was based on th e work o f B arber (1939)
who showed t h a t th e re were s t r i k i n g s i m i l a r i t i e s between L i s t e r i a and
E r y s ip e lo th r ix .
I t i s hoped t h a t th e s e a u th o rs w i l l confoim to th e newer
nom enclature in t h e i r n ex t e d i t i o n .
The sp e c ie s name monocytogenes i s
based on th e f a c t t h a t la rg e numbers o f monocytes a re o f te n found i n th e
p e r ip h e r a l blood o f b o th n a tu r a ll y and a r t i f i c i a l l y in f e c te d a n im a ls.
O ther synonyms in a d d itio n to th o se a lre a d y m entioned a r e :
B acterium
h e p a tis (H u lp h ers, 1911); L i s t e r e l l a monocytogenes hom inis (N y fe ld t,
1932) C orynebacterium parvulum (SchtjElz e t a l . , 1934) C ornybacterium
in f a n tis e p tic u m (D o te l, 195°) and L i s t e r i a i n f a n t i s e p t i c a ( P o t e l, 1952) .
L i s t e r i a monocytogenes i s a sm all gram p o s i t i v e , non-spore form ing
d ip h th e r o id - lik e r o d .
I t i s v ery e a s i l y confused w ith members o f th e
genus C orynebacterium . and no doubt o f te n has been d isc a rd e d a s a " con
ta m in a tin g d ip h th e r o id " •
S e v e ra l r e p o r ts in th e l i t e r a t u r e b e a r t h i s o u t
(A tk in so n , 1917t T e s d a l, 1934; M a rc e llu s e t a l . . 193&? Kennedy, 1947;
P o t e l, 1 9 50).
I t i s e x trem ely r e s i s t a n t to h e a t (Ozgen, 195I» Zink e t a l . .
1951) and d e s s i c a t io n , b u t i s k i l l e d by most o f th e common d i s i n f e c t a n t s
(K aboyashi, 1951)#
m ed ia.
I t i s e a s i l y c u ltiv a te d on th e common b a c t e r i a l
D ifco* tr y p to s e a g a r a f f o r d s an e x c e lle n t s u b s tr a te f o r growth
*Difco L a b o r a to r ie s , I n c . , D e t r o i t , Mich.
-
and p r e s e r v a tio n o f c u ltu re s *
4
-
I t i s a e ro b ic to m ic ro a e ro p h ilic and when
grown a t room tem p era tu re e x h ib i t s a p e c u lia r tum bling m o ti l it y which
u s u a lly i s a b s e n t when grown a t 37°
On blood a g a r i t i s b e ta hem o ly tic
and i s n o t known to produce e i t h e r en d o - o r ex o to x in s*
In tra v e n o u s i n
o c u la tio n o f r a b b i t s , guinea p ig s o r mice s tim u la te s th e fo rm a tio n o f a
h ig h p e rc en ta g e o f c ir c u l a t i n g monocytes*
Most s t r a i n s produce a c h a r a c te r
i s t i c c o n ju n c ti v i ti s and k e r a t i t i s when i n s t i l l e d in to th e c o n ju n c tiv a l
sa c o f th e ra b b it*
The m onocytosis and c o n ju n c ti v i ti s o f te n a re used f o r
q u ick i d e n t i f i c a t i o n o f s u s p e c t-c u ltu r e s *
The v a rio u s forms o f l i s t e r i o s i s a re w idely d i s t r i b u t e d and have been
re p o rte d from a l l c o n tin e n ts*
The d is e a s e i s m ost commonly found i n th e
tem p erate zone and has not been re p o rte d from th e tr o p ic s *
In th e U nited
S ta te s i t has been re p o rte d in a l l b u t a few so u th e rn s t a t e s .
i t has been r e p o rte d a s f a r n o rth a s th e Hudson Bay c o u n try .
cows, and g o a ts a p p ea r to be th e most s u s c e p tib le s p e c ie s .
I n Canada
S heep,
Man, c h ic k e n s,
r o d e n ts , p ig s , h o rse s and dogs fo llo w in t h a t o r d e r .
The epidem iology o f l i s t e r i o s i s in bo th an im als and man i s o b sc u re ,
and th e n a tu r a l r e s e r v o i r o f L* monocytogenes has n ev er been determ ined*
However, th e r e a re s tro n g in d ic a tio n s t h a t r o d e n ts , swine o r fo w ls may
p la y a p a r t i n tra n s m is s io n .
The r o l e o f i n s e c t s and p a r a s i t e s o f te n
has been s u g g e s te d , b u t s u p p o rtin g evidence i s la c k in g .
I t a ls o may be
t h a t t h i s b acteriu m i s w idely d i s t r i b u t e d in n a tu r e , perhaps in th e form
o f a s o i l b a cteriu m o r a s a sa p ro p h y te and becomes a pathogen o n ly when
th e h o s t i s su b je c te d to c e r t a i n p h y s ic a l o r p h y s io lo g ic a l s tr e s s *
L i s t e r i o s i s in ru m in an ts i s m ost p re v a le n t betw een November and A p ril
and seldom o c c u rs d u rin g th e summer.
The o n ly e x c e p tio n to t h i s i s th e
-5
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r e p o r t o f Khalimbekov (1952) who found t h a t in A z e rb a ija n the d is e a s e
i s m ost p re v a le n t d u rin g th e h o t summer months*
Records k e p t a t th e
M ichigan A g r ic u ltu r a l Experim ent S ta tio n in d ic a te t h a t th e number o f
o u tb re a k s in c re a s e s two to fo u r days fo llo w in g sudden d ro p s in tem pera
t u r e o r heavy snow f a l l s , s u g g e stin g t h a t c lim a te may p la y a p a r t .
U su al
ly o u tb re a k s f a l l o f f r a p id ly when an im als have a c c e s s to green p a s t u r e .
The fe e d in g o f e n s ila g e was su g g ested by O lafson (194°) a s a p re d is p o s in g
f a c t o r in s u s c e p t i b i l i t y .
However, in an i n v e s ti g a t i o n o f t h i s , Olson
e t a l . (1953) could draw no d e f i n i t e co n clu sio n s*
The p o s s i b i l i t y o f a
v ir u s as a " tr ig g e r " mechanism has been su g g ested b u t not confirm ed
(O lson e t a l . . 1951; S e e lig e r and Leinew eber, 1952; Geurden and D evor,
1954)#
The mode o f e n tr y in th e h o s t in a l l forms o f th e d is e a s e i s o b sc u re .
E n t e r i t i s o f te n i s found in e n c e p h a litic l i s t e r i o s i s , b u t i t i s u n lik e ly
t h a t food o r w a ter can be in c rim in a te d .
With th e e x c e p tio n o f m ic e , i t
i s alm ost im p o ssib le to i n f e c t a r t i f i c i a l l y an anim al by th e o r a l r o u te .
The o f te n p re s e n t r h i n i t i s su p p o rts th e p o s s i b i l i t y o f e n tr y through th e
upper r e s p i r a t o r y t r a c t .
P a lla s k e ( 1940)
"two o c ca sio n s s u c c e s s f u lly
i s o l a t e d L. m onocytogenes f rom th e n a s a l exudate o f ewes w ith l i s t e r i o s i s .
The c o n ju n c ti v i ti s o f te n found in l i s t e r i o s i s su g g e sts t h a t the eye may
p la y a p a r t in tra n s m is s io n (Van D r i e s t , 194®* S lo o f f , 1948; Beute e t a l , .
1948t Gray e t a l . . 1 951).
Graham e t a l . (1943) a a d Gray e t a l . (1948)
each re p o rte d one in s ta n c e where th e e n c e p h a litic form o f th e d is e a s e
developed in a p ig and r a b b i t r e s p e c tiv e ly , fo llo w in g o c u la r i n s t i l l a t i o n .
A sahi and Hosoda (1952) s u c c e s s f u lly produced symptoms and le s io n s o f
l i s t e r i c e n c e p h a l it i s in both g o a ts and r a b b i t s fo llo w in g o c u la r i n s t i l -
-
l a t i o n o f c u ltu re *
6
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Also p o s tu la te d i s e n tr y through s k in b rea k s and
s u r v iv a l in th e h o st u n t i l s t r e s s c r e a te s an environm ent fa v o ra b le f o r
th e d is e a s e ( S e e lig e r e t a l . , 1952)*
E v e le th et, a l . ( 1953) found t h a t when h e a lth y sheep a re im ported
from in f e c te d f lo c k s in to non in f e c te d flo c k s th e d is e a s e o f te n ap p ears
in th e new f lo c k a lth o u g h i t
observed#
may be s e v e ra l months b e fo re symptoms a re
Gray e t a l * (1951) re p o rte d t h a t cows from th e same so u th w e st
e rn fe e d in g ran g es developed th e d is e a s e a t about th e same tim e when
in tro d u c e d in to n o rth e rn fee d l o t s , even though th e l o t s were w idely
s e p a ra te d from each o t h e r , b u t s u b je c t to the same c lim a tic c o n d itio n s*
These fin d in g s s tr o n g ly su g g e st t h a t some anim als may be non c l i n i c a l
c a r r i e r s , and one sh o u ld use c a u tio n when in tro d u c in g new anim als in to
a f lo c k o r h e rd .
F u rth e r su p p o rt f o r t h i s i s th e f a c t t h a t many dom estic
an im als d is p la y h ig h a g g lu tin a tin g t i t e r s f o r th e b a cteriu m (B e lin , 194&»
Graham e t a l * . 1943? H ira to et, a l * , 1954? P o t e l, 1954)*
I t i s r e l a t i v e l y e asy to i s o l a t e JL. monocytogenes from th e l i v e r
o r o th e r organs in th e se p tic em ic o r a b o rtiv e form o f th e d is e a s e , p r o
v id ed a f a i r l y la rg e inoculum i s u se d , a s i t h as been found t h a t th e
number o f organism s p r e s e n t may be sm all*
For an u n e x p la in e d re a s o n ,
i t i s d i f f i c u l t to i s o l a t e th e b acteriu m from th e blood o f both n a t u r a l
ly and a r t i f i c i a l l y in f e c te d anim als*
T his t is s u e sho u ld n o t be used
f o r i s o l a t i o n a tte m p ts i f o th e r org an s a re a v a ila b le *
In a tte m p tin g to i s o l a t e L* mo no cy togene s from the e n c e p h a litic
fozm i t i s e s s e n t i a l t h a t c u ltu r e s a re p rep ared from th e m edulla oblon g ata*
S p encer a t a l * (1944)* Olson a t a l * (1953a )»
H ira to et^ a l* (1954)
-
7
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found th e number o f organism s in t h i s p o r tio n o f th e b r a in to be f a r
g r e a te r th a n i n any o th e r a r e a .
The method o f i s o l a t i o n found to be
m ost s u c c e s s fu l i s a m o d ific a tio n o f a te c h n iq u e d e s c rib e d by B ie s te r
and Schw arts (1939) and c o n s is ts i n a s e p t i c a l l y m a c era tin g a f a i r l y
la rg e p o r tio n o f th e m edulla i n a s t e r i l e m o rta r o r Waring b len d o r to
which has been added 10 to 15 ml s t e r i l e d i s t i l l e d w a ter o r broth*
A pproxim ately 0*2 ml o f th e r e s u l t i n g su sp en sio n i s p la te d on a try p to s e
a g a r p l a t e and in cu b a te d 18-24 h r a t e i t h e r 37° C o r a t room te m p e ra tu re .
With t h i s tech n iq u e i t i s r e l a t i v e l y easy to i s o l a t e th e m icroorganism
from sheep o r g o a ts , b u t o f t e n c u ltu r e s p re p a re d in t h i s manner from
th e bovine rem ain s t e r i l e *
In t h i s ev en t th e p rep ared m edulla su sp en sio n
should be r e f r i g e r a t e d f o r s e v e ra l days o r weeks and r e p l a t e d .
With th e
employment o f t h i s te c h n iq u e , Gray e t a l . (1951) were a b le to dem onstrate
th e b acteriu m i n la r g e numbers in a l l in s ta n c e s where th e d is e a s e was
su sp ec ted and where t is s u e s e c tio n s re v e a le d le s io n s c h a r a c t e r i s t i c o f
lis te rio s is *
These f in d in g s have been eonfiim ed by a number o f i n v e s t i
g a to rs (B ein , 1931* Van den Schaaf e t a l . , 1931* Zink ejb a l . , 1951?
S e e lig e r and L inzenm eier, 1933? Geunden and D evor, 1954)*
Th© number
o f c o lo n ie s which develop in i n i t i a l c u ltu r e s from th e bovine i s alw ays
sm all compared to th o se o f th e i n i t i a l i s o l a t i o n from th e ovine or
c a p r in e .
Gray e t a l . ( 1948) su g g ested th a t t h i s phenomenon may be a s s o c i
a te d w ith a b a c t e r i o s t a t i c f a c t o r in th e bovine b ra in which may account
f o r th e g r e a te r r e s i s t a n c e to th e d is e a s e d isp la y e d by cows.
T issu e
s e c tio n s r e v e a l th e b a c t e r i a to be in c a rc e r a te d in th e f o c a l le s io n s
and o f te n i n t r a c e l l u l a r l y in th e m acrophages and g l i a l c e lls *
T his
makes i t e s s e n t i a l t h a t th e m edulla be ground*
I k lla s k e (1 9 4 0 ), B e lin (1943) ©ad Olson e t a l . (1953) re p o rte d
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8
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th e s u c c e s s f u l i s o l a t i o n o f L. monocytogenes from th e l i v e r and sp leen
o f sheep w ith e n c e p h a litic l i s t e r i o s i s .
developed was alw ays s m a ll.
c a ttle .
The number o f c o lo n ie s which
No s im ila r f in d in g s a re re p o rte d f o r
E xcept i n man, antem ortem i s o l a t i o n i s u n re p o rte d .
Olson a t a l . (19531s) compared th e e f f ic a c y o f v a rio u s i s o l a t i o n
te c h n iq u e s i n a tte m p ts to i s o l a t e L. mono cytogenes from th e b r a in and
o th e r o rg an s o f bo th n a tu r a ll y and a r t i f i c i a l l y in f e c te d sh eep .
These
te c h n iq u e s in c lu d e d d u p lic a te c u ltu r e s from each o rg a n , in o c u la tio n o f
m ice w ith su sp en sio n s o f su sp ected t i s s u e , sto ra g e o f ground t is s u e in
g ly c e r in e , c e n tr if u g a tio n and s e le c tiv e m edia.
The b e s t r e s u l t s were
o b ta in e d by g rin d in g d u p lic a te sam ples o f f r e s h t i s s u e .
With t h i s
tech n iq u e i n a n o th e r stu d y Olson et. a l . ( 1953s1) s u c c e s s f u lly is o l a t e d
L. monocytogenes from th e b r a in o f 94% o f th e sheep b r a in s c u ltu r e d .
U n fo rtu n a te ly th ey d id n o t in c lu d e in t h e i r stu d y th e r e f r i g e r a t i o n
tec h n iq u e d e sc rib e d in th e p rec ed in g p arag rap h s which gave 100% i s o l a
t io n s from sheep b ra in s which showed p e r iv a s c u la r c u ffin g and f o c a l
n e c ro s is c h a r a c t e r i s t i c o f l i s t e r i c e n c e p h a l it i s .
(Gray e t a l . , 1951).
I n r e s p e c t to th e potassium t e l l u r i t e medium d e sc rib e d by Gray et. a l .
(1 9 5 0 ), Olson e t a l • (1953) found in h ib i t io n o f many in d iv id u a l s t r a i n s .
T his f in d in g has been confirm ed in unpublished s tu d ie s and l i m i t s to
a marked d eg ree i t s use in i s o l a t i o n a tte m p ts .
The in o c u la tio n o f r a b b i t s o r Guinea p ig s w ith tis s u e su sp en sio n s
o r body f l u i d s su sp ec ted o f h a rb o rin g L. monocytogenes has been em
ployed w ith su c c e ss in th e S o v ie t U nion.
With t h i s tech n iq u e Gudkowa
and S a c h a ro ff ( 1946) and B i l i b i n (1949) dem onstrated th e p resen c e o f
L. monocytogenes in th e blood o f p a ti e n t s w ith an in f e c tio u s mono
n u c le o s is - lik e syndrome and who showed r a th e r h ig h a g g lu tin a tin g a n t i
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9
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body t i t e r s a g a in s t th e s p e c i f i c b a c te riu m .
In view o f th e e x is ti n g
d i f f i c u l t y in s u c c e s s f u lly i s o l a t i n g L . monocytogenes from b lo o d , th e r e
i s a need f o r w id er a p p lic a tio n o f t h i s tech n iq u e in b o th human and
v e te r in a r y m e d ic in e .
At p re s e n t e n c e p h a litis in rum inants i s th e form o f l i s t e r i o s i s
m ost f r e q u e n tly rec o g n ize d and i s o f g r e a te s t economic im p o rtan ce.
I t g e n e r a lly o c c u rs d u rin g the p e rio d from l a t e November to th e f i r s t
o f May, and i s m ost p re v a le n t d u rin g F ebruary and M arch.
I t may a f f e c t
a n im als o f a l l a g e s a lth o u g h th e re a re in d ic a tio n s t h a t th e young a r e
more s u s c e p tib le th a n o ld e r a n im a ls .
E n c e p h a litis w ith o u t se p tic e m ia
i s unknown b e fo re th e rumen i s f u n c tio n a l.
I t may a f f e c t b o th sexes
b u t th e re i s evidence t h a t p reg n a n t ewes a re in f e c te d more f r e q u e n tly
th a n n o n -p re g n a n t.
d a ir y c a t t l e .
Beef c a t t l e a p p e a r to be more s u s c e p tib le th a n
The h ig h e r in c id e n c e in b eef c a t t l e may be e x p la in e d by
th e f a c t t h a t g e n e ra lly th ey a re exposed to a more v ig o ro u s w in te r
en v iro n m ent.
In sheep and g o a ts th e d is e a s e i s extrem ely a c u te and d e a th may
o ccu r in fo u r to 48 k r a f t e r appearance o f symptoms.
s e v e r a l days*
Some su rv iv e
Recovery i s r a r e in sheep and g o a ts t h a t show d e f i n i t e
symptoms o f i n f e c t i o n .
M o rta lity may range from th r e e to 30% o r m ore.
R ecen tly a t t e n t i o n has been fo cu sed on a more c h ro n ic n o n - f a ta l form o f
e n c e p h a litis in sh e e p .
more ty p ic a l fo rm .
This u s u a lly o ccu rs sim u lta n e o u sly w ith th e
Many o f th e s e anim als a re s a id to re c o v e r, b u t u n t i l
a b e t t e r antem ortem d ia g n o s is i s developed i t can n o t be s t a te d d e f i n i t e
l y w hether th e s e sheep have had l i s t e r i o s i s o r n o t.
I f they a re a c t u a l
ly in f e c te d w ith L. m onocytogenes. th e re i s th e p o s s i b i l i t y t h a t th ey
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10
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may a c t a s c a r r i e r s f o r long p e rio d s o f tim e .
needed to c o n f im o r deny t h i s h y p o th e s is .
F u rth e r re s e a rc h i s
For th e p re s e n t i t a p p ea rs
t h a t such rec o v e re d anim als should be e lim in a te d from th e flo c k a s q u ic k
ly a s p o s s ib l e .
In c a t t l e th e d is e a s e i s more c h ro n ic in n a tu re and most cows s u r
v iv e from fo u r to 14 d ay s.
Pounden e t a l . ( 194? ) re p o rte d one o f th e
few a c u te o u tb re a k s in c a t t l e in which d e a th s were sudden and a h ig h
p e rc e n ta g e o f th e herd was a f f e c t e d .
10% o f a herd i s a f f e c t e d .
U sually no more th an e ig h t to
Spontaneous re c o v ery may o f te n be observed
i n c a t t l e ( B ie s te r and S chw arte, 1941? Jensen and la c k e y , 1949)* b u t
many o f th e s e a n im a ls , and a ls o th o se which respond to th e ra p y , show
ev id en ce o f perm anent b r a in damage.
These anim als a r e u n s ig h tly and
unable to compete w ith the r e s t o f th e h e rd .
T his p re s e n ts no p a r t i c u l a r
problem in e i t h e r management o r p u b lic h e a lth in b e e f c a t t l e u n le s s
th e y co n tin u e a s c a r r i e r s , which i s n o t y e t e s t a b l is h e d , b u t does have
e s t h e t i c d isa d v a n ta g e s in d a iry h e rd s .
The symptoms in sh eep , g o a ts and c a t t l e a re s i m ila r and d i f f e r o n ly
in s e v e r i t y .
At th e o n se t th e in f e c te d anim al u s u a lly s e p a r a te s i t s e l f
from th e r e s t o f th e h e rd .
e n t to s u rro u n d in g s.
I t a p p ea rs d e p re sse d , confused and i n d i f f e r
Then fo llo w s in c o o rd in a tio n and t o r t i c o l l i s .
O ften l o c a l p a r a l y s i s o f th e f a c i a l and th r o a t m uscles i n t e r f e r e s w ith
sw allow ing and r e s u l t s in marked s a l iv a ti o n and d ro oping o f one o r bo th
e a rs.
I n t e r m it t e n t tw itc h in g o f th e s e m uscles i s o f te n o b se rv e d , b u t
fr a n k c o n v u lsio n s a re r a r e .
There i s o fte n s tra b is m u s , c o n ju n c ti v i ti s
and the anim al may appear b l i n d .
A marked n a s a l d is c h a rg e , a n o re x ia and
a tem p eratu re o f a s much a s 108° F o r more a re common.
In th e e a r ly
11
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s ta g e s th e anim al te n d s to crowd in to a c o rn e r o r le a n a g a in s t s t a ti o n a r y
o b je c ts a s i f unable to sta n d u n su p p o rte d .
I f th e anim al walks i t o f te n
moves in a c i r c l e , and i f i t c i r c l e s , i t i s alw ays in th e same d i r e c
tio n .
Only a sm all p e rc e n ta g e o f th e an im als w ith l i s t e r i c e n c e p h a litis
show t h i s symptom.
In th e te rm in a l s ta g e s th e a n imal f a l l s and can n o t
g e t up u n a s s is te d .
When
i t i s down th e r e
a im le s s ru n n in g m o tio n s.
The h e ad , n eck ,
a r e g e n e ra lly in v o lu n ta ry and
and f o re le g s u s u a lly a re
more te n s e th an th e p o s t e r io r p a r t o f th e body.
seen ex ce p t o c c a s io n a lly
V isc io u sn e ss i s n o t
in c a t t l e . I t i s u n lik e ly t h a t a l l th ese
symptoms would ap p ear in any one anim al*
Many cows show o n ly an i n a b i l i t y
to sw allow due to p a r a ly s is o f f a c i a l and th r o a t m u sc le s.
Antemortem d ia g n o s is o f l i s t e r i o s i s i s v i r t u a l l y im p o ssib le as
th e re i s no s a t i s f a c t o r y d ia g n o s tic t e s t .
L i s t e r i o s i s can be confirm ed
o n ly by i s o l a t i o n and i d e n t i f i c a t i o n o f L* m onocytogenes.
Some p ro g re s s
i s b e in g made toward d e v elo p in g a s e r o lo g ic a l t e s t , e s p e c ia lly in human
m e d ic in e , in Germany ( S e e lig e r , 1953)*
The blood p ic tu r e u s u a lly shows
no v a r i a ti o n from normal and th e p e r ip h e r a l m onocytosis o f te n found in
man, r o d e n ts , and a r t i f i c i a l in f e c tio n s seldom i s seen in in f e c te d
rum inants*
In man L. monocytogenes has been i s o l a t e d on numerous o c c a sio n s by
means o f s p in a l p u n c tu re .
T his technique a p p lie d to sheep and c a t t l e
has th u s f a r r e s u l te d in f a i l u r e .
I t has o f te n been dem onstrated t h a t c l i n i c a l symptoms a lo n e a re
n o t s a t i s f a c t o r y a s a d ia g n o s tic c r i t e r i o n .
Subsequent d e a th s fo llo w
in g s im ila r symptoms in th e same herd a f t e r p o s itiv e d ia g n o s is may be
presumed to be l i s t e r i o s i s .
However, even t h i s o f te n proves u n r e lia b le
- 12 -
p a r t i c u l a r l y i n sheep f lo c k s where l i s t e r i o s i s , en tero to x em ia and k e to s is
may o ccu r sim u lta n e o u sly and he d is tin g u is h e d o n ly a t necropsy*
In
l i s t e r i o s i s th e r e a re no d i s tin g u is h in g le s io n s such a s th e f a t t y l i v e r
in k e to s is and th e c h a r a c t e r i s t i c hem orrhages o f enterotoxem ia*
Acetone
may be found in u rin e o f an im als w ith l i s t e r i o s i s a s in k e to s is *
L i s t e r i o s i s in c a t t l e may o f te n be confused w ith r a b i e s , p o iso n in g ,
a c u te g a s t r o e n t e r i t i s , k e t o s i s , A u je sk y 's d is e a s e , o r v i r a l e n c e p h a l it i s .
The e n c e p h a litic f o m o f l i s t e r i o s i s o c c a s io n a lly may o c cu r in
swine*
I t has been re p o rte d by B ie s te r and Schwarte (1 9 4 °)• E v e le th
e t a l* (1953®) and in an a ty p ic a l form by Helmboldt ejt a l . (1951)*
In
m ost in s ta n c e s when p ig s show symptoms su g g e stiv e o f l i s t e r i o s i s c u ltu r e s
p rep a red from th e b ra in rem ain s t e r i l e even a f t e r long p e rio d s o f r e
frig e ra tio n .
The le s io n s found in th e m edulla o b lo n g ata o f th e s e p ig s
d i f f e r from th e u s u a l ones c h a r a c te r iz in g l i s t e r i o s i s , and more c lo s e ly
resem ble th o se o f one o f th e v i r a l i n f e c ti o n s .
I t i s v e ry p o s s ib le
t h a t th e se symptoms and le s io n s in d ic a te a n e u ra l m a n ife s ta tio n o f hog
c h o le r a •
U su ally th e re a re no d e te c ta b le g ro ss le s io n s in anim als t h a t d ie
w ith e n c e p h a litic l i s t e r i o s i s .
R arely s l i g h t c lo u d in g o f th e m eninges
o r p in - p o in t g re y is h w hite f o c i may be o b se rv e d .
There may be s l i g h t
c o n g e stio n o f th e b r a in and a few sheep b ra in s show marked congestion*
U su ally th e re i s an in c re a s e in th e amount o f c e re b ro s p in a l f l u i d .
L esions in o th e r v is c e r a a re r a r e b u t o c c a s io n a lly f a t t y l i v e r , d u o d e n itis
o r pulmonary edema a re se e n .
F a lla s k e ( 1940) r e p o rte d marked f o c a l
h e p a tic n e c r o s is i n an a d u lt sh e e p .
ed f o r c a t tl e *
These le s io n s have not been r e p o r t
-
13
-
I t has been w e ll e s ta b lis h e d t h a t th e m ic ro sco p ic le s io n s a re con
f in e d p r im a r ily to th e p o n s, m edulla and a n t e r i o r s p in a l c o rd .
th e w h ite and grey m a tte r may be in v o lv e d .
Both
I t i s f e l t t h a t th e prim ary
le s io n s develop in th e b r a in su b stan ce and t h a t th e m eningeal le s io n s
r e s u l t from e x te n s io n .
The predom inant le s io n s a re marked p e riv a s c u la r
c u ff in g w ith v a ry in g d e g re es o f f o c a l n e c r o s is .
The p e r iv a s c u la r c u ffs
c o n s is t m ainly o f m ononuclear c e l l s in a l l s p e c ie s o f ru m in a n ts .
How
e v e r, Gray and Moore (1953) re p o rte d marked d if f e r e n c e s betw een sheep
and cows in th e c e l l u l a r c o n s titu e n ts o f th e f o c a l l e s i o n s .
In sheep
and g o a ts th e f o c i c o n ta in a preponderance o f polym orphonuclear leuk o
c y te s and in some in s ta n c e s may ap p ear p u r u le n t.
Both O lafso n ( I 940) and
B allask e (1940) c a l l a t t e n t i o n to th e p u ru le n t n a tu re o f th e le s io n s in
sh e ep .
In some in s ta n c e s th e norm al parenchyma may be a lm o st com pletely
d is in te g ra te d .
There i s edema and hem orrhage, and neurons in the a f
f e c te d a re a s show v a rio u s s ta g e s o f d e g e n e ra tio n .
may be p r e s e n t .
Neuronaphagia a ls o
The blood v e s s e ls a re c o n g este d , f re q u e n tly c o n ta in
thrombi and show d e g e n e ra tiv e changes p a r t i c u l a r l y o f th e e n d o th e lia l
lin in g .
In c a t t l e th e p e r iv a s c u la r c u ff s a re much s m a lle r and th e f o c a l
le s io n s u s u a lly a re lim ite d to edema and sm a ll f o c a l c o ll e c t io n s o f
m ic r o g lia l c e l l s and lym phocytes.
re p o rte d f o r sh eep .
R arely a re le s io n s a s e x te n siv e a s
T his reem phasizes th e more ch ro n ic n a tu re o f th e
d is e a s e in c a t tl e *
L. mono cytogenes i s n ev er found in th e p e r iv a s c u la r c u ffs b u t
q u ite r e a d i ly may be d em onstrated in th e f o c a l le s io n s in s e c tio n s
s ta in e d by e i t h e r the G oodpasture o r Gram-Weigert method*
The b a c te r ia
-
14
-
o ccu r m ost f r e q u e n tly n e ar th e p e rip h e ry o f th e f o c a l le s io n and may
be e i t h e r e x t r a - o r i n t r a c e l l u l a r .
In c a t t l e they ten d to o ccu r s in g ly
o r in sm all clum ps, w hile in sheep they may form dense p la q u e s .
The known r e p o rte d o u tb re a k s o f l i s t e r i c e n c e p h a litis in sh e e p ,
g o a ts and c a t t l e a re shown in ta b le s 1 , 2 , 3 * pages 28 "to 31 #
S to ck to n et, a l . (1954) review ed th e s u b je c t o f a b o rtio n due to
L. m onocytogenes.
In a d d itio n to th e o u tb re a k s c ite d by th e s e a u th o rs
th e fo llo w in g r e p o r ts should be in c lu d e d .
Boppensiek ( 1944) i s o l a t e d
a gram p o s i t iv e rod which d i f f e r e d o n ly s l i g h t l y in c e r t a i n biochem ical
r e a c tio n s from L. m onocytogenes.
th e r a b b i t .
Jansen (1945) re p o rte d a b o rtio n in
Levy et, a l . (1952) d e s c rib e d two o u tb re a k s o f a b o rtio n in
c a t t l e , and van U lsen (1952 ) re p o rte d the i s o l a t i o n o f L. monocytogenes
from th r e e a b o rte d bovine f e t u s e s .
The known re p o rte d c a se s o f a b o rtio n
in a l l sp e c ie s i s shown in ta b le 4 * page
T h is form may be more p re v a le n t th an i s g e n e ra lly s u s p e c te d , and
p r e s e n ts a broad f i e l d f o r f u r t h e r r e s e a r c h .
Olson (1945) and Ferguson
( 1951) suggested t h a t t h i s b acteriu m may be a norm al in h a b ita n t o f th e
fem ale g e n it a l t r a c t , b u t t h i s has not been confirm ed.
Gray and McWade
( 1954) in one in s ta n c e were a b le to i s o l a t e t h i s a g e n t from the c e rv ix
o f a s o - c a lle d re p e a t- b r e e d e r cow showing symptoms o f nymphomania.
R epeated a tte m p ts to r e i s o l a t e th e b acteriu m from t h i s cow r e s u lte d in
f a i l u r e b u t i t does e s t a b l i s h L. monocytogenes a s a t r a n s i e n t in h a b ita n t
o f th e fem ale bovine g e n i t a l t r a c t .
Furtherm ore Levi ^ t, a l . (1945) on-
co u n te red le s io n s in th e u te r u s and sp le e n but n o t in th e c e n tr a l nervous
system o f one h e i f e r s la u g h te re d two and a h a lf months a f t e r a b o rtio n
and L . monocytogenes was reco v ere d from th e ovary on th e sid e where th e
-
15
-
pregnancy had o c c u rre d ; th u s in d ic a tin g t h a t th e b acteriu m may su rv iv e
f o r r e l a t i v e l y lo n g p e rio d s o f tim e in th e bovine re p ro d u c tiv e t r a c t .
In c a t t l e a b o rtio n u s u a lly o c cu rs between th e f o u r th and sev en th
month o f g e s ta tio n and th e b acteriu m can be i s o l a t e d from the l i v e r ,
b lo o d , f e t a l membranes and m ost commonly, from th e abomasal c o n te n ts o f
th e f e t u s .
Gray e t a l . (1954) re p o rte d a c o n d itio n in c a t t l e which a p p ea rs to
be s im ila r to th e o u tb re a k o f human a b o rtio n re p o r te d by P o te l ( 1953)*
and o th e rs ( t a b l e 4* page 33)*
They observed in s ta n c e s in th e same
h erd where th e c a lv e s were born a t o r n e ar term b u t liv e d o n ly a few
days i f born a liv e *
The l i v i n g c a lv e s alw ays were weak and some showed
a marked bloody d ia r r h e a .
L . mono cy togenes was i s o l a t e d in pure c u ltu r e
from th e l i v e r o f e ig h t c a lv e s and m icro sco p ic f o c a l h e p a tic l e s io n s
were dem onstrated in two c a lv e s not c u ltu r e d .
At necropsy th e m ost con
sp icu o u s a l t e r a t i o n was marked hem orrhagic g a s t r i t i s and e n t e r i t i s .
Some c alv e s showed a few p in p o in t n e c r o tic f o c i i n the l i v e r .
c o n d itio n was re p o r te d in one c a l f by Harbour (1 9 4 1 ).
A s im ila r
At no tim e was
L. mono c.vto ge nes is o l a t e d from th e dam, bu t i n t r a u t e r i n e in f e c ti o n was
s tr o n g ly s u g g e ste d .
A s im ila r c o n d itio n in lambs has been re p o rte d by
Jep sen (192j2) and Gray e t a l . (1949)*
The r o le o f L. monocytogenes in
th e e a r ly d e a th o f c a lv e s and lambs needs f u r th e r i n v e s ti g a t i o n .
It
may be t h a t t h i s b a cteriu m p la y s a f a r more im p o rtan t p a r t in d is o r d e rs
o f th e v e ry young than p re v io u s ly s u s p e c te d .
In sheep a b o rtio n o c c u rs a t o r near term and i s o l a t i o n s can be
made from th e same organs a s in the b o v in e .
As in the b o v in e , th e re
a r e no d is tin g u is h in g le s io n s to su g g est the e tio lo g y .
A b ortion and
-
16
-
e n c e p h a litis a re n o t known to o ccur sim u lta n e o u sly in th e same flo c k o r
herd*
With th e p r e s e n t p a u c ity o f in fo rm a tio n i t can n o t be s ta te d
w hether L* monocytogenes p la y s a prim ary o r secondary r o l e i n th ese a b o r
tio n s *
I t has been shown t h a t L* m onocytogenes can a tt a c k a wide range o f
a v ia n sp e c ie s*
B esides b e in g is o l a t e d from th e chicken ( t a b l e 5* P&ge 35)
i t has been i s o l a t e d from th e canary (B ig la n d , 1950? van d e r Schaaf and
de Jo n g , 1 9 5 1 ), duck and goose ( Z e l le r , 1949) w ild wood gro u se ( L ille e n gen* 192j2), and b lu e eag le (S c h u lz e , 195°)*
In fow ls th e d is e a s e o ccu rs
a s a se p tic e m ia and th e m ost conspicuous le s io n s a re m assive a re a s o f
m y o card ial d e g e n e ra tio n , p e r i c a r d i t i s and an in c re a se d amount o f p e r i
c a r d ia l f lu id *
O ther le s io n s may be f o c a l h e p a tic n e c r o s is , g e n e ra liz e d
edema, sp le n o m e g alia , p e r i t o n i t i s and e n t e r i t i s *
In a c u te c a se s the
n e c r o tic le s io n s a re l e s s marked and one o n ly f in d s th e le s io n s u s u a lly
a s s o c ia te d w ith a se p tic em ia*
The b a cteriu m i s e a s i l y c u lt i v a t e d from
th e abdom inal organs*
As in o th e r s p e c ie s , th e young fowl ap p ea rs to be more s u s c e p tib le
th an th e o ld e r b ird *
O utbreaks a re u s u a lly sp o ra d ic and m o r ta lity in
th e in d iv id u a l f lo c k may v a ry w ith in wide lim its *
symptoms i n chickens w ith t h i s d ise a se *
There a re no s p e c if ic
A dult b ird s u s u a lly d ie sudden
l y w hile i n th e young th e re may be a slow w a stin g b efo re death*
There
i s d isag reem en t a s to w hether th e re i s a c ir c u la t in g m onocytosis b e fo re
death*
I t has been su g g e ste d t h a t L* monocytogenes o f te n i s a secondary i n
vader*
I n f e c tio n s have been a s s o c ia te d w ith s a lm o n e llo s is , lym phom atosis,
and N ew castle d is e a s e (B a te rso n , 1937? B o lin and T urn, 1951) .
I t has
- 17 been i s o l a t e d from th e sp le e n o f a p p a re n tly normal ch ick en s (F e ls e n fe ld ,
1951)*
Geurden and Devos (1952) r e p o r te d , b u t w ith o u t s tro n g s u p p o rt,
t h a t l a t e n t i n f e c ti o n may be more fre q u e n t th an su sp ec ted and t h a t th e se
l a t e n t in f e c ti o n s may p la y an im p o rtan t p a r t in epidem iology*
They a ls o
showed t h a t sparrow s and pig eo n s could be in f e c te d a r t i f i c i a l l y and thus
may p la y a p a r t i n tra n sm issio n *
Pigeons p re v io u s ly had been re p o rte d
to be r e f r a c t o r y to a r t i f i c i a l in f e c tio n w ith L. mono cvtoge ne s (Verge
and G o re t, 1941? L esb o u y rie s, 1943)*
a p o s s ib le so u rce o f in fe c tio n *
These a u th o rs su g g e st the egg a s
They is o l a t e d L* monocytogenes from ex
te n s iv e n e c r o tic le s io n s in th e o v id u c t o f one hen*
U n fo rtu n a te ly , no
eggs were a v a ila b le f o r c u l t u r e , b u t egg in f e c ti o n does p r e s e n t an i n
t e r e s t i n g e p id e m io lo g ic a l im p lic a tio n *
They f e e l t h a t c o n ta c t p la y s on ly
a v ery sm all p a r t , i f a n y , in th e tra n s m iss io n o f th e d is e a s e i n p o u ltr y .
The p resen ce o f l a t e n t c a r r i e r s in a chicken f lo c k p re s e n ts a de
f i n i t e p u b lic h e a lth h a z a rd .
F e ls e n fe ld (1951) re p o rte d an o u tb re a k o f
l i s t e r i c c o n ju n c ti v i ti s in w orkers in a p o u ltry p ro c e ss in g p l a n t .
He
i s o l a t e d L* monocytogenes from th e sp le e n o f f iv e a p p a re n tly h e a lth y b ir d s
which were b e in g p ro cessed a t th e p l a n t .
I t was found t h a t th e s e b ir d s
o r ig in a te d from a p o u ltry farm where Graham et_ aJL. (1943) had diagnosed
l i s t e r i o s i s some y e a rs b e fo r e .
T his perhaps i s th e only in s ta n c e where
a d e f i n i te mode o f tra n s m is s io n could be e s ta b lis h e d , and a ls o the only
r e p o r t t h a t g iv e s d e f i n i t e in fo rm a tio n t h a t L. monocytogenes may p e r s i s t
on th e same prem ise f o r long p e rio d s o f time*
L i s t e r i c se p tic e m ia i s found most commonly in th e m o n o g astric a n im a ls.
I t has been r e p o rte d in p ig s (D eB lieck and Ja n se n , 1942? K e rlin and Graham,
1945)* r a t s (M a ch ia v ello , 1 9 4 2 ), groundmoles (Levy, 1948) , raccoons
- 18 -
(G iffo rd and J u n g h e rr, 1947)* Lemming (P lumme r and B yrne, 1950; B a r ra le s ,
1953)* c h in c h illa (Kennedy, 1947? McKay e t a l . * 1949; Shalkop, 1950?
S m ith , 1953)* fo x es (Cromwell e t a l » , 1939? Wramby, 1945? Jansen and
Peperkamp, 1947)* dom estic and w ild r a b b its (H u lp h ers, 1911; M urray e t
a l . . 1926; P a te r s o n , 1940; H e n ric so n , 1943? L e sb o u y rie s, 1943? Jansen and
van d e r Hurk, 1945; Gudkowa and S a c h a ro ff, 194&? Schoop, 194-6; V a lle e ,
1952^), guinea p ig s (Murray e t a l * . 1926; V iera and C a s te lo , 1944* Gudkowa
and S a c h a ro ff, 194&S Roine e t a l * . 1953)* and h o rse s ( G r in i, 1943? K rage,
1944; Svenkerud, 1949)•
The p r i n c i p a l le s io n i s f o c a l h e p a tic n e c ro sis*
In m ost in s ta n c e s a c i r c u l a t i n g m onocytosis can be dem onstrated i n th e
p e r ip h e r a l blood a s h o rt tim e b e fo re death*
With th e e x c e p tio n o f th e
r e p o r t o f Plummer and Byrne (1 9 5 ° ), no in fo rm a tio n i s a v a ila b le d e a lin g
w ith a l t e r a t i o n s in th e c e n tr a l nervous system in th ese cases*
They f a i l
ed to f in d m ic ro sc o p ic le s io n s in s e c tio n s p rep a red from the m edulla o f
one lemming*
T his form a ls o has been found in young lambs (Je p se n , 1942?
Gray e t a l * . 1949) anci c a lv e s (H arbour, 1941? Gray e t a l* . 1954) b e fo re
th e rumen i s f u n c tio n a l*
Gudkowa and S a c h a ro ff ( 1946) in d ic a te d t h a t ro d e n ts c o n s t it u t e a
r e s e r v o i r f o r th e i n f e c ti o n .
They claim t h a t L. monocytogenes i s w idely
d i s t r i b u t e d in m ic e , r a t s and r a b b i t s , and th a t th ey were a b le to i s o l a t e
th e b acteriu m from th e m asal mucous membrane o f 0 *5% o f a l l h e a lth y r a b
b its .
However, t h i s has n o t been confirm ed and b o th O lafson (1940) and
Graham .et a l * (1943) f a i l e d to i s o l a t e L* monocytogenes from any o f a
la r g e number o f r a t s caught on farm s where o u tb re a k s o f o vine l i s t e r i o s i s
were i n p ro g ress*
There i s no s a t i s f a c t o r y e x p la n a tio n a s to why lo c a liz e d e n c e p h a litis
-
does n o t o ccu r in th e s e anim als*
19
-
Gudkowa and S a c h a ro ff (194-6) p o s tu la te
a d if f e r e n c e in a d a p ta tio n in th e s t r a i n s which produce e n c e p h a litis and
th o se which produce e i t h e r se p tic em ia o r in f e c tio u s m ononucleosis and
t h a t th e p a r t i c u l a r d is e a s e produced i s a m a n ife s ta tio n o f th e s p e c if ic
a d a p ta tio n *
They r e p o r t t h a t passage through th e b ra in o f la b o ra to r y
an im als in c re a s e d th e v iru le n c e o f th e s t r a i n f o r b ra in t i s s u e .
Such
s t r a i n s may produce e n c e p h a litis fo llo w in g in tra m u s c u la r o r in tra v e n o u s
in o c u la tio n b u t soon lo s e t h e i r a f f i n i t y f o r b ra in t is s u e a f t e r r e p e a t
ed in o c u la tio n by th e s e two m ethods.
They found t h a t s t r a i n s p assed
through b ra in t is s u e lo s e t h e i r a b i l i t y to ferm ent su cro se and la c to s e
and th e fe rm e n ta tio n o f th e se su b sta n c es may be an index o f a f f i n i t y f o r
n e u ra l t i s s u e .
I t i s w e ll e s ta b lis h e d t h a t th e re i s wide s t r a i n d i f f e r
ence in a b i l i t y to ferm en t su cro se and la c to s e b u t f u r t h e r re s e a rc h i s
n e c e ssa ry to confirm th e r e l a t i o n s h i p ex p ressed by them*
A lso unexplained i s th e o ccu rren ce o f la rg e numbers o f monocytes in
th e c i r c u la t in g blood o f in f e c te d m onogastric an im als and the a p p a re n t
absence o f t h i s c e l l i n th e blood o f rum inants*
The developm ent o f a
m onocytosis fo llo w in g in tra v e n o u s in o c u la tio n in th e common la b o r a to r y
an im als i s such a s p e c i f i c r e a c tio n t h a t i t has been d e sc rib e d in th e
sp e c ie s name o f th e b a c te riu m ; *monocytogenes11 , g e n e ra to r o f monocytes*
However, Olson .et a l . (1950) f a i l e d to f in d an in c re a s e in the number o f
m onocytes in th e blood o f sheep fo llo w in g in tra v e n o u s ex p o su re.
They
found t h a t th e polym orphonuclear leu k o c y te was th e predom inant c e l l under
th e s e c o n d itio n s*
A lso most i n v e s ti g a t o r s have f a i l e d to fin d m onocytes
in th e blood o f ru m in an ts a f f e c te d w ith e n c e p h a litic l i s t e r i o s i s *
The monocyte p ro d u cin g f a c t o r (KEF) o f
monocytogenes was found
20
by S ta n le y (1949)
confirm ed
n o n -a n tig e n ic l i p i d f r a c t i o n
-
by G irard and Murray (1951) to be a
o f th e b a c t e r i a l c e l l .
T his MEF was found
to be l i b e r a t e d c h ie f ly in th e l i v e r and S e i t z f i l t e r e d s a lin e e x tr a c ts
o f n e c r o tic l i v e r produced m onocytosis in r a b b i t s .
S im ila r e x tr a c ts
p re p a re d from o th e r in f e c te d organs o r normal l i v e r f a i l e d to e l i c i t
re s p o n s e .
R e sista n c e in th e rum inant l i v e r to t h i s resp o n se may e x p la in
in p a r t a t l e s t , the
f a i l u r e o f L. monocytogenes to l o c a liz e in the l i v e r
o f a d u lt ru m in a n ts .
F a ilu r e to lo c a liz e in t h i s organ may r e s u l t in
l o c a l i z a t i o n in th e b r a i n .
However t h i s s p e c u la tio n r e q u ir e s f u r t h e r
in v e s tig a tio n a s v ery l i t t l e has been done in t h i s f i e l d to d a te .
S ep ticem ia in th e a d u lt rum inant has been re p o rte d b u t i s r a r e
( E a lla s k e , 1940* B e lin , 1943? Olson e t a l . . 1953®; H ira to e t a l . . 1954)*
The v a rio u s m edicam ents f o r the tre a tm e n t o f l i s t e r i o s i s were r e
view ed by Gray and Moore (1953) &nd L inzenm eier and S e e lig e r (1954) r e
p o rte d e x te n siv e jln v i t r o s tu d ie s o f th e e f f e c t o f th e v a rio u s sulfonam ides
and a n t i o b i o t i c s a g a in s t L. m onocytogenes.
The v a rio u s a n t i b i o t i c s and
su lfo n am ides d i f f e r g r e a t ly in t h e i r e ff e c tiv e n e s s on in d iv id u a l s t r a i n s
o f L. m onocytogenes.
P e n i c i l l i n was shown to be h ig h ly v a r i a b l e , and
may be m ost e f f e c t i v e when combined w ith one o f th e su lfo n a m id es.
Gray
e t a l . ( 1949®) found t h a t stre p to m y cin in low c o n c e n tra tio n in h ib ite d th e
grow th o f L . m onocytogenes. b u t th e m icroorganism v e ry q u ic k ly developed
a r e s i s t a n c e to i t .
I t i s p o s s ib le to produce a strep to m y cin -d ep en d en t
s t r a i n s o f t h i s b a c te riu m .
in e ffe c tiv e .
In v iv o s tu d ie s showed t h i s a n t i b i o t i c to be
Aureomycin and te rra m y c in were found to be h ig h ly e f f e c t i v e
in b o th i n v i t r o and in viv o s tu d ie s (Z ink e_t a l . « 1951; Norman et. a l . ,
1951) .
21
-
The r e p o r t o f Gray and Moore (1953) i s one o f th e few devoted to
t r i a l s o f an a n t i b i o t i c under f i e l d c o n d itio n s*
These a u th o rs t r e a te d
w ith aureom ycin 13 sheep and 25 cows su sp e c te d o f b ein g a f f e c te d w ith
lis te rio s is .
In th e dosage used (1.25 gm I.V* p e r day f o r fo u r days
f o r sheep and 2*50 gn I .V . p e r day f o r fo u r days f o r cows) th e a n t i b i o t i c
was i n e f f e c t i v e f o r sheep b u t d id have some th e ra p e u tic v a lu e f o r cows.
Only one sheep su rv iv e d th e fo u r day tre a tm e n t p erio d *
Many o f th e cows
which rec o v ere d fo llo w in g tre a tm e n t l a t e r showed symptoms su g g e stiv e o f
perm anent b ra in damage.
In s e v e ra l in s ta n c e s owners d isp o se d o f th e
an im als soon a f t e r trea tm e n t*
suddenly re la p s e d and d ie d .
Some anim als appeared to re c o v e r but
I t was p o s s ib le to dem onstrate m icro sco p ic
le s io n s in th e b ra in s o f s e v e ra l an im als se n t to s la u g h te r ap p ro x im ately
one y e a r a f t e r tre a tm e n t.
In sheep, b r a in damage ap peared to be too e x
te n s iv e f o r th e a n t i b i o t i c to be e f f e c t i v e .
In c a t t l e where a l t e r a t i o n s
in th e b r a in a re l e s s s e v e re , tre a tm e n t may be e f f e c t i v e i f i n i t i a t e d
e a rly .
The owner should be made aware o f th e f a c t t h a t symptoms may
p e r s i s t i f th e anim al s u r v iv e s .
T his i s no p a r t i c u l a r problem in b eef
c a t t l e b u t p r e s e n ts numerous d i f f i c u l t i e s in a d a iry h e rd .
The f a i l u r e o f th e ra p y once symptoms a re e v id e n t, em phasizes th e
need f o r a p ro p h y la c tic a g e n t i f liv e s to c k a re to be p r o te c te d a g a in s t
t h i s d is e a s e .
Graham ejt a l . (1940) found th a t both liv i n g and fo rm a lin
k i l l e d b a c te r in s had no e f f e c t on th e course o f in f e c tio n in sh e e p .
r e s u l t s o f Olson e t a l . (1951)
The
used bo th b a c te r in s and whole blood
from a rec o v ere d sheep were n o t very e n co u rag in g .
However, E v e le th e t
a l . ( 1952.) have employed a com m ercially made v a cc in e f o r th e p a s t s e v e ra l
y e a rs and r e p o r t some b e n e f i t from i t s u s e .
c a t t l e i s u n re p o rte d .
The use o f b a c te r in s f o r
However, f u r t h e r re s e a rc h i s n e ce ssa ry in t h i s a r e a .
-
22
-
The h ig h m o r ta lity o f l i s t e r i o s i s in man makes i t im p e ra tiv e t h a t
a l l su sp e c te d m a te r ia l be handled w ith c a u tio n .
T his i s e s p e c ia lly tr u e
d u rin g n ecropsy o f a b o rte d f e tu s e s and se p tic e m ic case s where the number
o f organism s i s a p t to be v e ry h ig h .
In th e e n c e p h a litic form the bac
t e r i a a r e g e n e ra lly con fin ed to th e b r a in and p re s e n t l i t t l e danger un
le s s th e b r a in i s removed.
P a lla sk e (1940)* B e lin (1943) Olson e t a l .
(1953) H ira to e t a l . (1954)» found th e bacteritiim in th e l i v e r and sp le e n
o f a few a d u lt sheep*
T his has never been re p o rte d in the cow.
However,
m eat from anim als showing evidence o f a c tiv e i n f e c tio n should n o t be
used f o r human consum ption.
On th e b a s is o f o b s e rv a tio n s in la b o ra to r y
an im als and f i e l d o b s e r v a tio n s , Gray and Moore (1953) suggested t h a t
re c o v ere d anim als a re s a fe f o r food a t l e a s t th re e weeks a f t e r abatem ent
o f symptoms.
Under la b o ra to ry c o n d itio n s L. monocytogenes seldom has
been found to p e r s i s t in an exposed anim al f o r more th an two weeks, and
u s u a lly not more th an f iv e o r s ix d a y s.
However, o c c a s io n a lly L. mono
cytogenes can be i s o l a t e d from a lo c a liz e d le s io n long a f t e r i n i t i a l
exposure (Levy e t a l . , 1952? Thompson, 1954)*
^
t h i s a ls o o c cu rs under
f i e l d c o n d itio n s , i t may account f o r the p resen ce o f c a r r i e r a n im a ls.
In man l i s t e r i o s i s u s u a lly o ccu rs a s m e n in g itis ( ta b le 6 , page 3 6 ) .
The in c id e n c e v ery l i k e l y i s much h ig h e r than p u b lish ed r e p o r t s i n d ic a t e .
There a re no d i s t i n c t i v e symptoms in the m eningeal form and i t e a s i l y
can be confused w ith m e n in g itis o f o th e r e tio lo g y .
However, a tte m p ts
to i s o l a t e L. monocytogenes from th e s p in a l f l u i d have been q u ite suc
c e s s fu l.
rum inants*
T his p re s e n ts a d e f i n i t e a id in d ia g n o s is which i s la c k in g in
The m o r ta lity due to t h i s form o f l i s t e r i o s i s i s about 70 %
and e i t h e r p h y s ic a l o r m ental d e fe c ts a re common se q u el to i n f e c t i o n .
Less s e r io u s i s th e c o n ju n tiv a l form .
T his i s u s u a lly a l o c a l in f e c tio n
- 23 fo llo w ed by com plete re c o v e ry ( F e ls e n fe ld , 1944; B i l i b i n , 1949; P le tn e v a
and S tik s o v e , 1950? Shm eleva, 1953)*
P le tn e v a and S tik so v e ( I 9 5 0 ) c a ll
a t t e n t i o n to the s i m i l a r i t y o f t h i s form o f l i s t e r i o s i s and the o c u la r
f o m o f tu la re m ia , b oth in th e le s io n which develops and th e f a c t t h a t
b o th ap p ear to be c a r r ie d by a n im a ls.
A low grade se p tic e m ic foim c h a ra c
t e r i z e d by c h i l l s , f e v e r , ra s h and sw e llin g o f the lymph nodes has been
r e p o rte d from th e S o v ie t Union by B i li b i n (1949) and P le tn e v a and S tik so v e
(1 9 5 0 ).
The r e l a t i o n o f L . monocytogenes to in f e c tio u s m ononucleosis has
been review ed by B enazet (1943)* S ta n le y (1949) and G irard and Murray
(1951)*
The r e s u l t s o f th e s e in v e s tig a tio n s in d ic a te t h a t th e r e l a t i o n ,
i f a n y , o f t h i s b acteriu m to the d is e a s e i s o b sc u re .
F u rth e r work i s
n e c e ssa ry a ls o in t h i s a r e a .
The r o le o f L. monocytogenes in human a b o rtio n has been touched
on in an e a r l i e r s e c tio n and i s expanded in th e work o f P o te l (1953)*
Erdmann (1953)* S ta rk (1953) and many o t h e r s .
The s i g n i f i c a n t fin d in g s
o f th e se r e p o r ts a re t h a t in a v a s t m a jo rity o f th e c a s e s , th e women
were from r u r a l a re a s and in clu d ed raw cow o r g oat m ilk in th e d i e t .
T his s tr o n g ly su g g e sts th e mouth a s th e avenue o f e n tra n c e in th ese
in s ta n c e s .
On one o c c a sio n P o te l (1953) is o l a t e d L. m onocytogenes from
an a ty p i c a l bovine m a s t i t i s .
A woman d rin k in g raw m ilk from t h i s cow
a b o rte d tw ins and L* monocytogenes was i s o l a t e d from th e l i v e r o f e a c h .
B a c te r io lo g ic a l exam ination o f s e v e ra l hundred o th e r m ilk sam ples f a i l e d
to y i e l d s i g n i f i c a n t grow th.
Wramby ( 1944) a ls o re p o rte d th e i s o l a t i o n
o f t h i s b acteriu m from a case o f bovine m a s t i t i s .
organism p re s e n ts a s e rio u s p u b lic h e a lth problem .
M a s t i ti s due to t h i s
The r e s is ta n c e s tu d ie s
o f Ozgen (1951) and Zink (1951) in d ic a te t h a t L. monocytogenes can su rv iv e
- 24 -
h ig h te m p e ra tu re s f o r lo n g p e rio d s o f tim e , and Ozgen (1951) in d ic a te d
t h a t th e b a cteriu m can su rv iv e p a s te u r iz a tio n *
T h is f a c t o r should be
c o n sid ered in case o f any a b o rtio n o r e a r ly i n f a n t d eath found to be
due to t h i s b a c te riu m .
L i s t e r i o s i s does n o t appear to be a s e rio u s problem in canine p o p u la
tio n s .
Graham ejt^ a l . (1943) were unable to i n f e c t t h i s s p e c ie s a r t i f i c i a l l y .
Cox (1945) re p o rte d th e i s o l a t i o n o f L. monocytogenes from th e m edulla
o b lo n g ata o f f o u r dogs which showed symptoms su g g e stiv e o f r a b i e s .
e v e r , a l l t e s t s f o r t h i s d is e a s e were n e g a tiv e .
i n & p e rio d o f s e v e r a l m onths.
How
A ll th e se o c cu rred w ith
In th e one case re p o rte d by Chapman (1947)
a d i f f e r e n t i a l leu k o c y te count made th re e days b e fo re d e ath showed 2 $%
m onocytes.
Necropsy re v e a le d m e n in g itis and a few c o lo n ie s o f L . mono
cy to g en es were o b ta in e d from th e m eninges, pons and l a t e r a l v e n t r i c l e .
These a p p ea r to be th e on ly re p o rte d in s ta n c e s o f l i s t e r i o s i s in th e dog.
N e v e rth e le s s , th e re i s rea so n to su sp e c t t h a t th e in c id en c e m ight prove
to be much h ig h e r i f the b ra in s o f a l l dogs showing nervous symptoms were
su b m itted f o r c u ltu r e o f t h i s b a c te riu m .
The horse a ls o a p p e a rs to be r e l a t i v e l y r e f r a c t o r y to in f e c tio n s
w ith t h i s m icroorganism .
B e lin (194&) re p o rte d th e i s o l a t i o n o f L.
monocytogenes from th e b r a in o f th re e h o rse s on the same farm w ith in a
p e rio d o f fo u r w eeks.
The symptoms and le s io n s a t necropsy were s im ila r
to th o se found in o th e r fo rn s o f m e n in g itis .
Z e l le r (1949) m entions
w ith o u t e la b o r a tio n two s t a in s o f L. monocytogenes i s o l a t e d from h o rse s
w ith borna d is e a s e .
Tabuchi ejt a l . (1952) d e sc rib e d what appeared to be
e n c e p h a litie o r m eningeal l i s t e r i o s i s in an a d u lt h o rs e .
No c u ltu r e s were
p re p a re d , but t is s u e s e c tio n s o f the m edulla re v e a le d p e riv a s c u la r c u f f -
- 25 in g , f o c a l n e c r o s is and la rg e numbers o f gram p o s itiv e b a c t e r i a .
Sep
tic e m ia in young f o a l s was d isc u sse d e a r l i e r under sep tic em ia in monog a s t r i c a n im a ls .
The re p p r te d a s s o c ia tio n o f L* monocytogenes w ith
p e rio d ic o p hthalm ia (Jo n e s, 1940) has never been confirm ed.
L. monocytogenes a ls o has been in c rim in a te d in s e v e ra l o th e r con
d i ti o n s which f u r t h e r emphasize the u b iq u ito u s n a tu re o f th e b a c te riu m .
F e ls e n fe ld (1948) reco v ere d th e m icrobe from th e blood o f a p a ti e n t
w ith a low -grade fe v e r and m oderate enlargem ent o f th e c e r v ic a l lymph
nodes fo llo w in g C esarean s e c tio n .
M orris and Norman (1950) i s o l a t e d i t
from th e l i v e r o f a p p a re n tly normal f e r r e t s .
S l a b o s p i t s 'k i l (1938)
i s o l a t e d the m icroorganism from p o x -lik e le s io n s in p i g s .
On two o c ca sio n s
i t has been i s o l a t e d from th e l i v e r o f p ig s t h a t o b v io u sly d ied from
o th e r ' c a u se s.
Rhoades and S u th e rla n d (1948) re p o rte d i t in c o n n ec tio n
w ith an o u tb re a k o f hog c h o le ra and B olin and E v e le th (1951) made i s o l a
tio n s from th e l i v e r o f p ig s which d ied o f ly e p o iso n in g .
In s ta n c e s such
a s th e se s tr o n g ly su g g est t h a t swine may p la y an im p o rtan t r o le a s c a r r i e r s .
T his may be s p e c u la tio n b u t does p re s e n t i n tr i g u i n g im p lic a tio n s in the
ep izo o lo g y o f l i s t e r i o s i s .
The accum ulated case r e p o r t s give l i t t l e
mode o f tra n s m is s io n under n a tu r a l c o n d itio n s .
su g g e stio n to th e a c tu a l
The f a c t t h a t in most
o u tb re a k s , e s p e c i a l l y in anim al p o p u la tio n s , more th an one in d iv id u a l
was in volved e s t a b l is h e s th e c o n tag io u s n a tu re o f the d is e a s e .
T his was
c l e a r l y b rought o u t in th e r e p o r t o f E v eleth (1952) who found t h a t when
a p p a re n tly norm al sheep from in f e c te d sheep f lo c k s were in tro d u c e d in to
new f l o c k s , th e d is e a s e o f te n appeared in the new f l o c k .
In t h i s manner
12 flo c k s became in f e c te d o v e r a nine y e a r p e rio d by in tro d u c tio n o f
sheep from a s in g le in fe c te d f l o c k .
F u rth erm o re, in a stu d y o f 44 sheep
- 26 f lo c k s where l i s t e r i o s i s had o c c u rre d , E v e le th (1952a) found th a t in 30
o f th e f lo c k s th e d is e a s e ap peared a f t e r in tr o d u c tio n o f anim als from
in f e c te d f l o c k s .
In th e rem ain in g 14 c ase s sheep from f lo c k s o f unknown
h is to r y had been in tro d u c e d d u rin g the y e a r p re c e d in g th e o u tb re a k .
Bop-
p in s ie k ( 1944) re p o rte d a b o rtio n in a ewe t h a t was thought to be due to
L. monocytogenes and t h a t su b seq u e n tly a cow g ra z in g on th e p a s tu re where
th e a b o rtio n o c cu rred d ie d from l i s t e r i c e n c e p h a l it i s .
Gray ejt a l . (1951)
re p o rte d an in s ta n c e where a sheep and a cow on the same p a s tu re both
d ied from l i s t e r i c e n c e p h a l it i s .
The p a ti e n t o f B erry (1950) who e x p ire d
w ith l i s t e r i c m e n in g o -e n c e p h a litis p o ssesse d a p e t chipmunk which became
s ic k and died j u s t s h o r tly b e fo re th e p a ti e n t became i l l .
Most o f th e
p a ti e n t s re p o rte d by Gudkowa and S a c h aro ff ( 1946) , B i li b i n (1 9 4 9 ), P l e t
neva and S tik so v e (195°) &nd Shmeleva (1953)
c o n ta c t w ith e i t h e r
r a t s , m ice, c a t , dogs o r o t h s r a n im a ls, but th e c o n tr ib u tio n o f th e se
anim als to in f e c tio n could n o t be d e term in ed .
a l . (1952) d e se rv e s s p e c ia l m en tio n .
The r e p o r t o f Odegaard e t
The p a ti e n t re p o rte d by them had
cleaned a sheep barn in which manure had accum ulated d u rin g th e w in te r .
A s h o rt tim e l a t e r when th e p a t i e n t s u ffe re d exposure a s a r e s u l t o f a
storm he became i l l and d ied o f l i s t e r i c m e n in g itis .
A s h o r t tim e l a t e r
one o f th e sheep in the l a t e p a tie n t* s sheep flo c k died o f l i s t e r i c
e n c e p h a l it i s .
When the rem ain in g sheep were s e n t to s la u g h te r , L.
m onocytogenes could n o t be is o l a t e d from s e v e ra l sheep b ra in s c u ltu r e d .
T h is case n o t o n ly im p lie s c o n ta g io n but a ls o t h a t exposure to ad v erse
w eath er c o n d itio n s may be a p re d is p o s in g f a c t o r in i n f e c t i o n .
In none o f th e above c a se s could the r o le o f c a r r i e r anim als be
d e f i n i t e l y e s t a b l is h e d , but the c ircu m stan ces demand t h a t f u r t h e r work
sh o u ld be u n d e rta k en in an a tte m p t to e s t a b l i s h th e p a r t t h a t c a r r i e r
an im als p la y in th e spread o f in fe c tio n #
U n til t h i s i s determ ined i t
w i l l be d i f f i c u l t to c o n tro l th e d ise a s e in bo th human and anim al popu
la tio n s #
The ta b le s which ap p ear on the fo llo w in g pages a re
in te n d ed o n ly a s re fe re n c e s to known o u tb rea k s o f
th e d ise a se #
Except f o r th e r e p o r ts in man no a t
tem pt was made to l i s t the number o f in d iv id u a ls
involved a s such an a tte m p t would be in a c c u ra te a t
b e s t and se rv e s no p a r t i c u l a r p u rp o se .
The d a te
in d ic a te d r e f e r s only to d a te o f p u b lic a tio n and
n o t to th e d a te o u tb reak s o c c u rre d as many r e p o r ts
covered s e v e r a l y e a rs o b s e rv a tio n .
The prim ary
purpose o f th e ta b le s i s to show th e wide d i s t r i b u
tio n o f L« monocytogenes and to p re s e n t a r e a d i ly
a v a ila b le source o f b ib lio g ra p h ic m a t e r i a l.
They
do n o t in c lu d e r e p o r ts in th e le s s commonly i n
fe c te d s p e c ie s which have been c ite d in th e t e x t .
N e ith e r in c lu d ed a re review a r t i c l e s nor those
d e a lin g w ith the c h a r a c t e r i s t i c s o f th e b acteriu m
a s su c h .
These w ill be in clu d ed in a l a t e r s tu d y .
-
28
-
T able 1
I s o l a t i o n o f L . m onocytogenes from sheep
A uthor
Year
Country
G ill
1931
Doyle
1932
USA
Ju n g h e rr
1937
USA
1938-1942
USA
1938
USA
1939.1944
USA
Graham e t a l . *
Morin
B ie s te r & Scbw arte
D o b b erstein**
1940
O lafson
1940
F a lla s k e
1940.1941.1943
New Zealand
Germany
USA
Germany
Cross
1941
USA
Henderson
19 h i
USA
1 9 4 1 ,1 9 4 2
USA
Jen sen & Gay
1941
USA
Math & M o r rill
1942
USA
Hoffman
B e lin & I a g r i f f o u l
1943,1944
F rance
G rin i
1943
Norway
Pomeroy e t a l .
1943
USA
Fothmann
1944
Spencer a t a l .
1944
van U lsen
1944
♦Summarized by Graham ejt a l . (1953)*
♦♦Found ty p ic a l le s io n s b u t made no i s o l a t i o n attem p ts*
Germany
USA
N eth erlan d s
*
29
-
Table 1 ( c o n t.)
I s o la t io n o f L. m onocytogenes from sheep
Author
Year
Country
1947
USA
1947*1951.1951a
USA
Founden & E dgington
1947
USA
R yff & Lee
1948
USA
Je n se n & Mackey
1949
USA
S to en n er a t a l .
1949
USA
E ie la n d & Finborud
1950
Norway
N aerland
1950
Norway
P le tn e v a & S tik so v e
1950
USSR
Fouska
1950
C zechoslovakia
Visw anathan & Ayyar
1950
In d ia
Kaboyashi
1951
Japan
B ain
1952:
Canada
G iffo rd & Ju n g h e rr
Gray et_ a l .
E v e le th
1952,1954
USA
Janos & Gyula
1952
Hungary
Khalimbekov
1952
A zerbai jan
Moll
1952
USA
Odegaard e t a l .
1952
Norway
H ira to et_ a l . *
1954
Japan
McGrath
1954
USA
♦ C ites fo u r re f e r e n c e s in Ja p an e se .
T ajim a, M, Y. Fujim oto and M. I s h ig u r o . 1951*
Y et. Med. ^6: 75*
Sugawa, A and K. M iy a ir i. 1951*
J&P* Y et. Med. A ssoc. 4* 8 0 .
Byogo, Y. 1951. J a p . J . V e t. S c i . 13: 362.
T ab u ch i, E, Y. Akiyama and T. Hosoda. 1952* Exp. Rep. Govt. Exp.
S t a t . Animal H yg., No. 2 5 * 93*
T o ta l r e p o r ts - 44
Table 2
I s o l a t i o n o f L. monocytogenes from g o a ts
Year
Country
King
1940
USA
O lafson
1940
USA
G iffo rd & E v e le th
1942
USA
Ja n se n & van den Hurk
1943
K aplan & Lager
1945
USA
G iffo rd & Ju n g h e rr
1947
USA
N aerland
1950
Norway
A sahi e t a l*
1951
Japan
Hyago & Kato
1951
Japan
Kaboyashi
1951
Japan
Gray e t a l *
1952
USA
Khalimbekov
1952
A z e rb a ija n
A uthor
T o ta l r e p o r ts - 1 2 .
N eth erlan d s
31 T able 3
I s o l a t i o n o f L* monocytogenes from cows
Year
Country
Matthews*
1928 ;
USA
Tones & L i t t l e
1 93 ^
USA
1938-1942
USA
O lafson
1940
USA
B ie s te r & Schw arte
19 41
USA
Schwarte & B ie s te r
194a
USA
Pomeroy el; a l*
1943
USA
Boppensiek
1944
USA
Wramby
1944
Sweden
B atch
1946
USA
Cole
1946
USA
B elin***
1946
F rance
Boucher
1946
USA
Pounden et, a l*
1947
USA
Thorp et^ a l*
1947
USA
Gray et, a l*
194 8-19 31
USA
A uthor
Graham et, a l * **
F is h & S chroder
1949
Canada
Jensen & Mackey
1949
USA
S e l l e r s e t a l*
1949
USA
Z e l le r
1949
♦Found ty p ic a l le s io n s b u t made no i s o l a t i o n a tte m p ts .
♦Sum m arized by Graham e t a l* (1 9 4 3 ).
♦ ♦♦Symptoms only*
Germany
- 32 -
Table 3 ( e o n t .)
I s o l a t io n o f L* m onocytogenes from cows
A uthor
B ain
C hivers
Year
1950
Country
Canada
1950,1931
DBA
Ward
1950
USA
Boyer et_ a l*
1951
USA
Zink e t a l*
1951
USA
van d e r S chaaf et, a l*
1951
Anderson
1952
van d e r Schaaf & de Jong
1952
N e th erlan d s
Geurden & Devos
195k
Belgium
McGrath
1954
T o ta l r e p o r ts - 30
N e th erlan d s
USA
USA
- 33 -
Table 4
I s o l a t i o n o f L« mono ey togene s from a b o rtio n s
A uthor
Y ear
Country
Ovine
B a te rso n
1940
England
B oppensiek
1944
USA
Olson
1945
Sweden
C h arles
1950
A u s tr a lia
E v e le th e t a l *
1953
USA
Graham et, a l *
1939
USA
Evans & Sawyer
1942
USA
Wramby
1942
Sweden
Olson
1945
Sweden
Ferguson e t a l *
1951
USA
Levi e t a l *
1952
Isra e l
van U lsen
1952
N e th e rla n d s
S to ck to n et, a l *
1954
USA
Gray e t a l * *
1954
USA
T o ta l r e p o r ts - 5
Bovine
*Some born p r e m a tu ra lly , o th e r s a t term b u t born dead*
T o ta l r e p o r ts - 9
- 34 -
Table 4 (co n t* )
I s o l a t io n o f L* m onocytogenes from a b o r tio n s
A uthor
Year
Country
A lex and P o te l
1953
DDR
?
Erdmann & P o te l
1953
DDR
10
Hagexnann & Simon
1953
DDR
14
L inzeum eier et, a l*
1953
Germany
1
L inzenm eier
1953
Germany
2
S ta r k
1953
DDR
4
Cases re p o rte d
Human
H o tel*
I 953/ I 954
DDR**
40
71 a s minimum
(alm o st a l l
a re f a ta l)
*Summary o f numerous p re v io u s r e p o r t s .
in fa n tis e n tic a *
**German D em ocratic R epublic*
C a lls b a cteriu m L i s t e r i a
- 35 -
T ab le 5
I s o l a t i o n o f L* monocytogenes from ch ick en s
A uthor
Year
Country
S easto ne
1935
USA
B a terso n
1937,1939
England
B e lin *
1941
F rance
Cole
1941
USA
H urt e t a l*
1941
USA
R a lla sk e
19U
Huffman & Lenarz
1942
Pothmann
1944
Germany
Wramby
1944
Sweden
Feperkamp & Jan sen
1947
N eth erlan d s
S p arap an i
1947
A rg e n tin a
Bi gland
1950
Canada,
P le tn e v a & S tik so v e
1950
USSR
B o lin & Turn
1951
USA
Chadkowski & Czarnowski
1951
Poland
Geurden & Devos
1952
Belgium
van U lsen
1952
N eth erlan d s
Thompson
1954
’•Pound ty p ic a l le s io n s b u t made no i s o l a t i o n a ttem p ts*
T otal rep orts - 18
Germany
USA
USA
36
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P o rz e c a n s k i, B. and C. De B a y g o rria . 1938. L i s t e r e l l a monocytogenes a
p ro p o s ito de su c o n sta c io n en e l Uruguay en una m e n in g o -e n c e fa litis
humana. A rch. Soc. B io l. 9 : 98-116.
P o sto n , M. A ., S . E. Upchurch and M. B ooth. 1937* L i s t e r e l l a m e n in g itis :
r e p o r t o f a d d it i o n a l case w ith necropsy f in d in g s . J . P e d ia t. 11:
515-517.
P o t e l , J . 1950* Die m o rphologie, k u ltu r und tie r p a t h o g e n it a t des
C orynebacterium in f a n tis e p tic u m . Z e n t. f . B a k t. A b t. I . Orig*
1 5 6 : 490 - 493 .
P O te l, J . 1953* A tio lo g ie der G ranulom atosis I n f a n t i s e p t i c a .
Z e i t . M a rtin L uther Univ. 2 : 341-364*
W issen.
-
51
-
P o t e l, J . 1954. Zur Epidem iology d e r L is te r io s e d e r Neugeborenen
( G ranulom atis i n f a n t i s e p t i c a ) . D uuts. G esundheit. 3 : 92 -9 5 .
Pothmann, E . 1944* L is te r e l l a - I n f e k ti o n e n b e i Schafen und Huhnern in
in O stp re u sse n . D tsc h . T i e r a r z t . Wochen. 13/14: 127-129.
Pounden, W. D ., D. S . B e ll and R* E . M a irs. 1947* An O utbreak o f
Acute Bovine L i s t e r e l l o s i s . J . Am. V et. Med. A ssoc. I l l : 128-129*
Pounden, W. D. and H. B. E dgington. 1947*
J . Am. V et. Med. A ssoc. 110: 107.
Pouska, F .
195°*
L is te re llo s is .
L i s t e r e l l o s i s in Ohio.
Gas. Gesko. V et. 5 : 57-58*
R hoades, H. E. and A. K. S u th e rla n d .
1948*
C oncurrent L i s t e r e l l a
J . Am. Vet* Med. A sso o m
m on ocytogen es and hog c h o le r a i n f e c t i o n s .
112: 451- 452.
R o in e, P . , A. R a itio and U. V a rtio v a a ra . 1953* L i s t e r i a in f e c tio n in
the guinea p ig caused by fe e d in g aureom ycin. N ature (London).
172 : 767 .
R y f f , J . F . and A. M. l e e . 1948* L i s t e r e l l o s i s - R eport o f an o u tb re a k
in Wyoming sheep and p re lim in a ry ex p erim e n tal th e ra p y in r a b b i t s .
Am. J . V e t. Res* 9: 147-151*
S av in o , E . 1940* O bservacion de l i s t e r o l i s i s humana y a ccio n t e r a p e u tie a d e l " dageman". Semana med. 1 : 336-339*
Schamesow, L. G. 1949*
(USSR). 11: 7 5 .
( L i s t e r i a in f e c ti o n in m an).
A rch. P a th .
Schm idt, V. and A. N y fe ld t. 1938* Ueber M ononucleosis i n f e c ti o s a und
M e n in g o e n c e p h a litis. Acta o to - la r y n g . 2 6 : 680-688.
Sch oop , G. 1 9 5 1 . L i s t e r i a m on o cy to g en es-, e in K r a n k h e itse r r e g e r u n sere
H a u s tie r e . D ts c h . T i e r a r z t l . Wschr. 5 8 : 293-294*
S c h u ltz , E. W., M. C. T e rry , A. T. B ric e , J r . and L. P . G ebhart. 1934*
B a c te r io lo g ic a l o b s e rv a tio n s on a case o f m en in g o -e n c e p h a litis*
P ro c . S o c . Exp. B io . & Med. 31: 1021-1023*
S ch u lze, W.
1950*
L i s t e r e l l o s e b e i einem A d le r.
S c h u lz e , M. L . , G. H. W ahle, J r . and J . B. W hite.
Mh. f . V et. Med. 5 : 2 0 0 .
1953*
M e n in g itis due
to L i s t e r i a m on ocytogen es in a c a se o f d is s e m in a te d lu p u s e r y th e m a to su s.
Am. J . C lin . P a th . 2 3 : 1 0 2 8 -1 0 3 0 .
S ch w a rte, L. H. and H. E , B i e s t e r .
1942.
L i s t e r e l l a i n f e c t i o n in c a t t l e .
Am. J . V e t. R es. 3 : 165- 176.
S e a sto n e , C. V. 1935* P ath o g en ic organism s o f the genus L i s t e r e l l a .
J . E xp. Med* 62 : 2 0 3 -2 1 2 .
- 52 S e d a ll i a n , P . , J* M oinecourt and R» M aral* 1948* I n f e c tio n s a
e l l a ” » B u ll, e t mem. Soc. Med. H o p it. de P a r i s . 346-350.
S e e li g e r , H. and R. L einew eber. 1952. L is te r is - M e n in g itis in
S ch w an g ersch aft. Munch. Med. Wochen. 94: 1 -3 .
L is te rd er
S e e li g e r , H. F . Ju n g . G. L inzenm eier and H. O denthal. 1952. Die
l i s t e r i o s e beim m enschen.
D tsch . Med. Wochen. 18: 1-14*
S e e li g e r , H. 1953* 2ur S e ro d ia g n o s tik d e r L is te r io s e m i t t e l a d e r
A g g lu tin a tio n s - und Komplementbindungs- R e ak tio n . Z e i t . f . Imm*
Exp. T h e ra . 110: 252-564.
S e e lig e r , H. and G. L in zen m eier. 1953* Die l i s t e r i o s e und ih re e r r e g e r .
(L i s t , m onocytogenes) . Z e its c h r . f . H ygiene. 136: 336-378.
S e l l e r s , A. F . , B. S. Pomeroy, J. H. S a r o t t e r , L. H. P in t and C. E.
S c h r a f e l. 1949* A ty p ic a l p se u d o rab ie s and l i s t e r i o s i s in c a t t l e .
J . Am. V e t. Med. A sso c. 114* 69- 7 3 *
S halkop, W. T. 1950* L i s t e r i a monocytogenes is o la te d from c h in c h il l a s .
J . Am. V et. Med. A ssoc. I l 6 : 447-448*
Shm eleva, V. V. 1953* S lu c h a i g la z o - z h e le z ia ta i forzny l i s t e r e l l e 2a .
V e st. O ft. 3 2 (1 ): 46-47*
Sim on, H. 1953* Ober d ie L i s t e r i e n z e p h a l i t i s .
9 0 : 353-359*
Z en t. A llg . P a th .
S l a b o s p i ts * k i l, T. P . 1938*
n o v ii m ikroorganizm v i d i l e n i i v id
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1 2 , 367 , 1942) .
S lo o f f , J . 1948* Nog een Geval van L is te r e lla - M e n in g itis .
v . Kenchogeneesk. l 6 : 109-110.
M aandschr.
Sm ith, H. C. 1953* I s o l a t i o n o f L i s t e r i a monocytogenes from c h in c h il l a s .
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S p a ra p a n i, J . C. 1947* L i s t e r e l l o s i s de l a s g u l li n a s .
A ir e s . 9s 107-115*
Gac. V e t. B.
S p e n c e r, G. R ., H. H. Hoyt, and C. K. W h iteh a ir. 1944* L i s t e r e l l o s i s
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S ta n le y , N. F* 1948. L i s t e r i a m e n in g itis : A d e s c r ip tio n o f a s t r a i n o f
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Wz 2 o 5 -2 o 8 .
S ta n le y , N. F . 1949. S tu d ie s on L is te r ia m onocytogenes. I .
I s o l a ti o n
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2 7 : 123-131*
S t a n le y , N. F .
1949* S t u d ie s on L is t e r ia m o n o cy to g en es. I I .
R ole o f
L i s t e r i a in i n f e c t i o n s m o n o n u c le o s is . A u s t. J . E xp. B io . & Med.
2 7 : 133 - 1 ^2 .
- 53 -
S ta r c k , C. 1953* Zur K lin ik d e r I n fe k tio n m it L i s t e r i a i n f a n t i s e p t i c a .
Zent* Gyn. 30: 1178-1182.
S to c k to n , J . J . , L isa Neu, W. S. C arp en ter and M. L. G ray.
1954*
The a s s o c i a t i o n o f L i s t e r i a mo no c y to ge ne s w ith a b o r tio n # Review o f
th e l i t e r a t u r e and r e p o r t o f a b o v in e c a s e . J . Am. V e t. Med. A ss o c .
124: 102- 105.
S to e n n e r, H. G ., F . R* Mensimer and R. S. F o s te r . 1949* Ano u tb re a k
o f ovine l i s t e r i o s i s in U tah. J* Am. V et. Med. A s s o c . 115: 174*
S v en k eru d , R. R. 1948* L i s t e r e l l a i n f e k s j o n e r , s p e s i e l t med h e n b lik k
pa d e r e s fo rek o m st h os h e s t e n . N orsk . V e t. T id s k r . 6 0 : 3 2 1 - 3 4 0 *
T ab u ch i, E . t Y. Akiyuma and T. Hosoda. 1952. A case o f equine meningo
e n c e p h a litis which was presumed l i s t e r i o s i s * E x p l. R eport No. 25
o f Gov*t. E x p ri. S t a t . f o r An. Hyg. Tokyo. 89-90.
T e s d a l, M. 1934* F a t a l m e n in g itis c au se d by a C orynebacterium .
Med. Scand. 8 3 : 351-358.
A cta
T h a tc h e r , L . H* F . 1938. A cute le p to m e n in g it is ? L i s t e r e l l a m onocytogenes
i n f e c t i o n . A rch . D i s . C h ild . 13: 285-286.
Thompson, C. H. 1954* Unusual p a th o lo g ic a l changes in a case o f fow l
l i s t e r i o s i s * Am. J . V et. R es. 15: 130-132.
Thorp, F . , J r . , M* L . G ray, W. F . R ile y and L. B. S h o ll. 1947*
o f bovine l i s t e r e l l o s i s . M. S . C. V e t. 8 : 33*
Tompkins, V. N.
1952 *
L i s t e r i a m o n o cy to g en es.
A case
N. Y. S t a t e De p t . H ea lth
B u l l e t i n . 5 s 49*
T raub, E . 1942* Uber ein e m it L is te r e lla - a h n lic h e n B a k te rie n v e r g e s e l ls c h a f te te Meningo±«Bncephalomyelitis d e r K aninchen. Z en t. B a k t.
A b t. I . ( G r i g .) . 149* 38-49*
V a lle e , A* 1952* Un cas de l i s t e r i o s e du l ie v r e en F ra n c e .
P a s te u r Ann. 8 3 : 8 3 2 - 8 3 3 *
In s t.
v . d . S c h a a f, A. and J . M. de Jo n . 1951* L i s t e r i a s i s ( L i s t e r e l l o s i s )
b i j een K a n a rie . T ijd sc h r* D ierg en eesk . 7 6 : 79- 80.
v* d . S c h a a f, A ., J . J* De Jong and J . M. De Jong. 1951* L i s t e r i a
mono cyto ge ne s a l s m o g elijk e verw ekker van een hersenaandoening b i j
h e t ru n d . T ijd s c h r . D ierg en eesk . 7 6 : 51-756*
v . d . Schaaf and J . M. De Jong.
e n c e p h a litis b i j h e t ru n d .
Van D r i e s t , G. W.
1948*
1952 * Nog Twee g e v a lle n van L i s t e r i a T ijd s c h r . D ierg en eesk . 7 7 : 904-907*
L i s t e r e l l a m o n o cy to g en es.
M aandschr. K in d e rg e n e e sk .
1 6 : 101- 109.
Van U ls e n , F . W*
D ie r g e n e e s k .
1952* I n f e c t i e s met L i s t e r i a m o n o cy to g en es.
77: 899-903*
T ijd s c h r .
- 54 -
V ie ra , 0 . and M. C a s te lo . 1948* L i s t e r e l l a monocytogenes en cobay.
Rev. Grancolom biana de Z o o t. H ig. y Med. V et. (C a ra c a s). 2 : 4^7.
V isw anathan, G. R. and V. V. A yyar. 195°* C irc lin g d is e a s e o f sheep
in th e Madras S ta te - E tio lo g y e s ta b lis h e d . In d ia n V e t. Jo u r.
2 6 : 395- 402.
Ward, D. E .
1950*
L i s t e r e l l o s i s in c a t t l e .
No. Am. V et. 31: 805 -So6.
Webb, R. A. and M. B a rb e r.
1937* L i s t e r e l l a in human m e n in g itis .
J . P a th . & B a c t. 45: 523-539.
1943* L i s t e r e l l a monocytogenes is o l a t e d from case o f
in f e c tio u s m o n onucleosis. l a n c e t . 2 : 5 - 10*
Webb, R. A .
Wenkebach, G. K. 1953* ( C u ltiv a tio n o f L i s t e r i a monocytogenes from
th e u r e th r a o f m en). R e is . d e l . Comun. 2 : S ec. 8 - 9 , 4 0 6 .
W in k ler, C. H ., M. F . James and R. D. C arter* 1952.
case r e p o r t . South Med. J o u r. 4 5 s 1181-1183.
L i s t e r i a m e n in g itis
Wramby, G. 0 . 1944* 0m L i s t e r e l l a monocytogenes b a k te r io lo g i och om
forekom st av L i s t e r e l l a i n f e c t i o n hos d j u r . Scand. V et. T id s k r.
3 4 : 278.
W right, H. A. and A. R. MacGregor. 1939* Case o f m e n in g itis due to
B acterium m onocytogenes. J . P a th . &Bact. 4 8 : 4 7 0 -4 7 2 .
Z e l l e r , M. 1949* D ie m o rp h o lo g isch en und k u l t u r e l l m achw eisbaren
b io lo g is c h e n E ig e n s c h a fte n d e r L is t e r i a m o n o cy to g en es . 33 PP*
D is s . G iessen.
Z ink, A ., G. C. de M e llo , and R. L. B u rk h a rt. 1951* L i s t e r i o s i s F ie ld and la b o ra to ry s t u d i e s , and aureom ycin a c t i v i t y * Am. J .
V et. R es. 12: I 94- I 98.
PART I I
COLONIAL DISSOCIATION
COLONIAL DISSOCIATION
INTRODUCTION
From th e l i t e r a t u r e r e v ie w in P a rt I , i t a p p ea rs th a t L. mono
c y to g e n e s i s e x tr e m e ly u b iq u ito u s and s u g g e s ts t h a t t h i s m icroorgan ism
i s a c t u a l l y w id e ly d i s t r ib u t e d i n n a tu r e , perhaps in th e form o f a
s o i l b a cteriu m o r a s a s a p r o p h y te , and th a t o n ly under s p e c i f i c c o n d i
t i o n s d oes i t o c cu r a s a p a th o g e n .
I t i s p o s s i b l e th a t f a i l u r e to
i s o l a t e t h i s organ ism from s o u r c e s o th e r than th e anim al body r e f l e c t s
a f a i l u r e to r e c o g n iz e i t in a n o n -p a th o g e n ic form .
A c c o r d in g ly an i n
v e s t i g a t i o n was c a r r ie d o u t to d eterm in e how many d i f f e r e n t c o l o n i a l
form s would d e v e lo p under normal la b o r a to r y c o n d it io n s .
The o c cu rr e n c e o f wide v a r i a t i o n in c o l o n ia l m orphology betw een
v i r u l e n t and non v i r u l e n t s t r a i n s w ith in th e same b a c t e r i a l s p e c ie s
was r e c o g n iz e d e a r ly in th e h i s t o r y o f b a c t e r io lo g y .
a s e a r ly as 1877 by N a g e li.
v ie w o f th e s u b j e c t .
v ie w o f Braun (1 9 4 7 ).
I t was s u g g e ste d
H adley ( 1 9 2 7 ) p u b lish e d an e x t e n s iv e r e
I t a ls o form ed th e b a s is f o r th e more r e c e n t r e
H uddelson e t a l . (1952) r e c o g n iz e d a t l e a s t 71
v a r ia t io n s w it h in th e th r e e s p e c i e s o f th e genus B r u c e lla .
That fe w er
v a r ia t io n s a r e r e p o r te d f o r o th e r g en era may be o n ly a r e f l e c t i o n o f
l e s s i n t e n s iv e s t u d y .
In s p i t e o f th e r a th e r e x t e n s iv e b ib lio g r a p h y a v a il a b le f o r L. mono
c y to g e n e s . th e r e i s a p a u c it y o f in fo r m a tio n r e l a t i n g to i t s c o l o n i a l
m o rp h o lo g y .
Webb and Barber (1937) appear to have been the f i r s t to
p u b lis h on t h i s s u b j e c t .
They r e p o r te d th e e x is t e n c e o f a t l e a s t two
c o l o n i a l form s d e s ig n a te d rough and sm ooth.
B arber (1939) i n a com parative
- 56 -
stu d y o f L . monoc.ytogenes and B ry s ip e lo th r ix rh u s io p a th ia e d e sc rib e d
sm ooth, in te rm e d ia te and rough c o lo n ia l forms in each s p e c ie s .
P a te rso n
(1940) i a a stu d y o f the p a th o g e n ic ity of L. monocytogenes f o r th e d ev elo p
in g ch ick en embryo found t h a t th re e "rough" s t r a i n s were non p a th o g en ic
w hile " smooth" s t r a i n s produced d eath in 48 h r .
G rini (1943) re p o rte d
t h a t an " a v iru le n t" s t r a i n f a i l e d to k i l l r a b b its when in o c u la te d i n t r a
venously b u t produced a marked m onocytosis in the c i r c u l a t i n g b lo o d .
m ention was made o f colony m orphology.
No
S o h ir ejt a l . ( 1948) is o l a t e d a
" noh pathogenic" s t r a i n o f L. monocytogenes from cooked b e e f , but d id
n o t m ention c o lo n ia l c h a r a c t e r i s t i c s .
S e v e ra l o th e r French a u th o r s ,
Verge and G o ret, 1941» B e lin , 1946} H a r rie r e t a l . , 1943; S e d a llia n e t a l . .
1948; c a lle d a t t e n t i o n to the p resen ce o f "rough" form s dev elo p in g in "smooth"
c u ltu r e s o f t h i s b a c te riu m .
Schoop (1951) observed "rough" c o lo n ie s
which c o n s is te d o f fila m e n to u s c e l l s in one c u ltu r e o f L, m onocytogenes.
S e e lig e r and L inzenm eier (1952) and H irato e t a]L. (1954) d e sc rib e d two
d i f f e r e n t c o lo n ia l ty p e s d e sig n a te d "smooth" and "rough" , but d id n o t
e la b o r a te on th e two ty p e s .
In g e n e r a l, a l l i n v e s tig a to r s who r e p o rte d
th e p resen ce o f rough form s c a lle d a t t e n t i o n to the fila m e n to u s n a tu re
o f th e c e l ls o f rough type c o lo n ie s and t h e i r s t r i k i n g resem blance to
r h u s io p a th ia e .
As f a r as can be determ ined none of th ese i n v e s tig a to r s employed
o b liq u e l i g h t i n g a s d e sc rib e d by Henry (1 9 3 3 )»
f o r s tu d ie s o f t h i s ty p e .
in v a lu a b le tech n iq u e
H unter ejt a l . (1950) o bserved 19 c o lo n ia l
v a r i a ti o n s by t h i s tech n iq u e in a c u ltu re o f L. monocytogenes exposed to
b e ta p a r t i c l e s from r^ d io ph o sp h o ru s.
They found t h a t only a few o f the
v a r i a n t c u ltu r e s d i f f e r e d e s s e n t i a l l y from the p a re n t s t r a i n in fe rm e n ta tiv e
a b i l i t y , b io ch em ical r e a c t i o n s , o r a n tig e n ic s t r u c t u r e .
In g e n e ra l the
- 57 v a r ia n t s showed g r e a t ly d e c r e a s e d p a t h o g e n ic it y f o r em bryonating c h ic k
en e g g s and w h ite m ic e .
No m en tio n was made o f th e a b i l i t y o f the v a r i
a n t s to produce a c i r c u l a t i n g m o n o c y to sis in th e in f e c t e d m ic e .
It
was fo u n d , how ever, th a t la r g e in t r a p e r it o n e a l in o c u la o f th e l e a s t
v i r u l e n t s t r a i n s f a i l e d to p r o t e c t w h ite m ice when s i x w eeks l a t e r th e y
were ex p o sed to a l e t h a l in o c u la t io n o f th e p a r e n t s t r a i n .
MATERIALS AND METHODS
Twenty c u ltu r e s of L. mo no c v to ge ne s were employed in t h i s stu d y .
These in c lu d e d s t r a i n s is o l a t e d from sh eep , cows, g o a ts and man.
They
were s tre a k e d on dry D ifco* try p to s e a g ar p l a t e s in such a manner th a t
both c o n flu e n t and w e ll i s o l a t e d c o lo n ie s developed.
A pproxim ately 20
ml o f a g a r were poured in to s t e r i l e P e t r i d is h e s , allow ed to harden and
in cu b a te d a t 37° C f o r 24 h r .
a t room te m p e ra tu re .
They were in cu b a te d an a d d itio n a l 24 h r
The su rfa c e o f th e a g a r , th e n , was f r e e o f con
d e n sa tio n and read y f o r u s e .
A sm all p latin u m loop ( 1 .0 mm d iam ete r)
was used to in o c u la te th e p l a t e s .
This loop was s u f f i c i e n t l y f le x ib le
to p re v e n t m arrin g th e a g a r s u r f a c e .
The c u ltu re to be examined was
s tre a k e d h e a v ily a lo n g a q u ad ran t o f th e p l a t e , the loop th en was flam ed ,
allow ed to cool and l i g h t l y touched in the p re v io u s ly in o c u la te d a re a
and th e rem ainder o f the p la te in o c u la te d w ith a l i g h t ra p id s e r i e s o f
w idely spaced s t r e a k s .
The colony d i s t r i b u t i o n o f an i d e a l l y in o c u la te d
p la te i s shown in f i g . 1 .
The p la te s were th en in cu b ated a t 37° C f o r
24 h r and examined w ith a b in o c u la r scanning m icroscope equipped w ith a
1 .5 o b je c tiv e and 10 X o c u la r s .
Oblique l ig h t i n g was used a t a l l tim e s.
S e v e ra l o f the c o lo n ia l ty p es were v i r t u a l l y in d is tin g u is h a b le from each
o th e r w ith o u t t h i s te c h n iq u e .
The r e l a t i o n o f the m icroscope to the
l i g h t so urce i s shown in f i g . 2 .
♦ D ifco L a b o r a to r ie s , I n c . , D e t r o i t , M ich ig a n .
F ig . 1 .
An i d e a l l y in o c u l a t e d a g a r p l a t e sh o w in g b o th c o n f lu e n t
and w e l l i s o l a t e d c o l o n i e s . S c u l t u r e a t 3 d a y s .
F ig * 2*
A rrangem ent o f l i g h t s o u r c e , m ir r o r and b in o c u la r
s c a n n in g m ic r o s c o p e . The lamp w as t i l t e d downward
a t a n a n g le o f a p p r o x im a te ly k0 ° a t a d i s t a n c e o f
12*5 cm from th e f l a t s u r f a c e d m ic r o s c o p e s u b s ta g e
m ir r o r p la c e d on a m ic r o s c o p e s l i d e box s e t i n f r o n t
o f th e m ic r o sc o p e * The g r e e n g l a s s m ic r o s c o p e s t a g e
was r e p la c e d by a p h o to g r a p h ic p l a t e from w h ich th e
e m u ls io n had b een rem oved* A l l c u l t u r e s w ere e x
am ined w it h th e P e t r i d i s h o p e n .
- 59 -
- 60 A f te r th e i n i t i a l in c u b a tio n a t 37° C, ih e p l a t e s were allow ed to
s ta n d a t room tem p eratu re u n t i l d is c a rd e d .
D uring th e f i r s t week they
were examined d a ily f o r th e p resen c e o f a ty p ic a l grow th.
T h e re a fte r they
were examined a t i r r e g u l a r i n t e r v a l s u n t i l the p l a t e was e i t h e r contam in
a te d o r too d ehydrated to p e rm it f u r t h e r c o lo n ia l developm ent.
The
ty p ic a l smooth type colony was d e sig n a te d MS *, and any secondary growth
o r s e c to r s o f d i s s i m i la r growth which appeared i n th e c o lo n ie s was a s
signed a number b eg in n in g w ith one and re in o c u la te d and in c u b a te d a s
d e sc rib e d above.
Mien any p a r t i c u l a r c o lo n ia l form f a i l e d to y ie ld
f u r t h e r d i s s i m i la r forms a f t e r s e v e r a l p la tin g s and long p e rio d s o f
in c u b a tio n , one o f the c o lo n ie s was tr a n s f e r r e d to a try p to s e a g a r s la n t
and s to re d in the r e f r i g e r a t o r a t 4° 0 u n t i l o th e r t e s t s were to be
c a r r ie d o u t.
In some in s ta n c e s the smooth c o lo n ie s were in o c u la te d in to tr y p
to se b ro th and allow ed to in c u b a te a s d e sc rib e d above.
At a p p ro p ria te
i n te r v a l s th e s e c u ltu r e s were s tre a k e d on a g a r p l a t e s , in c u b a te d and
examined f o r th e presen ce o f d is s im ila r forms*
S e v e ra l s t r a i n s o f b oth smooth and rough c u ltu r e s were p la te d on
try p to s e a g ar to which 2 , 3 * 5 - tr ip h e n y lte tr a z o liu m c h lo rid e was added*
-A 1*0% sto c k s o lu tio n o f t h i s s a l t was p rep a red in d i s t i l l e d w ater and
a u to c la v e d f o r 15 min a t 115° C,
One ml o f t h i s s o lu tio n was added to
100 ml o f m elted a g a r , mixed w e ll and p la te s p rep a red as d e sc rib e d above.
C u ltu re s a ls o were in o c u la te d in to try p to s e b ro th c o n ta in in g the same
c o n c e n tra tio n o f te tra z o liu m s a l t and p la te d a f t e r v a ry in g p e rio d s o f
in cu b atio n *
Smears o f c o lo n ie s r e p r e s e n ta tiv e of each o f the v a rio u s ty p es were
- 6l s ta in e d w ith e i t h e r th e Hucker m o d ific a tio n o f the gram s t a i n o r w ith
W rig h t's s t a i n .
C o n siste n c y -o f the c o lo n ie s was determ ined by s t r o k
in g o r pushing them w ith a sm all lo o p .
T his was done under the scan n in g
m icroscope*
The c o lo n ie s were photographed by p la c in g over the s ta g e o f th e
m icroscope a 5 1 7 Eastman C l in ic a l Camera equipped w ith a 2j2 mm m ic ro te s s a r le n s .
ment lamp*
The l i g h t source was a Bausch and Lomb 6 v o l t rib b o n f i l a
The r h e o s t a t was s e t a t the number 3 p o s i t io n .
p hotos were made on 2 1 /4 X 3 1 /4 s h e e t type B Ektachrom e.
was 1 1/2 seconds w ith th e le n s diaphram open*
The c o lo r
Exposure
Development was in E a s t
man Kodak Ektachrome p ro c e ss in g chem icals su p p lie d in k i t form .
c o lo r p r i n t s were made by Drewry P h o to co lo r C orp.*
The
The b la c k and w hite
ph o to s were made on 5 X 7 sh e et Isopan exposed f o r 1/2 second w ith the
le n s diaphram open*
They were developed f iv e m in u tes in Eastman Kodak
DK-11 and fix e d in Eastman Kodak r a p id f i x f o r 3 m in.
The m a g n ific a tio n
was th e same f o r a l l photos and was approxim ately 12 times*
The biochem ical and fe rm e n ta tio n re a c tio n s o f a number o f nohsmooth c u ltu r e s were compared w ith th e r e a c tio n s o f the p a re n t smooth
c u ltu re *
F erm en tatio n r e a c tio n s were c a r r ie d o u t on 20 ferm en tab le
su b sta n c e s a s in d ic a te d in r e s u l t s .
These compounds were added to a
b a s a l medium c o n ta in in g b a c to tr y p to s e , b eef e x t r a c t , sodium c h lo rid e
and d i s t i l l e d w a te r.
Bram c re s o l p u rp le was used as in d ic a t o r .
A ll
m edia were s t e r i l i z e d by a u to c la v in g a t 15 pounds p re s su re f o r 15 min
w ith th e e x c e p tio n o f d e x tr in and m alto se which were f i l t e r e d through a
f r i t t e d g la s s f i l t e r .
♦C h icago, 111
A ll r e a c tio n s were observed f o r 14 d a y s.
- 62 -
B iochem ical r e a c tio n s were c a r r ie d o u t u sin g media and methods
d e s c rib e d in "The Manual o f Methods f o r the Pure C u ltu re Study o f
B a c te ria " •
P a th o g e n ic ity s tu d ie s in r a b b i t s were c a r r ie d o u t w ith s e v e ra l
c u ltu r e s from non-sm ooth c o lo n ie s .
Each c u ltu re to be employed was
tr a n s f e r r e d to tr y p to s e agar s l a n t s and in cu b a te d a t 37° C fc>r approxim
a t e l y 18 h r .
The r e s u l t i n g growth was washed o f f w ith s t e r i l e d i s t i l l e d
w a ter and a d i s t i l l e d w ater su sp en sio n was p rep a red which corresponded
in d e n s ity to the number one tube o f the McFarland n ephelom eter.
Gener
a l l y a 0 .5 ml p o r tio n o f t h i s su sp en sio n was in o c u la te d in tra v e n o u s ly .
In a few In sta n c e s l a r g e r o r sm a lle r in je c tio n s were g iv e n .
Dosage
was based on u n p u b lish ed o b se rv a tio n s which re v e a le d t h a t t h i s approxim ated
a minimum l e t h a l d o se .
D i f f e r e n t i a l leukocyte counts were taken im m ediate
l y p rec ed in g in o c u la tio n and a t 24 h r i n te r v a l s f o r the n ext s e v e ra l
d a y s.
In some in s ta n c e s body te m p e ra tu re s a ls o were re c o rd e d .
To determ ine th e a b i l i t y o f some o f th e non-smooth c u ltu r e s to
produce a c o n ju n c ti v i ti s in the r a b b i t e y e, dense d i s t i l l e d w ater s u s
p en sio n s were p re p a re d from c u ltu r e s grown a t 37°G f o r a p p ro x im ately
18 h r .
With th e a id o f a c a p i l l a r y p ip e tte a few drops o f t h i s suspen
s io n were i n s t i l l e d in to the c o n ju n c tiv a l sac o f th e l e f t e y e .
The
r i g h t eye serv ed as c o n tr o l.
C u ltu r es from fo u r d i f f e r e n t c o l o n i a l ty p es were u sed to d eterm in e
p a t h o g e n ic it y in r a b b it s by th e o r a l r o u t e .
R - l l and R-14#
T hese were typ e S , R-7*
Each type was grown on try p to s e a g a r in a f l a t sid e d
a n tig e n b o t tl e f o r 24 h r a t 37° C.
The growth was washed o f f w ith ta p
w a ter and t h i s c o n s titu te d the o n ly source o f f l u i d in ta k e f o r the r a b b its
- 63 -
f o r a p e rio d o f two months*
The r e s u l t i n g su sp en sio n corresponded rough
l y to th e number 10 tube o f the McFarland nephelom eter when compared
in a p h o to lo m e te r* For a p e rio d o f two weeks th e mouth o f one r a b b i t
in each group was deeply la c e r a te d ev ery t h i r d day ju s t b e fo re the
c u ltu r e was given#
Two r a b b its in the group re c e iv in g type S and one
r a b b i t in each o f th e o th e r groups were fe d on ly e n s ila g e d u rin g the two
month p e r io d .
The rem ain in g r a b b its re c e iv e d K ellogg r a b b i t p e lle ts *
At i n t e r v a l s the fe c e s were c u ltu re d f o r the p resence o f the c o lo n ia l
type b ein g fed*
The f e c a l p e l l e t s were m acerated in a Waring b len d o r
to g e th e r w ith ap p ro x im ately 15 ml o f try p to s e b ro th c o n ta in in g 0 *15%
p o tassiu m t e l l u r i t e *
The r e s u l ti n g su sp en sio n was in c u b a te d o v e rn ig h t
a t 37° C and th en p la te d on try p to s e agar*
The p l a t e s were in cu b a te d
a t 37° C f o r 18 h r and examined under th e scanning m icroscope f o r th e
p resen ce o f the a p p ro p ria te c o lo n ia l type*
A g g lu tin a tio n r e a c tio n s were c a r r ie d o u t u sin g a n t i s e r a from
r a b b its which su rv iv e d th e in tra v en o u s in o c u la tio n s*
A ntigens were
p rep a red by growing th e v a rio u s c u ltu r e s to be s tu d ie d on try p to s e a g a r
s la n ts *
In c u b a tio n took p la c e a t 37
A
C f o r ap p ro x im ately 18 hr*
The
r e s u l t i n g growth was washed o f f and suspended in 0.85% s a lin e and s ta n d a r d
iz e d w ith 0*85% s a lin e to a d e n s ity co rresp o n d in g to th e number two
tube o f th e M cFarland nephelom eter*
The c u ltu re s were k i l l e d by i n
c u b a tio n f o r one h r in a w ater b a th a t 80° C*
D ilu tio n s were p rep a red
a s o u tlin e d in HThe Manual o f Methods f o r the Fure C u ltu re Study o f
B a cteria" * A fte r a d d itio n o f th e a n tig e n the tu b es were in c u b a te d f o r
two h r in a w a ter b a th a t 37° C and s to r e d in the r e f r i g e r a t o r o v e r
n ig h t*
A fte r rem oval from the r e f r i g e r a t o r the tu b es were allow ed to
rem ain a t room tem p eratu re f o r 30
a f t e r which the r e s u l t s were re a d .
-
64
-
As some non-smooth c u ltu r e s produced h ig h a g g lu tin a tin g t i t e r s
a g a in s t smooth c u l t u r e s , a tte m p ts were madte to v a c c in a te r a b b i t s w ith
th e se non-sm ooth c u ltu re s *
f o r in tra v e n o u s in o c u la tio n *
The c u ltu r e s used were p rep ared as d e sc rib e d
These were in o c u la te d su b cu tan eo u sly in
doses ra n g in g from 1*0 ml to 5*0 m l.
As e a r l i e r unpublished work r e
v e a le d t h a t a lo c a liz e d n e c r o tic le s io n developed a t the s i t e o f sub
cutaneous in o c u la tio n , 15 TR ( tu r b id ity - r e d u c in g ) u n i ts o f Wydase* were
added to some o f the su sp e n sio n s b efo re in o c u la tio n .
A f te r two months
th e r a b b i t s were c h alle n g ed w ith a l e t h a l dose o f v i r u l e n t smooth c u lt u r e .
RESULTS
E xam ination o f the a g a r p l a t e s w ith the scan n in g m icroscope r e v e a l
ed th e p resen ce o f a t l e a s t 19 d i f f e r e n t c o lo n ia l forms a r i s i n g e i t h e r
d i r e c t l y from th e p a re n t smooth c u ltu re o r from one o f i t s non-sm ooth
c o lo n ies*
D iffe re n c e s were d e te c te d by v a r i a ti o n s in t e x t u r e , c o n s is te n c y ,
c o lo r , o p a c ity , o r morphology o f th e r e s u l t i n g grow th.
I t o f te n was
d i f f i c u l t to d is tin g u is h two c o lo n ia l forms when th ey grew w idely s e p a r
a te d from each o th e r on the a g a r p l a t e , b u t i t was r e l a t i v e l y e asy to
d e te c t the more s u b tle d if f e r e n c e s in e i t h e r c o lo r o r te x tu r e when th ey
were a d ja c e n t to each o t h e r .
Fig* 14 and 25*
The d e n s ity and c h a r a c te r
o f th e p o p u la tio n had a marked e f f e c t on the appearance o f many o f the
c o lo n ia l ty p e s .
Dense mixed p o p u la tio n s produced marked changes in colony
s iz e and c o lo r , and the tr u e d e s c r ip tio n o f th e colony could be made only
i f i t could be grown in pure c u l t u r e .
Many o f th e c o lo n ie s were never
produced in pure f o m in s p i t e o f re p e a te d p l a t i n g s , but alw ays gave r i s e
to a m ix tu re o f c o lo n ia l forms*
*Wyeth I n c . , P h ila d e lp h ia , P e n n sy lv a n ia , brand o f h y a lu ro n id ase
- 65 -
I t sh o u ld be p o in te d o u t t h a t the term s " smooth" and " rough" as
a p p lie d to b a c t e r i a l c o lo n ie s do n o t always c a rry the same connotatio n *
As t h i s stu d y was devoted p r im a r ily to a r e c o g n itio n o f c o lo n ia l ty p es
th e two term s were employed r a t h e r lo o se ly *
S tu d ie s on a g g l u t i n a b i l i t y
in a c r i f l a v i n e (Braun and B o n e s te ll, 1947) and s t a b i l i t y in s a l t s o lu
t io n a s c r i t e r i a o f sm ooth, ro u g h , o r mucoid types were no t c a r r ie d out*
The fo llo w in g term s were a p p lie d to c h a ra c te r iz e the v a rio u s c o lo n ia l
ty p e s :
Smooth (S) - The sm all c i r c u l a r type colony w ith e n ti r e m arg in , f in e ly
te x tu re d su rfa c e and found th u s f a r o n ly in anim al tis s u e
o r body f lu id s *
H ighly p a th o g e n ic f o r r a b b its *
In te rm e d ia te ( I ) - The la rg e f l a t sp re a d in g type d au g h te r colony
a r i s i n g from smooth type c o lo n ie s and which alw ays g iv es
r i s e to a m ix tu re o f smooth and non-smooth c o lo n ie s , but
n ev er to a pure c u l t u r e .
P athogenic f o r r a b b its *
Rough (R) - The r e l a t i v e l y la rg e type colony w ith ero se to lo b a te edge,
c o a rs e ly te x tu r e d s u rfa c e .
Most fr e q u e n tly found as
s e c to r s o r d au g h te r c o lo n ie s d eveloping from th e i n t e r
m ediate ty p e .
O c c a sio n a lly found d i r e c t l y as s e c to rs o r
d a u g h te rs in smooth type c o lo n ie s .
o b ta in e d i n pure c u lt u r e .
Play o r may not be
Non p ath o g en ic f o r ra b b its *
In s p i t e o f e n ti r e m argin and smooth s u r f a c e , type 11
was in c lu d e d h e re on the b a s is o f developm ent.
Non-smooth (N-S) - Any form o th e r th an t h a t d e sc rib e d under .smooth*
C olor o f te n played an im portant p a r t in d is tin g u is h in g one c o lo n ia l
form from another*
As c o lo r i n t e r p r e t a t i o n i s o fte n l a r g e ly s u b je c tiv e
i t i s d i f f i c u l t to a s s ig n d e f i n i t e c o lo r v a lu e s to an o b je c t u n less i t i s
- 66 compared to some sta n d a rd such as th e "M unsell Book o f C o lo r, 19i|2n ,
a tec h n iq u e which Huddelson (1952.)employed*
com parison was made*
In t h i s stu d y no such
However, the most im p o rtan t c o lo n ia l ty p es are
rep ro d u ced by c o lo r p r in ts *
U n fo rtu n a te ly , due to the in h e re n t d i f f i
c u l t i e s o f c o lo r p r i n t i n g , th e c o lo rs a re not too e x a c t and in some,
most o f th e to p o g ra p h ic a l f e a tu r e s a re l o s t .
However, i t i s hoped th a t
th ey w i l l convey to a sm all degree some o f the more s u b tle f e a tu r e s of
th e v a rio u s colony ty p es which can be f u l l y a p p re c ia te d on ly when viewed
d i r e c t l y through th e m icroscope*
The o r i g in and d e s c r ip tio n o f the p r i n c i p a l colony ty p es i s given
o p p o site the photographs which appear on th e fo llo w in g pages*
Fig. 3.
Type S
Found i n a l l i s o l a t i o n s from an im al o r human t i s s u e o r
body f l u i d s .
Agar c o lo n ie s a t 2 4 h r e r e p u n tif o im , t r a n s l u c e n t ,
s l i g h t l y r a i s e d w ith a f i n e l y te x tu r e d s u r f a c e and
e n t i r e m a rg in , b lu e - g r e e n i n c o l o r . W atery i n con
s i s t e n c y . A f t e r f i v e to te n days w e ll i s o l a t e d
c o lo n ie s re a c h a d ia m e te r o f two to f i v e mm, a r e more
b lu e i n c o l o r , s l i g h t l y um bonate and ru b b e ry i n con
s i s t e n c y . May show e v id e n c e o f d a u g h te r c o lo n ie s
o r s e c t o r f o r m a tio n . C e lls s s h o r t ro d s 1 . 0 to 1 . 5 N
X 0 . 5 m ic ro n s . Some c o c c o id , c u rv e d , Y and T fo rm s .
P a lis a d e arran g em en t common. Few lo n g form s up to
7 . 0 m ic ro n s i n le n g t h .
Tum bling m o t i l i t y . H ighly
p a th o g e n ic .
F i g . 4*
Ten day o ld S c u lt u r e on a g a r c o n ta in in g 2 , 3 , 5 t r ip h e n y lt e tr a z o l i u m c h l o r i d e , show ing f o u r d i s t i n c t
grow th z o n e s ; deep p u r p le c e n t e r , r e d , p in k and p a le
g re e n a t p e r ip h e r y . I t h as n o t been d e te rm in e d i f
each zone g iv e s r i s e to a c h a r a c t e r i s t i c c u l t u r e .
- 67 -
Fig. 5*
Types 1-1, 1-2, R-4 and R-5.
F i r s t a p p e a r a f t e r ab o u t f i v e days in c u b a tio n o f S ty p e
s to c k c u l t u r e s . R a re ly d e v elo p i n f r e s h l y i s o l a t e d S
ty p e c u l t u r e s . L arge s p r e a d in g in te r m e d ia te ty p e , f l a t
c o a rs e s u r f a c e , t r a n s l u c e n t , fila m e n to u s m a rg in , s t r i n g y
in c o n s is te n c y .
1 -1 .
Are p a le to b r i g h t g re e n d ep en d in g on a g e .
1 - 2 . Are p a le to b r i g h t b l u e . P l a t i n g g iv e s r i s e to
c o lo n y ty p e s S , R-4* an&
i n d i f f e r e n t p r o p o r t io n .
1-2 g iv e s more S th a n d o es I->1.
R-4* A rise from p l a t i n g s o f 1-1 o r 1 - 2 . Agar c o lo n ie s
a t 2 4 h r a r e sm a ll i r r e g u l a r , s l i g h t l y o p aq u e, r a i s e d
to um bonate, c o a r s e ly grooved s u r f a c e , u n d u la te to lo b a te
m a rg in , b l u e , w a tery * C e l l s : S im ila r to type S b u t more
lo n g fo rm s . Tum bling m o t i l i t y . P a th o g e n ic .
R-5* A ris e from p l a t i n g s o f 1 -1 o r 1 - 2 . Agar c o lo n ie s
a t 2 4 h r a r e l a r g e r , i r r e g u l a r to f ila m e n to u s , opaque*
s l i g h t l y r a i s e d , f i n e l y te x t u r e d to d e e p ly grooved s u r
f a c e , lo b a t e to f ila m e n to u s m a rg in , cream to p a le y e llo w ,
w a te r y . C e lls : S im ila r to R-4 b u t more lo n g fo rm s.
May show few f i l a m e n t s . Tum bling m o t i l i t y . P a th o g e n ic .
T hese ty p e s c a n n o t be m a in ta in e d p u r e . P l a t i n g g iv e s
r i s e to ty p e S c o lo n ie s w hich v e ry q u ic k ly d e v elo p type
1 - 1 , 1-2 and R-3 d a u g h te r c o lo n ie s ; ty p e R-4 and R-5
c o lo n i e s ; o c c a s i o n a l l y ty p e R-3* R-7 and R - l l c o lo n ie s .
R-5 d i s s o c i a t e s more r a p i d l y th a n R-4* and type R-3
d a u g h te r c o lo n ie s can be se e n a s e a r l y a s 24 hr*
F ig . 6 .
Type R -3 .
A r is e a s d a u g h te r c o lo n ie s from f i v e to te n -d a y o ld type
S c o l o n i e s , o c c a s i o n a l l y from p l a t i n g s o f R-4 and R-5*
D evelop r a p i d l y i n some f r e s h l y i s o l a t e d S -ty p e c u lt u r e s .
A gar c o lo n ie s a t 24 h r a r e f ila m e n to u s to i r r e g u l a r ,
s l i g h t l y r a i s e d w ith opaque c e n t e r , c o a r s e ly rid g e d s u r
f a c e , e ro s e to fila m e n to u s m a rg in , g re e n c o l o r . A f te r
f i v e to te n d ay s c o lo n ie s r e a c h a d ia m e te r o f two to
f i v e am o r m o re. L i t t l e change i n c o lo r . Become ru b
b e ry i n c o n s is te n c y * C e l l s : M ostly r a t h e r lo n g s le n d e r
f i la m e n t s and lo n g ro d s t h r e e to sev en m ic ro n s in le n g th ,
few s h o r t fo rm s . Non m o tile * May be p a th o g e n ic *
- 68 -
At l e f t , te n day o ld S type colony from p l a t i n g non
pure R-3 colony showing la rg e and sm all 1-1 and 1-2
d a u g h te rs. At r i g h t , sm all b lu e , umbonate R-4 and
la r g e r f l a t cream to yellow R-5 c o lo n ie s . 24 h r
c u ltu r e from 1-1 p l a t i n g . Sm all R-3 d a u g h te rs can
be seen on th e R-5 c o lo n ie s . Also see f i g . 19 and 2 0 .
At l e f t , f iv e day o ld S type colony w ith type R-3
d a u g h te r. At r ig h t', 48
c u ltu r e r e s u l t i n g from
p l a t i n g o f R-3 d au g h ter showing m ixture o f S and
R-3 grows more slow ly th an type S . Can be p r o
duced pure a f t e r re p e a te d p l a t i n g s .
F i g . 7 an d 8 .
Type R-7#
A r is e a s s e c t o r s i n ty p e R -3 , R-4 o r R-5 type
c o lo n ie s o r from 1- 1 , 1 -2 , o r R-3 ty p e d a u g h te rs
d e v e lo p in g on S c o l o n i e s . O c c a s io n a lly a r i s e
d i r e c t l y from S ty p e c o lo n ie s which r e s u l t from
p l a t i n g o f 1 - 1 , 1 -2 , o r R-3 ty p e c o lo n ie s . Seldom
d e v e lo p s d i r e c t l y from S c o lo n ie s i s o l a t e d from
a n im a l t i s s u e . Not p a th o g e n ic .
A gar c o lo n ie s a t 24 h r a r e fila m e n to u s to i r r e g u l a r ,
t r a n s l u c e n t , s l i g h t l y r a i s e d d e e p ly rid g e d su rfa c e
w ith e ro s e to f ila m e n to u s m a rg in , p a le p in k in
c o l o r , w a te ry i n c o n s is te n c y . A f te r two to fo u r
d a y s th e co lo n y becomes more f l a t t e n e d w ith a
h ig h ly fila m e n to u s m a rg in and changes to a p a le
g re e n c o l o r . A fte r a b o u t fo u r days p in k to gold
f i l a m e n t s , d e s ig n a te d ty p e R-13» d e v elo p from
th e m a rg in , w hich when p l a t e d g iv e r i s e to type
R - l l and R-14 c o l o n i e s , o c c a s io n a lly to R-3*
C e l l s : u s u a l ly s h o r t plump ro d s 2 to 4 X 1 m icro n s,
o f t e n i n p a i r s o r s h o r t c h a in s , no p a li s a d in g o r
f i l a m e n t s , non m o t i l e .
-
6?
.
At l e f t , type S colony from p l a t i n g o f R-3 colony
a f t e r seven days in c u b a tio n . Large s e c to r s o f 1 -1 ,
1 -2 , and R-3* Conspicuous s e c to r o f R -7. S
colony a t c e n te r .
From l e f t to r i g h t , pure c u ltu re o f R-7 a t 24# 48
and 96 h r in c u b a tio n showing marked change in s i z e ,
c o n to u r and c o lo r . Same c o lo n ie s in each s e c t i o n .
F ig . 9 and 10.
Type R-ll.
A r i s e a s f i l a m e n t s from fo u r t o te n - d a y o ld R-7
c o l o n i e s . O c c a s io n a lly from s i m i l a r f ila m e n t s
d e v e lo p in g an o l d ty p e R -3 c o l o n i e s . R a r ely
d e v e lo p s d i r e c t l y a s a s e c t o r i n ty p e S c o lo n ie s *
N ot p a t h o g e n ic .
A gar c o lo n ie s a t 2 4 h r a re c i r c u l a r , opaque, f l a t
to convex w ith sm ooth s u rfa c e and e n t i r e m arg in ,
p a le to b r i g h t p in k i n c o lo r , w a te ry to s t r i n g y
i n c o n s is te n c y . A f t e r ab o u t f o u r to s ix days th e
c o lo n ie s a r e 2 - 4 mm i n d ia m e te r and a p p ea r v e ry
f l a t w ith e n t i r e to s l i g h t l y i r r e g u l a r m argin*
Kay o r may n o t d e v e lo p seco n d a ry f i la m e n t s .
C o lo n ie s w hich d e v e lo p no se co n d a ry fila m e n ts
r e t a i n th e p in k c o l o r , b u t when f ila m e n ts a r e
p ro d u ced th e c o lo n y becomes a p a le g re e n w ith
s l i g h t l y cream c e n t e r . T h is may r e p r e s e n t two
d i s t i n c t c o lo n y t y p e s , b u t has n o t been co n firm ed .
F ila m e n ts when p l a t e d g iv e r i s e to type R-14 and
R-15 c o lo n i e s . C e l l s : S h o rt plump ro d s 2 to 4
X 1 m ic ro n s i n p a i r s , c h a in s , o r p a l i s a d e s , many
c u rv e d , r a r e l y s h o r t f i l a m e n t s , non m o t i l e .
- 70 -
At l e f t , seven day old S type colony w ith con
spicuous R - ll secto r a t top . A ctually t h is i s
r a r e ly se en . At r ig h t , same colony a t 11 days.
R - ll se c to r now blu e-green w ith f a in t gold R-13
a t margin. 1-2 a t lower l e f t .
From l e f t to r ig h t, pure culture o f R - ll a t 2 4 ,
Zj.8 and 96 hr incub ation showing marked change
in s iz e but s im ila r ity o f margin and c o lo r .
At r ig h t , secondary filam en ts w ith R-13 a t mar
g in . Same c o lo n ie s in each s e c tio n . Contam
inant a t bottom.
1
F i g . 11 and 1 2 .
Type R -14#
A r is e a s f il a m e n t o u s d a u g h te r s from f i v e t o t e n d ay o l d ty p e R -7 o r R - l l c o l o n i e s . N ever d e v e lo p s
d i r e c t l y from ty p e S c o l o n i e s . Tendency to be
u n s t a b l e , b u t can be o b t a in e d i n pure fo r m .
Non p a t h o g e n i c .
A gar c o l o n i e s a t 2 4 h r a r e fila m e n t o u s t o i r r e g u l a r ,
t r a n s l u c e n t t o o p a q u e , s l i g h t l y r a i s e d , w ith
d e e p ly r id g e d and i r r e g u l a r s u r f a c e , e r o s e to
f ila m e n t o u s m a r g in , b r ig h t p in k , w a te r y . A f t e r
48 h r th e c o l o n i e s a r e f l a t , f il a m e n t o u s , sp r e a d
in g and become p a le g r e e n i n c o l o r . A f t e r s e v e r a l
d a y s may r e a c h 10 t o 15 mm d ia m e t e r . F ila m e n ts
when p l a t e d g iv e r i s e t o ty p e R-15 c o lo n y . C e lls :
Long plump r o d s 3 t o J X 1 m ic r o n s in c h a in s or
lo n g h e a v y f i l a m e n t s , many c u r v e d , no s le n d e r o r
S ty p e c e l l s . Non m o t i l e .
Mixture o f 5 day old S and R-7 c o lo n ie s from
p la tin g o f 1 -2 . At upper l e f t pink R-13 which
g iv e s r is e to R-14 and R-15 c o lo n ie s . At
bottom a few S c o lo n ie s w ith blue h a lo s .
ii
From l e f t to r ig h t, pure cu ltu re o f R-14 a t
2 4 , 48 and 72 hr showing marked change in
s i z e , contour and c o lo r .
each s e c tio n .
Same c o lo n ie s in
F ig * 13*
P l a t i n g o f R-10 c o lo n y a t 2 4 h r show ing m ix tu re of
R -3 and R-1% c o lo n ie s * D if f e r e n c e i n c o lo r and
t e x t u r e m ost pro n o u n ced i n c o n flu e n t grow th and a
few S c o lo n ie s can be s e e n .
F ig . 14.
P l a t i n g o f R-10 c o lo n y a t 48 h r show ing m ix tu re o f
R -3 , R-7 and R-14 c o l o n i e s . C o lo rs a re n o t e x a c tly
t r u e and th e i n d i v i d u a l c o lo n y ty p e s a r e d i f f i c u l t
to d i s t i n g u i s h on t h i s p r i n t .
- 72 -
P ig # 15 •
P~7 c o l o n i e s a t 16 d a y s on a g a r c o n t a in in g 2 , 3» 5 t r i p h e n y l t e t r a z o l i u m c h l o r i d e . P a le p in k d a u g h te r
a t f a r r i g h t g i v e s r i s e to m ix tu r e o f R -7 , R - l l , R -1 4 ,
R -l6 and R-17# P a le g r e e n s e c t o r s a t c e n t e r u s u a l l y
g i v e r i s e t o p u re R -3 o r m ix tu r e o f R-3 and R - l 6 .
R-16 d a u g h te rs ( n o t shown a r e a b r i g h t e r g re e n , more
c o a r s e l y t e x t u r e d , and a lm o st t r a n s p a r e n t . C e n te r
o f c o lo n y g iv e s r i s e to p u re R-7#
F ig . 16.
P l a t i n g o f R - l l a t f i v e days on a g a r c o n ta in in g 2 , 3*
5 - t r i p h e n y l t e t r a z o l i u m c h l o r i d e , show ing m ix tu re o f
r - 7 , R - l l and R-14 and d i f f e r e n c e i n a b i l i t y to reduce
th e s a l t . F in e p in k grow th i n background i s R -1 8 , a
p o o r ly u n d e rs to o d v a r i a n t which a p p e a rs o n ly in c u l
t u r e s grown on t e t r a z o l iu m s a l t . U s u a lly no grow th
when t r a n s f e r r e d to p l a i n ag ar*
- 73 -
17•
F ig . 1 8 .
P l a t i n g o f I —1 c o lo n y a t 48 h r show ing m ix tu re o f
R - l l (sm ooth s u r f a c e , e n t i r e m a rg in ) and R-7
( g r a n u l a r s u r f a c e , fila m e n to u s m arg in )*
Same c o lo n ie s a s i n F i g . 17 a f t e r f i v e days* R - l l
unchanged e x c e p t f o r s l i g h t i n c r e a s e i n s i z e . S t i l l
r e t a i n s p in k c o l o r . R-7 m arked in c r e a s e i n s i z e due
to e x te n s io n o f f ila m e n to u s R-13* Now p a le g re e n in
c o l o r . R-13 p in k to g o ld . Ms Id and two co n sp icu o u s
c o n ta m in a n ts i n f i e l d .
- 74 -
F ig # 19 #
R -4 and R-5 ®t t h r e e days#
Compare w ith F ig# 5
F ig# 20#
Same c o lo n ie s a s F ig# 19 a t sev en days show ing b o th
1-1 and 1-2 d a u g h te r s .
-
75
-
9
Ii
F ig * 21*
R -7 and o n e c o lo n y R - l l a t 10 d a y s* F ila m e n ts c o a le s c e d
an d f l a t t e n e d a p p e a r a n c e . L ig h t c o lo r e d c e n t r a l a r e a
m arks s i t e o f o r i g i n a l c o lo n y * R - l l h a s s m a ll R -13
d a u g h te r .
F ig . 2 2 .
Pure R - l l a f t e r 2 0 d a y s sh o w in g u n ifo r m d i r t y p in k t o
p a le g r e e n c o l o r and v e r y s m a ll f i l a m e n t o f R—13*
- 76 -
F ig * 2 3 #
R - l l from p l a t i n g R -12 c o lo n y a t 10 d a y s sh o w in g
d e v e lo p m e n t o f la r g e R -13 d a u g h t e r s . R - l l a r e
p in k and R -13 d a u g h te r s a r e g r e e n w it h g o ld m a r g in .
F i g . 2%.
Same a r e a a s F i g . 2 3 a t 2 1 d a y s sh o w in g u n ifo r m c o lo r
w h ic h i s now g r e e n . T h is i s a s t r i k i n g c h a r a c t e r i s t i c
o f a l l R t y p e s t h a t r e g a r d l e s s o f th e c o l o r o f young
c o l o n i e s a s t h e y a g e th e y become a p a le g r e e n s i m i l a r
t o o ld S c o l o n i e s . The o n ly e x c e p t io n i s R - l l w h ich
r e t a i n s th e p in k c o lo r when no o r o n ly s m a ll R -13
d a u g h te r s d e v e lo p . S e e F i g . 18 and 2 2 .
- 77 -
F i g , 25*
P l a t i n g of R-4 c o lo n y sh o w in g mixture of R-3* R-7
and R-ll colonies at 24 hrs. R-3 c©n
distinguished
from R-7 (pink) by lighter color (green) and smaller
size. R-ll can be distinguished by smooth surface
and entire margin. R-3
R-7 may be difficult to
distinguish as isolated colonies.
F ig . 2 6 .
Pure c u lt u r e o f R-17 a t s i x d a y s . T h is o r i g i n a t e d
from an R -3 c u l t u r e e x p o se d to 2 , 3» 5 “ t r i p h e n y l t e t r a z o l iu m c h lo r id e * S im ila r t o t h e on e shown i n
F i g , l 6 . T h is c o lo n y i s c h a r a c t e r iz e d by an a lm o s t
sm ooth p in k r a i s e d c e n t e r w h ich when y o u n g ( 2 4 - 4 ®
h r ) i s a lm o s t i d e n t i c a l t o R - l l e x c e p t t h a t i t i s
l a r g e r . A f t e r s e v e r a l d ays a c o a r s e l y t e x t u r e d h a lo
d e v e lo p s w h ich when p l a t e d u s u a l l y g i v e s r i s e t o R - l 6 .
T h is c o lo n y seld o m a t t a i n s a d ia m e te r o f more th a n
5 mm. I t a l s o may a r i s e from R-1& c u lt u r e s *
It
h a s n e v e r b e e n o b s e r v e d i n c u l t u r e s n o t e x p o se d t o
th e t e t r a z o l iu m s a l t . No p a t h o g e n i c i t y s t u d i e s .
F i g . 27*
P ure c u l t u r e of R-3 a t f i v e d a y s . T h is c u lt u r e i s
from p l a t i n g c e n t e r o f co lo n y shown b elow . N ote
i r r e g u l a r shape and fila m e n to u s to lo b a te m a rg in .
T h is co lo n y may re a c h 20 mm i n d iam ete r* Young
co lo n y shown i n F i g . 6 and 2 5 • Compare w ith R - l 6 f
F ig * 3 0 .
F ig . 2 8 .
R -3 c o lo n y a t 10 d a y s w it h w id e h a lo o f R - l 6 . T h is
u s u a l l y o c c u r r e d o n ly i n R -3 c u l t u r e s grown i n th e
p resen ce o f 2 , 3 , 5 - tr ip h e n y lte tr a z o liu m c h lo r id e .
The h a lo d e v e lo p e d i n th e f i r s t t r a n s f e r to a g a r
n o t c o n t a in in g th e t e t r a z o l iu m s a l t . At tim e s
s i m i l a r a p p e a r in g h a lo s g i v e r i s e t o R -17 c o l o n i e s
a s shown i n F ig * 2 6 o r to a m ix tu r e o f R - l6 and R -17*
F ig * 29*
P ure c u lt u r e o f R -l6 a t Z|£ hr* S im ila r to R-3 in c o lo r
and t e x t u r e b u t much l a r g e r in siz e * See Fig* 6*
U s u a lly d e v e lo p s from e i t h e r S o r R c u l t u r e s exposed
t o 2 , 3* 5 - t r ip h e n y l t e t r a z o l i u m c h lo r id e b u t may
o c c u r w ith o u t t h i s s a l t * T h is c u ltu r e can be m ain
ta in e d pure* Non p a th o g e n ic .
F i g . 30*
P -l6 a t 11 d a y s . Can be d is tin g u is h e d from R-3*
F i g . 27* by l a r g e r s iz e * r a i s e d f i n e l y t e x tu r e d c e n te r
and heavy fila m e n to u s to lo b a te m a rg in .
- 80 -
F ig * 31*
Pure c u l t u r e o f R-15 a t f o u r days* Showing f i n e l y
te x tu r e d s u r f a c e and fila m e n to u s m argin* T h is
c u l t u r e i s s i m i l a r to R-1 4 b u t b r i g h t g re e n in
c o lo r e s p e c i a l l y i n th e c e n te r* May re a c h 20 mm
i n d iam ete r* Can n o t be m a in ta in e d pu re* P l a t i n g
g iv e s r i s e to R-14* R-15 and R-7* No p a th o g e n ic ity
s tu d ie s *
F ig* 32*
R-15 co lo n y a t e ig h t days showing l a r g e s e c t o r o f R-7
a t rig h t.
„ 31 »
F ig * 33*
P o s s ib le **re v e rte d " S c o lo n y a t 2j£ h r from R-1&
co lo n y m a in ta in e d on tr y p to s e a g a r c o n ta in in g 2 , 3*
5 - t r ip h e n y l t e t r a z o l i u m c h lo r id e * Very s i m i l a r to S
b u t s l i g h t l y l a r g e r and more c o a r s e ly t e x tu r e d a t
m argin* Compare w ith F i g , 3« No p a th o g e n ic ity
s tu d ie s *
F ig* 34*
F iv e day o ld R-6 c o lo n ie s from p l a t i n g o f 1 -1 show ing
f ila m e n ts and p i t t e d a p p earan ce* Found in 10 to 20
day o ld S c u l t u r e s . C o lo n ie s a r e l a r g e , up to 10 mm
d iam ete r* T h is a c t u a l l y i s a h ig h ly m o tile form o f
S co lo n y and p i t t e d a p p e a ra n c e was due to th e org an ism s
grow ing i n t o th e a g a r . These c o lo n ie s o f t e n gave
r i s e to la r g e 1 -1 * 1 -2 o r R-3 d a u g h te rs w hich were
i n d is ti n g u i s h a b le from th e c o rre s p o n d in g ty p e s from
o t h e r S c o lo n i e s . No p a th o g e n ic ity s tu d ie s * T his
i s an i n t e r e s t i n g co lo n y and needs f u r t h e r i n v e s t i g a t i o n .
Compare w ith F ig* 6 and 7*
F ig * 35•
F i f t e e n day o ld S c o lo n ie s w ith sm a ll R-19 d w a r f - lik e
se co n d a ry c o lo n ie s w hich a p p e a r a s m in u te w h ite p ro
j e c ti o n s * C a re fu l p ic k in g and p l a t i n g alw ay s g ive
r i s e to w hat a p p e a r to be p u re S c o lo n ie s * No
p a th o g e n ic ity s tu d ie s *
F ig . 3 6.
S eventy day o ld S co lo n y w ith c o n sp ic u o u s R-19 d w a rfl i k e se c o n d a ry c o lo n ie s * At r i g h t R-3 d a u g h te r*
V iab le c e l l s o n ly a t m arg in o f co lo n y and i n R-19
c o lo n ie s .
- 83 -
F ig # 37*
S ix day o ld S c o lo n ie s c o m p le te ly su rro u n d e d by h a lo
o f fila m e n to u s g ro w th . T h is o c c u rre d r a r e l y and in
o n ly a few c u ltu r e s # P l a t i n g u s u a l ly gave r i s e to
m ix tu re s o f R-3* R-8
R - l 6 . I t h a s n o t b een d e
te rm in e d i f c u l t u r e s w hich show t h i s h a lo c o n s t i t u t e
a d i s t i n c t group# A lso se e F ig# 11*
F ig . 38*
S c u ltu r e a t 48 h r show ing a few p a le b lu e c o lo n ie s
w hich u s u a lly g iv e r i s e to p u re c u l t u r e s o f R-7 when
p la te d #
” 8fy *
F ig # 39*
M ix tu re o f B c o lo n ie s a t 2 4 h r from b r o th c u lt u r e o f
S colony#
Fig* 4 0 .
M ixture o f H c o lo n ie s a t e i g h t days from b r o th c u lt u r e
o f S colony*
- 85 -
F ig * 41*
C e lls from a n S ty p e c o lo n y show ing s h o r t ro d s in
p a lis a d e a rra n g e m e n t, p a i r s , Y and T form s and a few
lo n g c e l l s c h a r a c t e r i s t i c o f a l l S c u l t u r e s o f L*
m onocytogenes* A ll th r e e f i g approx* 1000X, gram
s ta in
F ig* 42*
C e lls from a non-sm ooth c o lo n y type (R -5) show ing
m ix tu re o f s h o r t and lo n g s le n d e r ro d s and a few
s h o r t s le n d e r fila m e n ts *
F ig* 43♦
C e lls from a non-sm ooth c o lo n y type (R -14) show ing
lo n g heavy curved fila m e n ts *
r< 7
***
V *
- 87 O ther c o lo n y ty p e s w hich d ev elo p ed but w hich co u ld n ev er be m ain
t a in e d i n pure c u ltu r e a r e l i s t e d below*
Type R -8.
Found m ost commonly i n p la t in g s from S c u lt u r e s m a in ta in ed fo r
s e v e r a l w eeks in b roth *
and R—5*
O c c a s io n a lly d e v e lo p e d from p l a t i n g s o f R -4
Agar c o lo n ie s a f t e r 24 hr a re v e r y s im ila r to R-7 e x c e p t
t h a t th e y a r e g o ld i n c o lo r *
o f R-3* R-? and R - ll*
P la t in g alw ays g iv e s r i s e to a m ix tu re
No p a t h o g e n ic it y s t u d i e s .
Type R-9*
Found m ost commonly in p la t in g s from S c u ltu r e s m a in ta in e d fo r s e v e r a l
w eeks i n b ro th *
O c c a s io n a lly d ev elo p ed from p j a t in g s o f R-4 and R-5 •
Agar c o lo n ie s a f t e r 2 4 hr are v e r y s im ila r to R - l l e x c e p t th a t th e y a re
p a le y e llo w to cream c o l o r .
th an s e v e r a l days*
Can n ev er be m a in ta in ed pure f o r more
P l a t in g s g iv e r i s e to R -3 # R-7* R-9 and R -ll*
No p a t h o g e n ic it y s t u d ie s *
Type R -1 0 .
S im ila r i n a l l c h a r a c t e r i s t i c s to R-9 e x c e p t th a t th e s u r fa c e o f th e
c o lo n y was c o a r s e ly t e x t u r e d .
R -1 0 , R - l l and R-14*
P l a t in g s g iv e r i s e to R-3* R-7* R-9*
No p a t h o g e n ic it y s t u d i e s .
Type R-12*
S im ila r i n a l l c h a r a c t e r i s t i c s to R - l l e x c e p t th ey have a g o ld sheen*
P l a t in g s g iv e r i s e to R - l l , R-14 and R-15*
No p a t h o g e n ic it y s t u d i e s .
S c o l o n i e s in o c u la t e d in t o tr y p to s e b r o th and in c u b a te d a t 3 7 ° C
f o r 18 h r and a t room tem p eratu re f o r i n d e f i n i t e p e r io d s o f tim e , when
s tr e a k e d on agar p la t e s f a i l e d to produce c o lo n ia l forms o th e r than
th o s e w h ich d e v e lo p e d when th e c o lo n ie s w ere p la te d d i r e c t l y on agar
p la t e s *
R a r e ly one o f th e R form s was found a s a pure i s o l a t e d c o lo n y .
- 88 However th e o c cu rren c e o f the u n sta b le type colony was much more common
th a n in p la tin g s from ag ar c u ltu re s *
When 2 , 3* 5 “ tr ip h e n y lte tr a z o liu m c h lo rid e was in c o rp o ra te d in
th e a g a r i t was p o s s ib le to d is tin g u is h one new c o lo n ia l from which had
n ot p re v io u s ly o ccurred*
T his was type R-17.
Fig* 2 6 .
In a d d itio n th e
d if f e r e n c e in a b i l i t y o f some c o lo n ia l ty p es to reduce t h i s s a l t g r e a tly
enhanced t h e i r d e te c tio n *
Type 16 f a i l e d to reduce t h i s s a l t and the
c o lo n ie s appeared a r a t h e r p a le
g reen in c o lo r w hile a l l o th e r types
v a rie d from p a le pin k to a deep re d o r p urple*
F ig . 15*
A fte r about a
week o f in c u b a tio n th e S colony was c h a ra c te riz e d by a d ark re d c e n tr a l
a re a which approached b la c k .
F ig . 4*
T his in te n s e c e n tr a l a re a a ls o
developed in some R c o lo n ie s , p a r t i c u l a r l y R-7#
F ig . 7#
The degree to which the in d iv id u a l colony type reduced the t e t r a zolium s a l t was r e f l e c t e d in the c o lo n ie s which develop from tr a n s f e r s
to p l a t e s w ith o u t the s a l t .
T his was e v id e n t on ly in th e f i r s t t r a n s f e r .
N otable in t h i s re s p e c t was type R-15 which took on a much deeper pink
th an b e fo re exposure to th e s a l t .
As type R-7 and R-14 d id n o t reduce
th e s a l t as r e a d i l y as d id R-15* th ey could v ery e a s i l y be d is tin g u is h e d
from each o t h e r , p a r t i c u l a r l y when th ey were a d ja c e n t.
T his e f f e c t un
d o u b ted ly was due to a sm all r e s id u a l o f te tra z o liu m s a l t c a r r ie d o v er
on the f i r s t t r a n s f e r and was an amount s u f f i c i e n t to enhance the c o lo r
o f th o se c o lo n ie s which a lre a d y d isp la y e d a tendency toward deep p in k .
There were no d if f e r e n c e s in e i t h e r fe rm e n ta tiv e o r biochem ical
c h a r a c t e r i s t i c s d isp la y e d by any o f the N—
S c o lo n ia l types when compared
w ith th e p a re n t S ty p e .
A ll c o lo n ia l forms te s te d produced a c id but
no gas a f t e r 24 h r in g lu c o s e , le v u lo s e , tr e h a lo s e and sa la c in *
Acid
- 89 p ro d u c tio n was slow and v a r ia b le in a ra b in o s e , ea l a c t o s e , l a c to s e ,
m a lto s e , s u c ro s e , d e x t r i n , s o r b i t o l , g ly c e r o l, and m e le z ito s .
There was
no fe rm e n ta tio n o f x y lo s e , r a f f i n o s e , i n o s i t o l , i n s u l in , d u l c i t o l , m an n ito l
o r a d o n ito l*
There were some s l i g h t v a r ia tio n s in the r a te o f fe rm e n ta tio n
b u t no s i g n i f i c a n t d iffe re n c e s *
The v a r ia tio n s appeared to be o f th e
same o r d e r one would ex p ec t to f in d in any la rg e number o f sto c k c u ltu r e s
R* m onocytogenes exposed to the same ferm en tab le substances*
None o f th e R ty p es te s te d reduced n i t r a t e s , o r produced indole*
G e la tin was n o t liq u e fie d *
In g e n e ra l th e R types produced e i t h e r no
hem olysin o r d isp la y e d a much s m a lle r zone than did the p a re n t S ty p e .
By th e m ethods employed, c u ltu r e s from N-S colony ty p es 1 -1 , 1 -2 ,
and R-3 were p a th o g e n ic f o r r a b b i t s .
However, a l l ty p es te s te d produced
marked m onocytosis and a v a ry in g degree o f a g g lu tin a tin g a n tib o d ie s in
th e p e r ip h e r a l b lo o d .
T ypical r e s u l t s , fo llo w in g in tra v en o u s in o c u la tio n ,
to g e th e r w ith a g g lu tin a tin g a n tib o d y t i t e r f o r the homologous type and
S type c u ltu r e s a re shown in the p ro to c o ls which ap p ear on pages 9s -96#
At no tim e d id any anim als which re c e iv e d c u ltu r e s from R c o lo n ie s d i s
p la y any marked c l i n i c a l in d ic a tio n o f i l l n e s s .
A few r a b b its showed a
s l i g h t r i s e in tem p eratu re on th e second o r t h i r d p o st in o c u la tio n day.
C u ltu re s p re p a re d from the l i v e r , sp le e n and h e a rt blood of r a b b its s a c r i
f ic e d from s e v e ra l days to s e v e r a l weeks a f t e r in o c u la tio n , f a i l e d to
y i e l d s i g n i f i c a n t b a c t e r i a l grow th.
R ab b its in o c u la te d in tra v e n o u s ly
w ith c u ltu r e s from R c o lo n ie s v e ry o fte n su rv iv ed a subsequent l e t h a l
dose o f S c u ltu re *
When c u ltu r e s from R type c o lo n ie s were i n s t i l l e d in to the conjunc
t i v a l sa c o f r a b b i t s , l i t t l e o r no r e a c tio n fo llo w ed .
Two r a b b its i n
- 90 s t i l l e d w ith ty p e R -7 showed marked la c r im a tio n on th e second p o s t i n
s t i l l a t i o n d a y , hu t appeared normal t h e r e a f t e r .
I n s t i l l a t i o n o f c u ltu r e s
from typ e S c o l o n i e s r e s u lt e d in a marked c o n j u n c t i v i t i s and k e r a t i t i s
in a l l i n s t a n c e s .
R a b b its in o c u la te d w ith c u lt u r e s from c e r t a in R ty p e c o l o n i e s ,
p a r t i c u l a r l y ty p e s R-3 and R-7 produced h ig h a g g lu t in a t in g t i t e r s a g a in s t
typ e S c u ltu r e s *
T h is su g g e ste d th a t th e s e ty p e s m ight be s u c c e s s f u l l y
em ployed in a v a c c in e th a t cou ld c o n v e n ie n tly be a d m in is te r e d by the
su b cu ta n eo u s r o u t e .
The r e s u l t s in d ic a t e d th a t in th e amount g iv e n , th e s e
ty p e s d id produce f a i r l y h ig h a g g lu t in a t i n g t i t e r s f o ll o w i n g subcutaneous
in o c u la t io n *
I t was fu r t h e r shown th a t when 15 TR u n it s o f Wydase p er
ml were added to th e b a c t e r i a l s u s p e n s io n a n tib o d y t i t e r wb^s enhan ced.
The a d d it io n o f Wydase a ls o p r e v en te d the developm ent o f th e l o c a l i z e d
n e c r o t ic l e s i o n th a t d e v e lo p e d a t th e s i t e o f in o c u la t io n when c u lt u r e s
from typ e S c o lo n ie s were g iv e n su b c u ta n e o u sly *
H owever, in r a b b it s
in o c u la te d w ith c u lt u r e s from ty p e R c o l o n i e s , c o n g e s tio n and sm a ll su p e r
f i c i a l hem orrhages were th e p r in c ip a l l e s i o n s o b s e r v e d , and n e c r o s i s ,
i f p r e s e n t , was lim it e d to s m a ll d i s c r e e t a r e a s w hich seldom measured more
than two to th r e e mm in d ia m e te r .
T hese may have r e s u l t e d from the
p r e se n c e o f p a th o g e n ic S typ e c e l l s in th e c u ltu r e *
A l l r a b b it s exp osed su b c u ta n e o u sly w ith c u ltu r e s from e i t h e r typ e S
or typ e R c o lo n ie s d e v elo p ed a marked m o n o cy to sis in th e c i r c u l a t i n g
b lo o d .
The m o n o c y to sis d e v e lo p e d m ost q u ic k ly and reached th e h ig h e s t
v a lu e i n th e r a b b it s exp osed to c u ltu r e c o n ta in in g Wydase.
There was no
e v id e n c e e i t h e r _in v i t r o o r in v iv o th a t Wydase e x e r te d any u n fa v o ra b le
e f f e c t on th e c u l t u r e s .
When th e se r a b b i t s were exposed fiv e to 10 weeks l a t e r to a known
l e t h a l dose o f c u ltu r e from S type c o lo n ie s , d e ath o c cu rred in two to
th r e e d a y s.
A ll u n v accin ated c o n tro ls died w ith in th e same p eriod*
At
n ecro p sy a l l r a b b i t s showed le s io n s ty p ic a l o f se p tic e m ic l i s t e r i o s i s ;
f o c a l h e p a tic n e c r o s is and splenom egalia*
Type S c u ltu r e s were reco v ered
in pu re c u ltu r e from th e l i v e r , sp le en and o c c a s io n a lly from the h e a r t
blood o f a l l anim als which d ie d .
This s t r i k in g l y dem onstrated t h a t th e
p resen ce o f a g g lu tin a tin g a n tib o d ie s in th e blood serum was not a measure
o f immunity to in f e c ti o n w ith L* m onocytogenes.
These r e s u l t s a re sum
m arized in ta b le 1 and ty p ic a l p ro to c o ls a re given on page 97%
- 92 F ate o f two r a b b its in o c u la te d in tr a v e n o u sly
w ith 0 .3 ml cu ltu re 1 2 2 5 5 -S *
Rabbit 15 9
D i f f e r e n ti a l leu k o cy te count
Day
L
M
B
E
29
65
0
6
0
1
61
17
19
2
1
2
29
32
3-8
1
0
3
38
7
35
0
0
4
Dead* F ocal h e p a tic n e c ro sis and splenom egalia
L. monocytogenes is o la te d from h e a r t blood and
liv e r.
R ab b it 158
D i f f e r e n t i a l leukocyte count
Day
p
%
M
B
E
0
23
72
0
0
0
1
45
32
2
1
0
2
49
12
38
1
0
3
Dead* S p lenom egalia.
from liv e r *
L. monocytogenes is o la te d
♦All c u ltu r e s sta n d a rd iz e d to No* 1 tube McFarland n ephelom eter.
♦ ♦ In o c u la tio n day*
L
M
B
E
.
-
polym orphonuclear leu k o cy te
lymphocyte
monocyte
b a so p h il
e o s in o p h il
Same symbols a re employed in subsequent ta b le s *
- 93 -
T y p ical r e s u l t s fo llo w in g in tra v en o u s
in o c u la tio n o f 0 .3 ml c u ltu r e 12255 B-3*
B abbit 153
D i f f e r e n t i a l le u k o c y te count
Day
L
M
B
E
0**
38
6o
0
2
0
1
63
28
2
6
1
2
6o
5
35
0
0
3
47
9
44
0
0
4
40
30
28
2
0
7
45
47
8
0
0
T it e r a f t e r 100 days
C u ltu r e
1:20
1:40
1:80
1 : l 60
1:320
1:640
s
////
////
I
-
-
-
R-3
////
////
1
-
1:1280
-
C h a llen g e a t 106 days w ith 1.0 ml 12255-s I .V .*
c ount
D i f f e r e n t i a l le u k o c y te ■
Day
P
L
M
B
E
0
49
42:
5
3
1
1
54
42
4
0
0
2
62:
17
21
0
0
3
51
13
35
0
1
6
47
25
28
0
0
10
56
31
11
0
2
S u rv iv e d
•
For e x p la n a t io n o f sym bols s e e page 92*
1:2560
- 94 T y p ica l r e s u lt s fo llo w in g intravenou s
in o c u la tio n o f 0 . 3 nil cu ltu re 1 2 2 5 5 R-7 *
R a b b it 12j2
D i f f e r e n t i a l leukocyte count
paM‘*
L
M
B
E
0 **
31
66
1
0
2
1
83
15
2
0
0
2
42
18
40
0
0
3
31
23
46
0
0
4
37
26
36
0
1
7
44
52
4
0
0
I s 160
1:320
1:640
-
-
-
////
////
////
Day
T i t e r a f t e r 100 days
C u ltu re
1:20
S
/
R-7
////
1:40
1:80
-
//#
////
-
C hallenge a t 106 days w ith 1.0 ml 12255-S I.V .*
D i f f e r e n ti a l leu k o cy te count
Day
P
L
M
B
E
0
58
32
8
1
1
1
70
30
0
0
0
2
59
3a
2
1
0
3
52
12
35
1
0
6
63
17
18
0
2
10
51
27
19
1
2
S u r v iv e d .
For e x p la n a tio n o f symbols s ee page
9 2
.
1:1280
//
1 t 2560
- 95 T y p ica l r e s u lt s fo llo w in g in travenou s
in o c u la tio n o f 0 .3 ml c u ltu r e 1 2 2 5 5 R - ll*
Rabbit 143
D i f f e r e n t i a l le u k o c y te count
Day
L
M'
B
E
0**
29
63
2
4
2
1
58
41
0
0
1
2
5^
6
37
1
0
3
35
20
44
0
1
4
42
17
40
0
1
(Boor s l i d e )
7
T it e r a f t e r 100 days
1 :20
1 :40
S
////
AV
R -ll
AW
////
1:80
«S>
////
l:l6 o
1:320
1 :6/|.0
1:1280
-
-
-
-
////
////
////
-
C h a llen g e a t 106 days w ith 1 .0 ml 12255-S I . V . *
D i f f e r e n ti a l leukocyte count
Day
P
L
M
B
E
0
45
47
2
3
3
1
51
46
1
1
1
2
53
24
20
3
0
3
47
27
23
2
1
6
46
20
29
4
1
10
30
59
8
l
2
S u r v iv e d .
For e x p la n a tio n o f symbols see page 9 2 .
1 :256 o
- 96 -
T y p ic a l r e s u lt s fo llo w in g intravenous
in o c u la tio n o f 0 .3 ml cu ltu re 1 2 2 5 5 R- 1 4 *
R abbit 154
D i f f e r e n t i a l le u k o c y te count
Day
M
B
E
o**
31
68
0
1
0
1
62
22
11
3
2
2
38
17
44
0
1
3
27
36
34
0
3
4
48
27
25
0
0
7
29
63
7
1
0
T it e r a f t e r 100 days
C u ltu re
1:20
1:40
1:80
1 :l6 o
S
AW
AW
MM
AW
R-15
AW
MM
MM
MM
1:320
1:640
*
I
I
M
1:1280
C hallenge a t 106 days w ith 1.0 ml 12255“s 1*7.*
D i f f e r e n t i a l leu k o cy te count
Day
p
L
M
B
E
0
26
62
0
0
0
1
67
26
2
1
4
2
37
3
60
0
0
3
25
9
66
0
0
6
27
39
31
3
0
10
37
37
22
4
0
S u rv iv e d .
For e x p la n a tio n o f sym bols see page 92*
1:2560
- 97 -
Table 1
Condensation of a vaccination trial
C u ltu re
T it e r
S
R
1007X
R-7 w ith Wydase
1*320
R-7 w/ ° Wydase
Monocytes
% on 6 th day
S u rv iv a l a f t e r
ch alle n g e in days
32
2**
10
2
S w ith Wydase
1 :2560
1:320
43
3
S w/o Wydase
1:640
1 : l 60
18
3
R-3 w ith Wydase
1:80
1 :40
27
R-3 w/o Wydase
1 :40
R«*l6 w ith Wydase
1:320
R -l6 w/o Wydase
1 :320
12255
*»i
25
3
1 :80
19
3
1:320
18
3
Immunising d o se : 0*1 ml sta n d a rd iz e d to No* 9 tube McFarland nephelom eter.
C hallenge d o se ; 0*5 ml S c u ltu r e std * to No* 1 tube McFarland nephelom eter*
Four r a b b i t s in each group* P ercentage o f monocytes r e p r e s e n ts h ig h e s t
v a lu e i n each g roup. T ite r s a re average o f anim als b le d .
* T ite r a t two w eeks.
**Challenge a t f iv e w eeks.
* * * T iter a t seven weeks.
^ '^ C h a lle n g e a t 10 w eeks.
- 98 DISCUSSION
The r e s u l t s r e v e a le d th a t a t l e a s t 20 d i f f e r e n t c o lo n ia l form s o f
L* m on ocytogen es cou ld be r e c o g n iz e d on tr y p t o s e agar p l a t e s exam ined
by the o b liq u e l i g h t i n g tech n iq u e*
Of t h e s e , s i x co u ld be m a in ta in ed in
s t a b le form w ith o u t fu r t h e r d is s o c ia t io n *
R - l l , R -14 and R - l6 .
T hese were ty p es S , R-3* R-7»
Type S was th e t y p ic a l smooth c o lo n ia l form ch a r
a c t e r iz e d by a sm a ll c i r c u l a r , b lu e - g r e e n , s l i g h t l y r a is e d c o lo n y w ith
a f i n e l y t e x t u r e s s u r fa c e and e n t ir e m argin*
F i g . 1*
I t was th e ty p e
commonly found i n i n i t i a l i s o l a t i o n s from in f e c t e d m a te r ia l*
C u ltu r es o f
t h i s typ e w ere h ig h ly p a th o g e n ic f o r r a b b its *
In g e n e r a l o n ly fo u r s t a b l e c o lo n y ty p e s d ev elo p ed d i r e c t l y from S
c o lo n ie s .
T hese w ere R -3 , R-7* R - l l and R - l6 .
However ty p es R -7 , R - l l
and R-1& o n ly r a r e l y d e v elo p ed d i r e c t l y from S ty p e c u lt u r e s .
However,
th e y c o u ld be dem on strated q u ite r e a d i ly in p la t in g s from 1 - 1 , 1 - 2 , R-4
o r R-5 c u ltu r e s *
F i g . 5* 19 an& 2D .
R-3 was th e m ost common typ e and
u s u a lly app eared as dau gh ter c o lo n ie s on w e ll i s o l a t e d S c o lo n ie s a f t e r
ab ou t f i v e days in c u b a tio n *
F ig . 6 .
R a rely d id i t o ccu r in c o n flu e n t
S typ e c u l t u r e s .
R-7 u s u a ll y d ev elo p ed a s a daughter but o c c a s io n a lly d e v e lo p e d a s
a s e c t o r in S c u l t u r e s .
a l l y from S c o lo n ie s *
F i g . 7 and 8 .
Type R -l6 d ev elo p ed o n ly o c c a s io n
In S c u lt u r e s i t alw ays appeared as a dau ghter and
p la t in g s o f young R -l6 d a u g h ters v e r y o f t e n gave r i s e to S o r R-3 t y p e s .
I t o f t e n d e v e lo p e d a s i s o l a t e d c o lo n ie s from s e v e r a l o f th e u n s ta b le R
fo r m s.
F i g . 2 8 , 29 and 3 0 .
R - l l was un iqu e in th a t i t r a r e ly d e v elo p ed in S c u lt u r e s , but i f
i t d id , i t alw a y s appeared a s a v e ry c o n sp ic u o u s smooth s u r f a c e d , pin k
- 99 t o g o ld s e c t o r ,
F ig * 9 and 10*
Many o f th e s e s e c t o r s , i f c a r e f u l l y
p ic k e d , gave r i s e on f i r s t t r a n s f e r to pure c u ltu r e s o f P.• 1 1 ,
O cca sio n
a l l y p l a t i n g s were m ixed w ith ty p e s S and R -7, but n ev er w ith R -3 .
The
young R—11 c o lo n y was so d i f f e r e n t from any o f th e o th e r ty p e s th a t i t
f i r s t was th ou gh t to be c o n ta m in a tio n .
I t was th e o n ly R type w hich
d e v e lo p e d in t o a round c o m p le te ly smooth su r fa c e d c o lo n y .
However a f t e r
s e v e r a l days in c u b a tio n th e c o lo n y became more f la t t e n e d and d ev elo p ed
a f a i n t g r e e n is h t i n t .
F ig . 1 0 ,
Some R - l l c o lo n ie s r e ta in e d t h e ir
c i r c u l a r form and e n t i r e m argin w h ile o th e r s d ev elo p ed in t o a more lo b a t e
fo r m .
S t i l l o th e r s d e v e lo p e d lo n g g o ld c o lo r e d f il a m e n t s , R- 1 3 ,
2 1 , 2 2 , 23 and 24*
F ig .
A nother d i s t i n c t i v e c h a r a c t e r i s t i c o f the R - l l
c o lo n y was i t s f l a t t e n e d appearance a f t e r s e v e r a l days in c u b a tio n con
t r a s t e d to i t s i n i t i a l convex form .
C o lo n ie s w hich r e t a in e d t h e ir c i r c u l a r
form a l s o tend ed to r e t a i n th e p in k c o lo r w hich made t h i s typ e e a sy to
i d e n t i f y in o ld m ixed c u lt u r e s .
F i g . 17 and 1 8 ,
Type R -14 was th e o n ly o th e r s t a b le R c o lo n y .
F ig . 12.
I t n ev er
d e v e lo p e d d i r e c t l y from S c u l t u r e s , but was found o n ly in p la t in g s o f
th e g o ld c o lo r e d lo n g fila m e n to u s seco n d a ry growth or d a u g h te r s, R-13»
w hich d e v e lo p e d on o ld R—7 and some o ld R—11 c o l o n i e s .
F i g . 8 , 18 and 23*
In young c u lt u r e s R -14 and R -7 c o lo n ie s were v e r y s im ila r and cou ld v e r y
e a s i l y be c o n fu s e d .
R -14 was a more in t e n s e p in k than R-7 and some
c o l o n i e s appeared to be a lm o st r e d .
Compare F i g . 8 w ith F i g . 1 2 .
Twenty
fo u r hr c o lo n ie s were a p p ro x im a te ly 1 to 2 mm o r more in d ia m eter w hereas
R«»7 seld om m easured more than 1mm a t 2 4 h r .
With co n tin u ed in c u b a tio n
th e developm ent o f R -14 was much more r a p id than th a t o f R-7 and some w e ll
i s o l a t e d c o lo n ie s r ea c h e d a d ia m eter o f 20 to 30 h1331*
ed a d ia m eter o f more than 20 mm.
^"7 seldom a t t a i n
Compare F i g . 8 w ith F ig . 1 2 .
One
o f th e o u t s ta n d in g d if f e r e n c e s was in th e c e l l s o f th e two c o l o n i e s .
R—7 c e l l s were u s u a ll y lo n g plump ro d s in p a ir s or s h o r t c h a in s s im ila r
to th o s e shown i n F i g .
2 , w hereas c e l l s o f R -14 were u s u a lly curved and
in lo n g h eavy f i l a m e n t s .
F i g . 43.
R—16 was un iqu e in th a t i t s developm ent appeared to be enhanced in
c u lt u r e s exp osed to 2 , 3* 5 * t r ip h e n y lte tr a z o liu m c h lo r id e .
I t s p r e se n c e
had b een s u s p e c te d in c u lt u r e s n o t exposed to t h i s s a l t but b eca u se o f
i t s c l o s e resem b lan ce to R-3 i t was d i f f i c u l t to e s t a b l i s h i t a s a
d i s t i n c t typ e*
F i g . 6 , 27* 28*
t a in in g th e te tr a z o liu m s a l t i t
29 and 30*
However* on agar p l a t e s con
appeared a s l a r g e , tr a n s lu c e n t* green
d a u g h ter c o lo n ie s w h ile th e p a ren t c o lo n y , u s u a lly 1 - 1 , 1 - 2 , R -4 or R -5 ,
o r one o f th e u n s ta b le N-S c o l o n i e s , appeared a b r ig h t p in k to deep red
in c o lo r .
R-3 a l s o f a i l e d to red u ce th e te tr a z o liu m s a l t but th e app ear
ance o f th e R-3 d a u g h te r s was q u it e d i s t i n c t i v e from the R - l6 , b e in g more
e v en t e x t u r e d , more opaq ue, and d id n ot have th e g l i s t e n and s p a r k le o f
R-1&.
F ig . 1 5 .
I t ap p eared th a t th e rem a in in g 14 c o l o n ia l forms c o n s is t e d o f e i t h e r
a h ig h ly u n s ta b le p o p u la tio n w hich d is s o c ia t e d in t o two or more o f th e
s t a b le N-S (non sm ooth) fo r m s, o r th a t th ey were a c t u a ll y a m ix tu re o f
s e v e r a l grow th fo r m s.
S ta in e d sm ears prepared from the u n sta b le c o lo n ia l
form s r e v e a le d a w ide v a r ia t io n
in c e l l u l a r m orphology•
c e l l s ranged from s m a ll c o c c o id
form s to lo n g fila m e n ts w ith many i n t e r
m e d ia te fo r m s.
The b a c t e r i a l
No a ttem p t was made to determ ine th e r a t io o f th e s e v e r a l
com ponents o f th e u n s ta b le c u l t u r e s , but th e f a c t th a t th e r a t i o and th e
c o l o n i a l typ e w h ich r e s u lt e d from subsequent p l a t in g s cou ld a lm o st alw ays
be p r e d ic te d s u g g e s te d th a t th e c o lo n ie s co n ta in ed a r a th e r c o n s is t e n t
p r o p o r tio n o f th e in d iv id u a l c e l l t y p e s .
U n sta b le R -typ es n ev er d ev elo p ed
- 101 d i r e c t l y from S c o lo n ie s on agar p la t e s *
However th e y were found in
o ld b r o th c u lt u r e s o f S c o lo n ie s and in p l a t in g s o f N-S c o lo n ie s *
S c o l o n i e s seldom d ev elo p ed from p la t in g s o f e i t h e r th e s t a b le o r
u n s t a b le R c u ltu r e s *
Y et when R c u lt u r e s m a in ta in ed f o r lo n g p e r io d s o f
tim e in tr y p t o s e b r o th c o n ta in in g 2 , 3 . 3 * tr ip h e n y lte tr a z o liu m c h lo r id e ,
w ere s tr e a k e d on a g a r p l a t e s o n ly S c o lo n ie s d evelop ed *
The number o f
c o lo n ie s w hich d e v e lo p e d on th e s e p l a t e s was alw ays sm all#
T h is su g
g e s t e d t h a t in th e c o n c e n tr a tio n u s e d , the te tr a z o liu m s a l t e x e r te d a
t o x i c e f f e c t on N-S ty p e s a f t e r exp osu re o f 10 days or m ore, but to a
l e s s d e g r e e in th e S typ e*
The t o x i c e f f e c t o f 2 ,
3 * 3**
t r ip h e n y lte tr a z o liu m on the R c o lo n y
ty p e s was a l s o e v id e n t on agar p l a t e s c o n ta in in g t h is s a l t .
When t r a n s
f e r s were attem p ted from c o lo n ie s w hich had in cu b a ted f o r lon g p e r io d s
o f t im e , 2 0 days o r m ore, th e in o c u la te d p l a t e s rem ained s t e r i l e or o n ly
v e r y few c o lo n ie s d ev elo p ed *
O ften a few S c o lo n ie s were ob served on
t h e s e p l a t e s , a g a in s u g g e s tin g th a t a l l R c o lo n ie s c o n ta in e d a few c e l l s
c a p a b le o f p ro d u cin g S c o l o n i e s .
B oth S and R c o lo n ie s on agar p la t e s
w ith o u t te tr a z o liu m rem ained v ia b l e f o r lo n g p e r io d s o f tim e , up to two
months o r m ore.
Growth f a i l e d to o ccu r when t r a n s f e r s were made to
f r e s h p l a t e s | o n ly when th e agar was d e s ic c a te d to th e p o in t th a t i t was
hard and b r i t t l e ? th u s i n d ic a t in g th a t p rolon ged exposure to the te tr a z o liu m
s a l t e x e r te d a l e t h a l t o x ic e f f e c t on the R t y p e s .
T his e f f e c t was l e s s
marked in th e S c u l t u r e s , and grow th o ccu rred even a f t e r lo n g exposure
to th e s a l t .
The v a r i a t i o n in c o lo r o f th e mature c o lo n ie s on agar c o n ta in in g
te t r a z o liu m s a l t s in d ic a t e d th a t th e r e were d if f e r e n c e s in th e r e d u c in g
- 102 enzyme sy stem s o f th e v a r io u s c o lo n y t y p e s .
The red c o lo r w hich d e v e lo p s
i n l i v i n g c e l l s in th e p r e se n c e o f te tr a z o liu m s a l t s i s th o u g h t to d e
pend on th e p r e se n c e o f r e d u c in g enzymes (d eh yd rogen ases w ith or w ith o u t
coenzym es I o r XI) w hich red u ce th e c o l o r l e s s compounds to c o lo r e d f o r m azans.
That a l l R c o lo n ie s c o n ta in e d a few S typ e c e l l s was a g a in su g
g e s t e d by th e l e s i o n s w hich d ev elo p ed in r a b b it s a f t e r subcutaneous i n
o c u la t io n o f R c u lt u r e s p a r t i c u l a r l y th o se c o n ta in in g Wydase.
At n ecro p sy
o f many o f th e s e a n im a ls v e r y s m a ll ( 1 mm) d i s c r e t e g rey f o c i o f n e c r o s is
w ere o b serv ed in an area s e v e r a l cm in d ia m eter surrounding th e in o c u la
tio n s i t e .
As la r g e in o c u la o f R c u ltu r e s a d m in iste r e d by v a r io u s r o u te s
had shown no p a th o g e n ic e f f e c t i t was f e l t th a t th e s e i s o l a t e d n e c r o t ic
f o c i r e s u lt e d from & typ e c e l l s d is p e r s e d by the sp r ea d in g a c t io n o f
Wydase.
R a r ely a sm a ll area o f n e c r o s is was found a t the in o c u la t io n
s i t e in a n im a ls i n w hich Wydase had n ot been added to th e c u lt u r e .
When
pure S c u lt u r e s were in o c u la te d by t h i s r o u te a la r g e w e ll l o c a l i z e d
n e c r o t ic l e s i o n d ev elo p ed a t th e s i t e o f in o c u l a t i o n .
When Wydase was
added to S c u lt u r e s la r g e (5 -1 0 mm) d is c r e t e n e c r o t ic f o c i were found in
a wide a r ea su rro u n d in g th e in o c u l a t io n s i t e .
These f in d in g s s u g g e ste d
th a t a l l R c u lt u r e s c o n ta in e d a few S c e l l s w hich in th e proper e n v ir o n
ment c o u ld produce l e s i o n s .
A p p a re n tly the number o f S c e l l s in R c u ltu r e s in o c u la te d in t r a
venously* o r i n s t i l l e d
e v id e n c e o f i n f e c t i o n .
in t o th e c o n ju n c tiv a l sa c was to o sm a ll to produce
I t has been found th a t f a i r l y la r g e in o c u la o f
S c u lt u r e s m ust be u sed to produce i n f e c t i o n under la b o r a to r y c o n d it io n s ,
and th e number o f S c e l l s in th e R c u lt u r e s may have been too s m a ll to
- 103 in c it e a c l i n i c a l resp onse.
Whether the m onocytosis which developed in rab b its inoculated
in traven ou sly w ith R cu ltu res resu lted from the presence o f S c e l l s ,
or whether the R cu ltu res a ls o contain the monocyte producing fa cto r
(MEF) has not been determ ined.
Grini (1943) found that a non pathogenic
s tr a in o f L. monocytogenes produced a m onocytosis when inoculated in tr a
venously in to r a b b its, but unfortunately gave no d e scrip tio n o f the
colony*
Stan ley (1949) showed and Girard and Murray (1951) confirmed
that the MEF o f L, monocytogenes was found in a non-antigenic lip id fr a c
tio n o f the b a c te r ia l c e l l , and was found to be lib erated c h ie fly in the
l iv e r o f in fe c te d anim als.
The R cu ltu res may apparently a lso contain
t h is l ip i d MEF but fu rth er work i s necessary to confirm t h i s ,
None o f the rab b its which received dense suspensions o f e ith e r S or
R cu ltu res in the drinking water showed any c lin ic a l in d ic a tio n of i l l
ness during the two month observation p eriod .
The rab b its which received
e n sila g e disp layed marked em aciation due to th e ir refu sa l to accept th is
r a tio n .
L aceration o f the mouth made no con trib u tion to s u s c e p t ib ilit y
to in f e c t io n .
N either did lo c a l le s io n s develop at the la ce ra tio n s i t e s .
Cultures prepared from the fe c e s as described by Gray et_ al,. (1950) gave
in co n clu siv e r e s u lt s and i t could not be determined whether the cu ltu res
added to the drinking water survived passage through the d ig e stiv e tr a c t
or n o t.
Further in v e s tig a tio n revealed that the p a rticu la r str a in o f
cu ltu re ( 1 2 2 5 5 * is o la t e d from the brain of a c a lf with l i s t e r i c encephali
t i s com plicated by vitam in E d e fic ie n c y ) was in h ib ited by the potassium
t e l l u r i t e in the concen tration (0,05%) employed.
fin d in g s o f Olson e t a l .
This confirmed the
(1953) who found that certa in s ta in s o f L,
monocytogenes were s e n s itiv e to 0 , 05 ^ potassium t e l l u r i t e .
- 104 -
With the excep tion o f mice ( J u lia n e lle , 1941 ) n o st in v e stig a to r s
f a i l e d to in fe c t a r t i f i c i a l l y e ith e r ruminant or monogastric animals by
the o ra l route*
Roine <3t a l , (1953) reported death of guinea p igs fed
a r a tio n con tain in g aureomycin due to in fe c tio n with I,. mono cy to gen es.
These authors expressed the opinion that aureomycin suppressed other
b a c te r ia l in h a b ita n ts of the in t e s t in a l tr a c t and favored the p r o life r a
tio n o f L. mono cy togenes and that in fe c tio n was esta b lish ed as a r e s u lt
o f th is *
For the present th is must be viewed with reservation s a s , as
in d ica ted in part one o f th is t h e s i s , L* monocytogenes has been shown to
be h ig h ly su sc e p tib le the a n tib a c te r ia l a c tio n o f aureomycin*
The r e s u lt s obtained in th is study confirmed the in a b ilit y o f
e it h e r S or R cu ltu res o f L. monocytogenes to in fe c t by way o f the in
t e s t i n a l tr a c t even when the animals were subjected to emaciation and
la c e r a tio n ,
Asahi and Hosoda (1952* 1953) suggested that L. monocytogenes may
use the trigem in al nerve es an avenue of entrance in the en c ep h a litic
form o f the d isease*
They succeeded in in fe c tin g both mice and young
goats (2 -4 mo o ld ) by sc a r ify in g the mucous membrane o f the mouth and
the cutaneous t is s u e surrounding the mouth and nose before exposure to
L.
monocytogenes*
N either Hirato e_t a l . (1954) nor
present study
could confirm these fin d in gs*
At the presen t very l i t t l e i s known concerning the nature o f the
u n sta b le R c u ltu r e s .
Because they could not be maintained in pure form
they were not used in p ath ogen icity stu d ies or te ste d for biocnem ical
r e a c tio n s .
However i t did appear that they were composed of a d e f in it e
r a tio o f the variou s c e l l types end on th is b a sis can be considered as
- 105 d i s t i n c t c o lo n y t y p e s ; p o s s ib ly a s in te r m e d ia te s b ecau se o f t h e ir r a p id
d i s s o c i a t i o n in t o th e o th e r s t a b le R fo r m s.
As t h i s stu d y was d ir e c t e d
p r im a r ily toward th e d e t e c t io n and r e c o g n it io n o f new c o lo n ia l form s in
th e genus L i s t e r i a no in t e n s iv e s tu d y has been made to d a te to fu r th e r
c h a r a c t e r iz e th e s e t y p e s .
The numerous p u b lic a t io n s o f Huddelson and
Braun on d i s s o c i a t i o n s t u d ie s in th e genus B r u c e lla e m p h a tic a lly em phasize
t h a t s t u d ie s o f t h i s nature are n ot q u ic k ly e x h a u ste d .
I t i s hoped th a t
t h i s p r e s e n t stu d y i s o n ly th e b e g in n in g o f s im ila r s t u d ie s on th e genus
L is te r ia .
From th e d a ta now a t hand, com piled from s e v e r a l hundred s e p a r s te
p l a t i n g s , a t e n t a t i v e d i s s o c i a t i o n schem e, shown on th e f o ll o w in g p a g e ,
can be d e r iv e d f o r th e p r in c ip a l s t a b le R t y p e s .
I t i s a lm o st c e r t a in
th a t fu tu r e work w i l l show t h i s scheme to be in co m p lete and many r e v is io n s
may be n e c e s s a r y .
In work o f t h i s nature where c o lo r v a lu e s , s i z e and
te x tu r e o f c o lo n ie s p la y so im p ortan t a p a r t in d i f f e r e n t i a t i n g one
c o lo n y ty p e from a n o th e r , i t i s o f t e n d i f f i c u l t to f r e e o n e s e l f from
s u b j e c t iv e p r e j u d ic e s w hich m ight le a d to m is in t e r p r e t a t io n o f the tru e
p ic tu r e .
In s p i t e o f a l l p o s s ib le p r e c a u tio n s th e p r e se n t scheme may
be c o lo r e d by such m is in t e r p r e t a t io n s and th e r e fo r e sh o u ld be c o n sid e r e d
a s o n ly t e n t a t i v e u n t i l i t
a to r s.
can be confirm ed or r e v is e d by o th e r i n v e s t i g
I t was f e l t th a t i t s in c lu s io n would p o r tr a y th e d i s s o c i a t i o n
p a t t e r n in a more g ra p h ic manner than an ex ten d ed d e s c r ip t io n .
In th e 20 c u lt u r e s ob serv ed in t h i s stu d y i t was noted th a t s e v e r a l
c u lt u r e s d i s s o c i a t e d more r a p id ly than d id o t h e r s .
C e rta in c u lt u r e s a l s o
ten d ed to produce a predom inance o f a p a r t ic u la r R type w h ile o th e r s
showed a predom inance o f a n o th e r ty p e .
S t i l l o th e r c u ltu r e s d is p la y e d
a lm o st no ten d en cy toward d i s s o c i a t i o n w hereas some d i s s o c ia t e d so r a p id ly
F i g . 44*
D i s s o c i a t i o n schem e sh o w in g o r i g i n o f th e p r i n c i p a l
s t a b l e R c u l t u r e s . 1 - 1 , 1 - 2 , R -4 &nd
a r e no^
s t a b l e b u t a r e in c lu d e d a s m o st R fo rm s d e v e lo p e d
from th em . R-3 was th e o n ly s t a b l e R ty p e to com
m only d e v e lo p d i r e c t l y from S c u l t u r e s . B roken
l i n e s i n d i c a t e l e s s common d e v e lo p m e n t. R -1 4
a c t u a l l y d e v e lo p s from f i l a m e n t s (R -13 on R -7 and
some R - l l c o l o n i e s . R-1& i s n o t shown a s i t r a r e l y
d e v e lo p e d i n th e a b se n c e o f 2 , 3* 5 “ t r i p h e n y l t e t r a
z o liu m c h l o r i d e .
1 -1
I-E
R-4
R -5
R-7
R-3
R -ll
R-14
- 107 t h a t i t was d i f f i c u l t to r ec o r d i t s d ir e c t io n a c c u r a te ly *
The scheme
shown on p a g e 106 r e p r e s e n t s th e m ain tr e n d shown by a l l th e c u lt u r e s a s
a group*
No doubt c e r t a i n s t r a i n s , i f tak en i n d i v i d u a l l y , would p r e s e n t
a d i f f e r e n t p ic t u r e and c e r t a in o f th e R typ e c o lo n ie s w ould n ev er ap
p ear*
At t h i s tim e th e s i g n i f i c a n c e o f th e w ide v a r ia t i o n in r a te o f d i s
s o c i a t i o n o f th e v a r io u s c u lt u r e s cqn o n ly be c o n je c tu r e d *
Whether c u lt u r e s
i s o l a t e d from p a r t i c u l a r d is e a s e p r o c e s s e s showed a d i s t i n c t i v e d i s s o c i
a t i o n p a tte r n h as n o t y e t been a c c u r a t e ly d eterm in ed to d a t e .
l e s s th e r e were s t r o n g in d ic a t io n s t h a t t h i s m ig h t be tru e*
N e v e r th e
In g e n e r a l
i t ap p eared t h a t th r e e s t r a i n s (1007X , 1192X, 1197X) i s o l a t e d from the
l i v e r o f c a lv e s w h ich d ie d s h o r t l y a f t e r b i r t h (Gray e t a l * . 1954) anc^
two s t r a i n s (455X , 9^720 i s o l a t e d from th e s p in a l f l u i d o f man (b o th un
r e p o r t e d ) showed a f a r more r a p id d i s s o c i a t i o n th an d id any o f th e o t h e r
ty p e s *
A lso th e s e p a r t ic u l a r s t r a i n s produced many more R-3t R-7» R-8
and R-9 c o lo n ie s th a n d id any o f th e o th e r c u l t u r e s .
C u ltu r e s i s o l a t e d
from th e b r a in o f s h e e p and cows w ith l i s t e r i o s i s showed a r a th e r a v e ra g e
c o n s i s t e n t p a t te r n and r a t e o f d i s s o c i a t i o n .
However, a few c u lt u r e s
showed a lm o st no d i s s o c i a t i o n o th e r th an th e developm ent o f sm a ll 1 -1
and 1 -2 d au gh ter c o l o n i e s .
C u ltu r e s f r e s h ly i s o l a t e d from th e v is c e r a o f
a r t i f i c i a l l y i n f e c t e d r a b b it s (n o t in c lu d e d in th e o r i g i n a l 20 c u lt u r e s )
d e v e lo p e d a predom inance o f typ e R-3 d a u g h te r s, and 1 -1 and 1 -2 ty p e s
w ere r a r e ly s e e n in th e s e c u l t u r e s .
Some o f th e s e c u lt u r e s n ev er gave
r i s e to o th e r th an R-3 c o lo n ie s w h ile o th e r s produced a v a r ie t y o f th e
se v e r a l ty p e s.
Two c u lt u r e s o f L i s t e r i a i n f a n t i s e p t i c a (new s p e c ie s q u e s tio n e d )
r e c e iv e d from P o t e l and i s o l a t e d from a b o rted in f a n t s in the German
- 108 D em o cra tic R e p u b lic , and a s im ila r c u lt u r e r e c e iv e d from S e e l i g e r ,
H ygiene I n s t i t u t e , F r ie d r ic h -W ilh e lm s U n iv e r s it y , Bonn, Germany, showed
a w ide v a r i e t y o f R fo im s on i n i t i a l p la t in g s s im ila r to th o se se e n in
th e c u lt u r e s i s o l a t e d from c a lv e s *
T hese c u lt u r e s were r e c e iv e d to o
l a t e f o r e x t e n s iv e o b s e r v a t io n s to be in c lu d e d i n t h i s r ep o rt*
More
o v e r i t was found th a t th e s e c u lt u r e s w ere n o t a g g lu t in a t e d by r a b b it
a n t i s e r a p rep ared a g a in s t a s t r a i n o f L* m on ocytogen es i s o l a t e d from a
b o v in e b ra in *
Whether th e r e i s a c o n n e c tio n betw een the d if f e r e n c e in
d i s s o c i a t i o n and th e f a i l u r e to a g g lu t in a t e can n ot be s t a t e d a t t h i s
tim e , b u t i t d oes p r e s e n t an i n t e r e s t i n g s u b j e c t f o r f u r t h e r stud y*
A s t r a i n o f L . m on ocytogen es i s o l a t e d from th e i n t e s t i n a l t r a c t o f
a g u in e a p i g by R oine a t a l * (1 9 5 3 ) i n F in la n d showed a w ide v a r ie t y o f
R form s on i n i t i a l p l a t i n g s and i s p r e s e n t ly under f u r t h e r stud y*
As i t
was fou nd t h a t a l l pure R form s were non p a th o g e n ic f o r th e r a b b it th e r e
i s a s u g g e s t io n t h a t th e s e c u lt u r e s w h ich show a w ide v a r i e t y o f R form s
may be l e s s p a th o g e n ic th an th o s e w hich show o n ly S form s on i n i t i a l
i s o l a t i o n a s do th e s t r a i n s i s o l a t e d from sheep and cows w ith l i s t e r i c
e n c e p h a lit is *
There i s o f co u rse th e p o s s i b i l i t y t h a t d i s s o c i a t i o n o f
t h e s e c u lt u r e s o c c u r r e d d u rin g t r a n s i t *
However, many o f th e s t o c k c u l
tu r e s o f L* m on ocytogen es m a in ta in e d i n t h i s la b o r a to r y f o r lo n g p e r io d s
o f tim e w ith o u t t r a n s f e r f a i l to show th e r a p id and marked changes a s
th e s e c u lt u r e s from l e s s " ty p ic a l"
d is e a s e p ro cesses*
("T ypical"
is
used h ere on th e q u e s tio n a b le assu m p tion th a t e n c e p h a li t is o f rum inants
i s th e m ost c h a r a c t e r i s t i c and d i s t i n c t i v e form o f i n f e c t i o n due to t h i s
b a c te r iu m ).
S o h ie r e t a l* (1 9 4 $ ) r e p o r t the i s o l a t i o n o f a non p a th o g e n ic
s t r a i n o f L . m on ocytogen es from cooked b e e f*
As non p a th o g e n ic s t r a in s
o f t h i s m icrobe a re r a th e r r a r e , i t would p rove to be an i n t e r e s t i n g
- 109 s t r a in fo r fu rth er stu d y.
I t s a r r iv a l i s a n tic ip a te d s h o r tly .
The
f iv e s t r a in s is o la te d by Wenckebach (1953) from the u reth ra l exudate o f
f iv e men w ith gonorrhea should a ls o prove in te r e stin g #
At the present
s t a t e o f our knowledge i t seems more than coincidence that str a in s which
d is s o c ia t e slow ly are a ss o c ia te d w ith acute d isea se p rocesses and the
more r a p id ly d is s o c ia tin g s tr a in s are a sso c ia te d w ith a more chronic type
o f d is e a s e .
A s t r ik in g ex cep tio n to t h is g e n e r a liz a tio n i s the s tr a in
r e c e n tly is o la t e d by Gray and McWade (1954) from the cer v ix o f a s o c a lle d rep eat breeder cow which showed no c li n i c a l in d ic a tio n s o f in f e c
t io n .
This cu ltu re has shown very l i t t l e tendency toward d is s o c ia t io n .
Therefore i t appears th a t many more comparisons w i l l be required before
a d e f in it e trend can be e s ta b lis h e d .
The r o l e o f S and R c u lt u r e s in th e e p id e m io lo g y o f v a r io u s b a c t e r i a l
d i s e a s e s h a s b een th e s u b j e c t o f numerous i n v e s t i g a t i o n s .
b een summarized by H adley ( 1927) and Braun (1947)*
T hese have
The r e l a t i o n s h i p b e
tw een d i s s o c i a t i v e c h a n g e s , accom panied by changes in v ir u le n c e , to
ep id e m ic waves i s s t i l l a m a tte r o f s p e c u la t io n .
Braun (1947) c i t e s
s e v e r a l in s t a n c e s in w hich S c u lt u r e s o f v a r io u s b a c t e r i a l s p e c ie s were
i s o l a t e d i n th e a c u te s t a g e s o f a d is e a s e and th%t a s th e d is e a s e becom es
more c h r o n ic , th e i s o l a t e d organism s showed a marked ten d en cy toward R
fo r m s•
A ls o c i t e d was an e p id em ic o f S h i g e l l a d y s e n te r y in w hich S form s
were i s o l a t e d i n th e e a r l y s t a g e s o f th e e p id e m ic , SR form s a s th e e p id e m ic ,
Saned and R form s tow ards th e end o f th e e p id e m ic .
C u ltu r e s from c h r o n ic
c a r r i e r s o f ty p h o id b a c i l l i and E r y s ip e lo t h r ix r h u s io n a th ia e have y ie ld e d
R fo r m s .
I t i s contend ed th a t th e s e changes in th e b a c t e r i a l p o p u la tio n
a r e in t im a t e ly a s s o c i a t e d w ith changes in th e s e l e c t i v i t y o f th e h o s t e n
v ir o n m e n t.
Thus normal serum a p p ea rs to c o n ta in f a c t o r s w hich su p p re ss
- 110 th e e s t a b lis h m e n t o f R form s and f a v o r s th e d evelopm ent o f 3 fo r m s.
But a s a n t ib o d ie s ( a g g l u t i n i n s ) a r e produced a g a in s t th e S form , i t i s
s u p p r e s se d and an environm ent i s e s t a b li s h e d w hich f a v o r s th e developm ent
of
R form s*
When th e R typ e h a s been e s t a b lis h e d in d is e a s e c a r r i e r s ,
th e r e f o ll o w s a d e c r e a s e in S a n t ib o d ie s w hich may a l t e r th e environm ent
by p e r m itt in g th e n a tu r a l a n t i R f a c t o r s to e x e r t t h e i r e f f e c t on th e R
form , and a c o n d it io n fa v o r a b le to th e r e e s ta b lis h m e n t o f 3 form s r e s u l t s .
F ib r in o u s e n c a p s u la t io n s , a s found i n many b a c t e r i a l d i s e a s e s , may
a l s o s e r v e to remove th e S form s from th e t o t a l body en v iro n m en t.
If
i n th e s e e n c a p s u la t io n s th e r e i s an S to R tr a n s fo r m a tio n , when th e R
form s a r e r e le a s e d in t o th e b od y, c o n d it io n s would fa v o r th e e s t a b l i s h
m ent o f S form s*
T h is p o s s i b i l i t y p r e s e n t s i n t r i g u i n g im p lic a t io n s f o r
a d i s e a s e , such a s t h a t produced by L* mo no cv to g e ne s where th e l e s i o n s
a r e a lm o st alw a y s l o c a l i z e d and c o n c e iv a b ly cou ld and would be e n c a p s u l
a te d i f th e d is e a s e w ere l e s s a c u te *
S e d a ll ia n e_t a l .
( 1 9 4 8 ) r e p o r te d
th e i s o l a t i o n o f what th e y c o n s id e r e d an R form o f L. m onocytogenes from
a l o c a l i z e d lu n g l e s i o n in a 45 y e a r o ld man.
The a u th o r s do n o t d e s c r ib e
th e c o lo n y but s t a t e d th a t s t a in e d sm ears showed la r g e ro d s to f ila m e n t s
w hich were gram n e g a t iv e , m o t ile and r e q u ir e d g lu c o s e f o r g ro w th .
f a i l e d t o k i l l r a b b it s and to produce a c o n j u n c t i v i t i s .
It
However i t d id
k i l l m ice and g u in e a p ig s w hich showed a m o n o c y to sis and f o c a l h e p a t ic
n e c r o s is a t n e c r o p s y .
H elm boldt et. a l . (1951) r e p o r te d th e i s o l a t i o n
o f L . m on ocytogen es from a l o c a l i z e d a b c e s s i n th e b r a in o f a p ig w hich
d ie d a f t e r show ing symptoms o f a n erv o u s d is o r d e r .
w it h l i s t e r i c e n c e p h a l i t i s were n o t o b s e r v e d .
L e sio n s a s s o c ia t e d
U n fo r tu n a te ly no m en tio n
was made o f c o l o n i a l c h a r a c t e r i s t i c s o f t h i s c u lt u r e .
Old l o c a l i z e d
f i b r o t i c l e s i o n s have o f t e n been o b se rv e d in v a r io u s v i s c e r a , p a r t i c u l a r l y
- I ll i n th e u te r u s and h e a r t o f r a b b it s th a t s u r v iv e d in tr a v e n o u s ex p o su re*
On c u lt u r e t h e s e l e s i o n s a r e o f t e n s t e r i l e *
I n a fe w i n s t a n c e s S —li k e
c u l t u r e s o f L* mo no c y t o g e ne s h a v e b een i s o l a t e d .
o f th e
A t no tim e h ave any
ty p e c u l t u r e s i d e n t i f i e d i n t h i s s tu d y b e e n i s o l a t e d ,
A n o ta b le exam ple o f a l o c a l i z e d l e s i o n due to L . m on ocytogen es i s
th e e n c a p s u la te d n e c r o t ic fo c u s w hich d e v e lo p s a t th e s i t e o f su b cu tan eou s
in o c u l a t io n o f c u lt u r e s from S c o l o n i e s .
T h is l e s i o n p r e s e n t s i n t e r e s t i n g
p o s s i b i l i t i e s f o r S t o R o r R to S s t u d ie s *
At th e p r e s e n t tim e t h i s
h as b een s u b j e c te d to o n ly a few e x p lo r a to r y o b s e r v a t io n s , and i n s u f
f i c i e n t d a ta have been accu m u la ted to draw p o s i t i v e c o n c lu s io n s *
From
some o f th e s e l e s i o n s w hich d e v e lo p from S c u lt u r e s i t was p o s s i b l e to
r e i s o l a t e what ap p eared to be S c u ltu r e s *
H ow ever, a f t e r 2 4 hr in c u b a
t i o n t h e s e c o l o n i e s , e s p e c i a l l y th o s e w e ll s e p a r a t e d , appeared to be
la r g e r and have a more w r in k le d s u r fa c e th an d id th e o r i g i n a l S c o lo n ie s
a f t e r th e same in c u b a tio n p e r io d .
F i g . 33*
A fte r l±Q hr in c u b a tio n th e y
were i n d is t in g u is h a b le from th e o r i g i n a l S c u lt u r e .
At t h i s tim e i t can
n o t be d e f i n i t e l y s t a t e d w h eth er th e s e c o lo n i e s r e p r e s e n t th e s o - c a l l e d
" r e v e r te d 9* o f H adley ( 1 9 3 7 ) , S p ic e r (193&) and M ellon ejb a l • (1 9 4 4 )*
" R everted S" i s a term a p p lie d to S - l i k e c o lo n ie s w hich a r i s e from R
c u lt u r e s and appear to be an R to S r e a c t io n but a c t u a l l y i s an £ to R
to S1 , S1 b e in g th e r e v e r t e d S .
The r e v e r te d S o f S p ic e r (1 9 3 6 ) and
M ello n eib a l . (1944-) showed d i s t i n c t d i f f e r e n c e s from th e normal £ fo rm .
Braun ( 1 9 4 6 ) i n dji v i t r o s t u d ie s w ith B r u c e lla a b o r tu s found o n ly an
in c r e a s e d grow th r a t e f o r r e v e r te d S fo r m s, a l l o th e r c h a r a c t e r i s t i c s
were i d e n t i c a l to th e norm al S .
T h is a g a in p r e s e n t s th e q u e s tio n o f
w h eth er th e r e i s tr u e R to S r e v e r s io n o r w hether i t i s o n ly " app arent
r e v e r sio n " .
H uddelson jet a l * ( I 952) f a i l e d to f in d e v id e n c e o f R to £
r e v e r s io n e i t h e r in i n v i t r o o r in v iv o s t u d i e s w ith B r u c e l la .
Braun
( 1946 a ) claim s true rev ersio n i s p o ssib le providing the environment i s
such, that S forms which develop in R cu ltu res are able to m an ifest them
s e lv e s *
This i s seldom p o ssib le under in v itr o condions but may occur
in v iv o .
In t h is study there was no evidence that true R to 3 rev ersio n o c
curred e ith e r in v it r o or in. v iv o * S forms which developed from R cu ltu res
m aintained fo r long p eriod s o f time in tryp tose broth containing 2 , 3» 5 “
trip h e n y ltetr a zo liu m ch lorid e or from the sm all lo c a liz e d n e c r o tic le s io n s
th a t developed in r a b b its a fte r subcutaneous in o c u la tio n o f R c u ltu r es
to which Wydase had been added, were presumed to be the r e s u lt of S type
c e l l s in the R c u ltu r es or reverted S ty p e s.
ported th at p la tin g s o f
Webb and Barber (1937) r e
c o lo n ie s o f L* mono cy to ge ne s r e su lte d each time
in approxim ately equal numbers o f both S and R fonsns, in d ic a tin g that
they may a c tu a lly have had an I form rather than a true R.
S e e lig e r and
Linzenmeier (1953) f a i le d to fin d evidence o f 3 forms in R c u ltu r es even
a f t e r 18 mo o b serv a tio n .
version*
H irato e t a l * (1954) did not mention R to S r e
However, from these meagre data i t appears that there i s no
true R to S r ev e rsio n in c u ltu r e s o f L. monocytogenes.
From the photographs o f R c o lo n ie s o f L. monocytogenes published by
Webb and Barber (1937)* Barber (1 9 3 9 ). S e e lig e r and Linzenmeier (1953)
and H irato e t a l , (1954)* one can o n ly sp eculate as to which p a r tic u la r
colony type they were d e a lin g .
None of the photographs appear to have
been made w ith o b liq u e ly r e fle c te d l i g h t .
The R c o lo n ie s described by
Webb and Barber (1937) no doubt were type R-Zj.*
This would ex p la in the
alm ost equal numbers o f S and R forms which developed from su cc essiv e
p la t in g s .
Other R forms which may have developed perhaps escaped d e tec
tio n due to the fa c t th at oblique lig h tin g was not employed.
Only Barber
- 113 -
(1939) m entions the occurrence o f interm ediate form s.
appeared to be an R-4 ty p e.
The one p ictured
The R c o lo n ie s shown may have been R-3*
T h is was s u g g e s te d by t h e i r ir r e g u la r m argins and lo n g s le n d e r fila m e n to u s
c e lls .
The c e l l s o f th e in te r m e d ia te ty p e were a m ix tu re o f rod s and
fila m e n ts .
In t h i s r e s p e c t t h e i r f in d in g s were s im ila r to th o s e r e p o r te d
here.
The c o lo n ie s shown by H ir a to et. ad. (1954) appear to be R-3*
p h otom icrograp h te n d s to con firm t h i s .
The
The c o lo n ie s shown by S e e l i g e r
and L in zen m eier (1954) were i n t e r e s t i n g b eca u se th e y a r e th e o n ly o n es
to show e v id e n c e o f s e c t o r fo r m a tio n .
O ther than th a t i t would be d i f
f i c u l t to d eterm in e w ith w hich s p e c i f i c R form th e y were w orking a s t h e i r
c u lt u r e s were grown on b lo o d a g a r .
Ih terson (1940)* i*1 stu d ie s o f the p a th o g en icity o f L. mono cy to genes
fo r the embr3ronating chick en , did not d escrib e the R forms w ith which he
worked.
N eith er are there su g g estio n s as to which p o ssib le R forms the
various French workers had encountered.
(Verge and Goret, 1941* B e lin ,
1946? Harvier et, a l • , 1943? S e d a llia n ejb a d ., 1948)*
I t i s remarkable th at o f the se v er a l d octoral th eses (Z e lle r , 1949?
B o ek els, 1950; Schu lz, 1950; Ozgen, I 95 I 5 Beinhauer, 1953) presented a t
the Justus L ieb ig V eterinary High School, G iessen, Gennany, none o f the
authors mentioned the occurrence o f R forms; in s p ite of e x te n siv e stu d ies
on c u ltu r a l c h a r a c te r is tic s in clu d in g c o lo n ia l morphology.
Only Z e ller
( 1 9 4 9 ) m entions the presence o f halos on some co lo n ie s a fte r sev era l days
in cu b a tio n .
The fa ilu r e to recognize R forms must have r e su lte d from
f a ilu r e to employ o b liq u ely r e fle c te d l i g h t .
There i s a need f o r a more e f f e c t i v e v a c c in e a g a in s t l i s t s r i c
- 114 -
e n c e p h a litis than i s p resen tly a v a ila b le .
Tfte use o f n on -viru len t varia n ts
o f s e v e r a l b a c te r ia l sp ec ies as immunising agents i s w e ll e s ta b lis h e d .
The most notable type perhaps i s the M form o f B ru cella abortus (Huodelson,
1946)*
However in the genus L is t e r ia i t has been found that a g g lu tin a t
in g a n tib o d ies are not a measure o f p ro tectio n a g a in st in fe c tio n .
J u lia n e lle (1941) on stu d ie s in mice was the f i r s t to su ggest t h i s .
Hunter e t a l . (1950) found th at mice immunized w ith v a ria n ts o f L .
monocytogenes produced by exposure to rad io a ctiv e phosphorus, gave no
p r o te c tio n when the mice were challenged with a le t h a l dose o f cu ltu re s ix
weeks l a t e r .
I t has been found in unpublished stu d ie s th at rab b its im
munised w ith S c u ltu r es o f L. monocytogenes and which showed high a g g lu t
in a tin g antibody t i t e r s , (1 :1 2 8 0 or more) a g a in st S cu ltu res w ere not
always p rotected from a le t h a l exposure to the homologous c u ltu r e .
This
was again confirmed in the attem pts to immunize ra b b its w ith R c u ltu r es
as reported h ere.
I t i s obvious th at some other means to measure immunity
must be devised fo r t h is bacterium .
Various in v e stig a to r s (B e lin , 194&?
Graham a t a l . , 1943; Kaboyashi, 1951; Zink et. a l . ,
; P o te l, 1954?
Ehrenhardt, 1954) found th at many animal sp e c ie s show high a g g lu tin a tin g
t i t e r s a g a in st L* m onocytogenes.
H irato et. a l . (1954) reported wide f l u c
tu a tio n s in a g g lu tin a tin g t i t e r s of in d iv id u a l sheep during an outbreak
o f lis te r io s is *
U n til the sig n ific a n c e o f th ese t i t e r s i s determ ined, they
serve no purpose as an in d ic a tio n o f e ith e r previou s exposure or p o te n tia l
r e sis ta n c e *
Hunter e t a l . (1950) and S e e lig e r and Linzenmeier (1953) are the
o n ly in v e s tig a to r s to mention a g g lu tin a tio n stu d ie s w ith R c u ltu r e s ,
Two o f the 19 v a r ia n ts is o la te d by Hunter .et ad. (1950) a g g lu tin a ted
spontaneously in s a lin e when a g g lu tin a tio n r e a c tio n s were attem pted.
No mention was made o f a u to a g g lu tin a tio n in the remaining 17 v a ria n ts
- 115 s t u d ie d .
S e e l i g e r and L in zen m eier (1 9 5 3 ) found th a t R c u lt u r e s showed a
s t r o n g ten d en cy toward a u t o a g g lu t in a t io n .
Only by th e employment o f
u l t r a s o n i c s were th e y a b le to p rep are s t a b l e s u s p e n s io n s o f R c u l t u r e s .
By t h i s m ethod th e y w ere a b le to d em o n stra te s p e c i f i c a g g lu t in a t io n o f
R c u ltu r e s .
A u to a g g lu tin a tio n d id n o t p r e s e n t a p a r t i c u l a r problem in
th is stu d y .
I t s o c c u r r e n c e was i n c o n s i s t e n t and a t tim es in v a lid a t e d
a p p a re n t r e s u l t s .
B o e c k e ls (1950 s u g g e s te d th a t i f a 0 .2 ^ s a l i n e s o l u
t i o n w ere em ployed f o r a g g lu t in a t i o n r e a c t io n s w ith L. m on ocytogen es more
c o n s i s t e n t r e s u l t s c o u ld be o b ta in e d than w ith 0.85% s a l i n e .
T h is has
n o t been u sed w ith R c u lt u r e s b u t may prove u s e f u l in th e f u t u r e .
T h is r e p o r t e s t a b l i s h e d t h a t in th e genus L i s t e r i a a t l e a s t 2 0 d i s
t i n c t c o l o n i a l form s o c c u r , s i x o f w hich can be m a in ta in e d in s t a b le
fo r m .
Of t h e s e 2 0 fo r m s , one i s sm ooth and th e o th e r s r o u g h .
Whether
any o f th e R form s o c cu r i n n a tu re e i t h e r w it h in or o u t s id e th e an im al
b od y, o r o c c u r a s common b u t u n re c o g n iz ed s o i l organism s o r sa p r o p h y tes
rem ain s to be d e te r m in e d .
However w ith th e d e t e c t i o n and r e c o g n it io n o f
th e s e 20 t y p e s a tte m p ts can be i n i t i a t e d to d eterm in e th e n a tu r a l r e s e r
v o ir o f t h i s m ic r o o rg a n ism .
SUMMARY
Twenty d i f f e r e n t c o l o n i a l form s o f L i s t e r i a m on ocytogen es co u ld be
d e t e c t e d by means o f o b li q u e ly r e f l e c t e d l i g h t and a b in o c u la r s c a n n in g
m ic r o s c o p e .
One o f th e s e foim s was th e n a t u r a lly o c c u r r in g smooth form
fou nd in i s o l a t i o n s from in f e c t e d human o r anim al t i s s u e .
T h is typ e was
c h a r a c t e r iz e d by a s m a ll c i r c u l a r , s l i g h t l y r a i s e d , f i n e l y t e x t u r e d , tr a n s
l u c e n t , b lu e -g r e e n c o lo n y .
T h is gave r i s e e i t h e r d i r e c t l y or i n d i r e c t l y
t o th e rem a in in g 19 ty p e s d e s ig n a te d a s ro u g h .
They were c h a r a c te r iz e d
- il6 -
by a v a r i e ty o f c o lo n ia l forms which d if f e r e d in t e x t u r e , c o n fig u r a tio n ,
o r c o lo r from th e smooth ty p e .
Only th e smooth form was p ath o g en ic f o r
r a b b i t s when in o c u la te d in tra v e n o u s ly o r i n s t i l l e d in to th e e y e .
th e 19 rough forms could be m ain ta in e d in s ta b le form .
i n d ic a tio n s t h a t rough ty p es r e v e r te d to sm ooth.
S ix o f
There were no
On th e b a s is o f f e r
m e n ta tiv e and b io ch em ical r e a c tio n s the rough ty p es were i n d is ti n g u i s h
a b le from the p a re n t smooth ty p e , b u t d id d i f f e r in a n tig e n ic s t r u c t u r e .
C e rta in rough form s which produced hig h a g g lu tin a tin g an tib o d y t i t e r s
a g a in s t th e smooth form f a i l e d to p r o te c t r a b b its a g a in s t a subsequent
l e t h a l dose o f smooth c u lt u r e .
I t was suggested t h a t a n tib o d ie s found
in th e blood o f many anim als which a g g lu tin a te d th e smooth type c u ltu r e
were n e it h e r an i n d ic a t i o n o f exposure nor o f im m unity.
Because o f th e
u b iq u ito u s n a tu re o f l i s t e r i o s i s in anim als and man, th e re i s a p o s s i b i l i t y
t h a t some o f th e s e rough form s may e v e n tu a lly be found i n e i t h e r c a r r i e r
a n im als o r su b sta n c e s such a s s o i l o r w a te r.
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M it c h e ll L. Gray
L i s t e r i a m o n o cy to g en es:
A Review and D i s s o c ia t io n S tu d ie s
An A b s tra c t
The t h e s i s i s d iv id e d in to two p a r t s .
P a r t one c o n s t it u t e s a review of th e w o rld 1s l i t e r a t u r e w ith
em phasis on th e v a rio u s d is e a s e p ro c e sse s w ith which th e b acteriu m
L i s t e r i a m onocytogenes. has been a s s o c ia te d .
E n c e p h a litis in sh eep ,
cows and g o a ts was th e m ost p re v a le n t form .
Then fo llo w ed se p ticem ia
w ith m e n in g itis in many se p tic e m ia w ith o r w ith o u t m e n in g itis in monog a s t r i c a n im a ls , e s p e c ia lly ro d e n ts ; a b o rtio n in man; a b o rtio n in an im als
and c o n j u n c t i v i t i s in man*
The accum ulated r e p o r ts f a i l e d to re v e a l
c l e a r c u t e p id e m io lo g ic a l tre n d s and th e s p e c i f i c mode o f tra n s m is s io n
was o b s c u re .
The blood o f many normal anim als was found to c o n ta in h ig h
a g g lu t i n a t in g a n tib o d y t i t e r s f o r L. m onocytogenes.
In no in s ta n c e was
th e b a c te riu m found o u ts id e th e anim al body y e t th e wide d i s t r i b u t i o n and
m u l t i p l i c i t y o f d is e a s e p ro c e s s e s w ith which i t has been a s s o c ia te d su g
g e s ts t h a t th e b a cteriu m i s a c t u a l l y w id ely d i s t r i b u t e d in n a tu r e , perh ap s
in an u n reco g n ized form .
P a r t two i s a s tu d y devoted to th e re c o g n itio n and i s o l a t i o n o f
"rough" c o lo n ia l v a r i a n ts which develop in "smooth" c u ltu r e s m ain tain ed
on a r t i f i c i a l media under la b o r a to r y c o n d itio n s .
By means o f o b liq u e ly
r e f l e c t e d l i g h t and a b in o c u la r sc an n in g m icroscope 20 d i f f e r e n t c o lo n ia l
form s o f L. m onocytogenes could be d e te c te d .
One o f th e se forms was th e
n a t u r a l l y o c c u rin g smooth form found in i s o l a t i o n s from in f e c te d human
o r an im al t i s s u e .
T h is was c h a ra c te riz e d by a sm all c i r c u l a r , s l i g h t l y
r a i s e d , f i n e l y te x tu r e d , tr a n s lu c e n t , b lu e -g re e n colony which gave r i s e
e i t h e r d i r e c t l y o r i n d i r e c t l y to th e rem ain in g 19 rough ty p e s .
These were
M it c h e ll L. Gray
c h a r a c te r iz e d by a v a r i e ty o f c o lo n ia l forms which d i f f e r e d in t e x t u r e ,
c o n f ig u r a tio n , o r c o lo r from th e smooth ty p e .
p a th o g e n ic f o r r a b b i t s .
in pu re form .
Only th e smooth form was
Six o f th e 19 rough forms could be m ain tain ed
Rough ty p e s were in d is tin g u is h a b le from th e p a re n t smooth
ty p e i n fe rm e n ta tiv e and biochem ical r e a c t i o n s .
C e rta in rough form s
w hich produced h ig h a g g lu tin a tin g an tib o d y t i t e r s a g a in s t the smooth
form f a i l e d to p r o t e c t r a b b i t s a g a in s t a subsequent l e t h a l dose o f smooth
c u l t u r e , s u g g e stin g t h a t a n tib o d ie s found in th e blood o f many anim als
which a g g lu tin a te d smooth type c u ltu r e s v/ere n e ith e r an in d ic a tio n o f e x
p o su re nor im m unity.
Because o f th e u b iq u ito u s n a tu re o f l i s t e r i o s i s
i n an im als and man, th e r e i s a p o s s i b i l i t y t h a t seme rough forms may
e v e n tu a lly be found in e i t h e r c a r r i e r an im als o r su b sta n c e s such a s s o i l
and w a te r.
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