Antibody response to oral and subcutaneous administration of Escherichia coli

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ANTIBODY RESPONSE TO ORAL AND SUBCUTANEOUS ADMINISTRATION OP ESCHERICHIA COLI

A T hesis P resented to the F a c u lty

of

th e D epartm ent of B a c te r io lo g y

U n iv e rsity

of

S outhern C a lif o r n ia

In P a r tia l F u lfillm e n t of t h e R e q u ire m e n ts f o r t h e Degree M aster

of

Science

by K e i t h M o r r i s Cowan May 1 9 5 0

UMI Number: EP55010

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

Dissertation Publishing

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,3a /S'0

CKn^t

This thesis, written by ....................................

under the guidance of h..?:3.. Faculty Com m ittee, and a p proved by all its members, has been presented to and accepted by the Council on Graduate S tu d y and Research in partial fulfill­ ment of the requirements fo r the degree of

.................^STEH... OF... SCIENCE.............................

De an Dau.

June 1 9 5 0 ______

Faculty Committee

TABLE OP CONTENTS CHAPTER I. II. III.

PAGE

INTRODUCTION

. . . . .

1

REVIEW OP L I T E R A T U R E ........................................................

.

MATERIALS AND P R O C E D U R E S .................................................. P r e p a r a tio n of A ntigen f o r

In jectio n

. . . .

3 11. 11

P re p a ra tio n ' of A ntigen f o r O ral A d m in istratio n

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

P r e p a r a tio n of B ile f o r O ral A d m in is tra tio n

12

.

13

I m m u n i z a t i o n P r o c e d u r e s ....................................................

14

P r e p a r a tio n of A ntigen f o r the A g g lu tin a tio n T ests IV.

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

15

E X P E R I M E N T A L ............................................................ '.....................

17

B lood A g g l u t in a t io n T i t e r s

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

17

P ecal A g g lu tin atio n T ite rs

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

17

R E S U L T S ................................................................................................

20

Serum A g g l u t i n i n R e sp o n se

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

20

P e c a l A g g lu tin in Response

.

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

26

V I.

D I S C U S S I O N ......................................................................................

29

V II.

SUMMARY AND CONCLUSIONS.......................................................

32

V.

B I B L I O G R A P H Y ........................................................

33

LIST OP TABLES TABLE I*

PAGE P e c a l a g g l u t i n i n re sp o n se to o r a l l y and subcu tan eo u sly ad m in istere d E. (zuerich)

c o li

a n t i g e n ...................................................................

27

.L I S T OP FIGURES FIGURE 1.

PAGE The a g g l u t i n i n r e s p o n s e o f r a b b i t s r e c e i v i n g E.

2.

c o li a n tig e n by v a rio u s

Serum a g g l u t i n i n t i t e r s an tig en of ra b b its w ith E.

3.

co li

21

w i t h hom ologous

im m unized s u b c u t a n e o u s l y

(zu erich )

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

22

Serum a g g l u t i n i n t i t e r s w i t h h o m ologous an tig en of ra b b its b ile

4.

r o u t e s .........................

and E • c o li

im m unized o r a l l y w i t h

(zu erich )

Serum a g g l u t i n i n t i t e r s a n tig en of rab b its E . co li

a n t i g e n ...................

24

w i t h hom ologous

imm unized o r a l l y w i t h

( z u e r i c h ) a n t i g e n ...............................................

25

CHAPTER

I

INTRODUCTION A n tig en ic su b sta n c es are o r d i n a r i l y in tro d u c e d in to t h e b o d y a s a r e s u l t of* a n a c c i d e n t a l i n v a s i o n of* t h e h o s t b y some p a r a s i t i c m i c r o - o r g a n i s m o r v i r u s . how ever, be in tr o d u c e d a r t i f i c i a l l y d u c in g im m unity.

Im m unizing d o ses

A n t i g e n s m ay,

f o r th e purpose of p ro ­ of a n tig e n s have been

a d m i n i s t e r e d i n many d i f f e r e n t f o r m s a n d b y s e v e r a l d i f f e r ­ ent ro u tes. C on sid erab le c o n tro v e rsy has a r i s e n in the p a st con­ c e rn in g the a d v an tag e s of o r a l a d m i n i s t r a t io n of a n tig e n o v e r s u b c u t a n e o u s a n d o t h e r m o d es o f i n j e c t i o n . t h e s t u d i e s made,

the c r i t e r i a

f o r d e te rm in in g the degree of

i m m u n i t y p r o d u c e d was t h e b l o o d a g g l u t i n i n o b tained. it

W hile t h i s

I n m ost o f

t i t e r t h a t was

i s n o t an a b s o l u t e m e a su re o f im m unity

Is a tec h n iq u e t h a t r e a l l y dem o n strates a n tib o d y p ro ­

d u ctio n . B urrow s, H avens, and E l l i o t th e presence

(1947) have d e m o n s tra te d

of a g g lu tin a tin g a n tib o d ie s

in the

feces of

g u in e a p i g s t h a t h av e b e e n i n f e c t e d o r im m unized w i t h V i b r i o comma.

The p o s s i b i l i t y t h a t f e c a l a n t i b o d i e s # h a v e t h e i r

o r i g i n in a n tib o d y form ing c e l l s

of the

in te stin e ,

suggests

t h a t a n t i g e n g i v e n b y t h e o r a l r o u t e may b e m o r e e f f e c t i v e # T hese have b e e n te r m e d " c o p r o a n t i b o d i e s " by Burrov/s, e t a l

2

in s tim u la tin g f e c a l an tib o d y p ro d u ctio n th an a n tig e n given by a subcutaneous

in jectio n *

D eterm in atio n of co p roan tib od y

p r o d u c t i o n may p o s s i b l y b e a n o t h e r m e a n s o f m e a s u r i n g immu­ n ity to e n te ric In th is

in fec tio n s* study,

a n a t t e m p t was made t o p r o d u c e a n d

c o m p a r e b o t h s e r u m a n d f e c a l a g g l u t i n a t i n g a n t i b o d i e s when a stra in

o f E s c h e r i c h i a c o l i was a d m i n i s t e r e d b y b o t h t h e

subcutaneous and o ra l r o u t e s .

CHAPTER I I REVIEW OF THE LITERATURE A ttem pts to produce a r t i f i c i a l in je ctio n ,

im m un ity by s u b c u t a n e o u s

o r a l in g e s tio n or su rfa c e a p p lic a tio n of the a n t i ­

gen d a te back to a n t i q u i t y .

N ativ es

of th e e a st co ast of

A fric a v a c c in a te d th em selv es a g a in s t snake p o iso n by a p p ly ­ ing a p a ste

c o n ta in in g the p o iso n to s k in lesio n s*

C h in ese and Siam ese i n t r o d u c e d v a r i o l o u s trils

and s k in le s io n s

The

scabs in to the nos­

f o r p r o t e c ti o n a g a i n s t sm allp o x .

O ral v a c c in a tio n has a ls o been u t i l i z e d

in th e p a s t.

M ith rid ates,

im m unized h i m s e l f

K ing o f P o n t u s

(120 B . C . ) ,

by d r in k in g th e b lo o d o f ducks t h a t had b e en t r e a t e d w ith t o x i c s u b s t a n c e s ; h e a l s o a t e p o i s o n o u s mushrooms i n s m a l l q u a n titie s. liv ers

The Roman, P l i n y t h e E l d e r ,

o f mad d o g s a s a c u r e f o r r a b i e s .

recom mended t h e (F inder and

S im m o n s, 1 9 3 2 ) About s i x t y y e a rs ago, E h r li c h

(1 8 9 3 ) showed he c o u ld

d e v e lo p a h i g h d e g r e e o f im m unity i n m ice a g a i n s t r i c i n and a b r i n by f e e d i n g them c ak es c o n t a i n i n g t h e s u b s t a n c e s .

Since

t h a t t im e , a g r e a t d e a l o f work h as b e e n done w i t h o r a l a d ­ m in istra tio n

of a n tig e n .

T here has b e en c o n s i d e r a b l e co n ­

tro v e rs y as to th e e f f ic ie n c y and a l s o

of o r a l l y a d m in is te re d a n tig e n

c o n c e r n in g th e b e s t m ethod o f o r a l a d m i n i s t r a t i o n .

M o s t o f t h e w o r k d o n e on o r a l v a c c i n a t i o n h a s b e e n d o n e w i t h

4 th e ty p h o id organism and th e r e l a t e d M etchnikoff and B esredka a g a in s t ty p h o id by th e (1914) a l s o were a b le by t h i s

ro u te.

(1914)

o ral ro u te; to

S alm o n e lla and S h i g e l l a . im m unized c h im p a n z e e s

Lum iere an d C h e v r o t i e r

immunize g u i n e a p i g s a g a i n s t t y p h o i d

I n 19 1 9 B e s r e d k a sh o w e d t h a t

not su sce p tib le

t h e r a b b i t was

t o i n f e c t i o n by m o u th w i t h t h e

p a ra ty p h o id group.

ty p h o id -

By t h e u s e o f b i l e h e was a b l e

down t h e

i n t e s t i n a l b a r r i e r to in f e c tio n .

S o letsk y

(1920) were u n a b le to c o r r o b o r a t e t h e s e

Z in g h er and

a lth o u g h th e y fo llo w ed B e sre d k a fs tec h n iq u e to

immunize r a b b i t s

to break

resu lts

in attem p tin g

o r a l l y w ith o x - b ile and p a r a - ty p h o id

v accin e• Kanai

(1921)

im m unized r a b b i t s

th e y a c q u ire d a c e r t a i n degree in o cu latio n

o f B. d y s e n t e r i a e .

o r a l l y and found

of re s ista n c e

to in trav en o u s

He o b t a i n e d a d e g r e e

im m u n ity f r o m s u b c u t a n e o u s v a c c i n a t i o n however,. th at b ile

d id not in crease

th e e f f ic ie n c y

of

He f o u n d

o f o r a l im m u n i ­

z a tio n b u t n o ted t h a t B esredka had a ls o p o in te d out t h i s f a c t in the

case

o f B. d y s e n t e r i a e .

He c o n c l u d e d t h a t o r a l

a d m i n i s t r a t i o n o f a n t i g e n wa s i n f e r i o r t o t h e

subcutaneous

ad m in istratio n . Burke a n d B a rn e s

(1926) fo u n d t h a t a g g l u t i n i n s a p ­

p e a r e d i n t h e b l o o d o f some o f t h e r a b b i t s f o l l o w i n g t h e i n tr o d u c t i o n - o f s t a n d a r d ty p h o id v a c c in e by m outh. felt

th at b ile

They

d id not enhance th e p ro d u c tio n of blood

5 ag g lu tin in s,

a lso

was m ore e f f e c t i v e

i t wa s f e l t

th a t subcutaneous v a c c in a tio n

in th a t h ig h er tite r s .w e r e

o b tain ed th an

w ith o r a lly a d m in istere d v accin e, H o f f s ta d t and co-w orkers

(1929) a d m i n i s t e r e d t r i p l e

t y p h o i d b a c t e r i n t o h u m a n s i n l i q u i d and- c a p s u l a r f o r m . rep o rted th a t b ile

T hey

p r e p a r e d i n d i v i d u a l s w e r e show n t o h a v e

g r e a t e r im m unity t h a n n o n - b i l e p r e p a r e d i n d i v i d u a l s a s i n d i - . c a te d by h ig h e r b lo o d a g g l u t i n i n t i t e r s . B e sre d k a sum m arized t h e a series

to p ic

of o ra l v a cc in a tio n in

of p u b l i c a t i o n s w hich c u lm in a te d i n h i s book "L o cal

Im m unization"

(1927).

I n t h i s w ork he s e t s

c e p t o f l o c a l im m unity as b e i n g due t o t h e of b a c te ria c ells

f o r c e r t a i n organs

he c a l l s

mucous s u r f a c e s h is

recep to rs.

en teric

i n w hich r e s i d e

of th e body as w e ll as i n th e

lin e

organism s,

w hich g iv e r i s e

th a t the

o f defence a g a in s t

or the

sk in .

of

Prom

i n t e s t i n a l mucosa in v a s io n of the

to acute i n t e s t i n a l c o n d itio n s.

organism s T his c o n ­

o b se rv a tio n th a t the f i r s t

i n g e s t i o n o f a n tig e n gave r i s e body in th e b lo o d b u t t h a t

a ffin ity

groups

to x ic products o f th ese

t e n t i o n was s u p p o r t e d b y t h e

to the appearance of a n t i ­

su b se q u e n t ones d id n o t .

p e r i o d when h e was a b l e t o d e m o n s t r a t e m als,

selec tiv e

con­

The l a t t e r a r e f o u n d * i n t h e

o b se rv a tio n s, B esredka f e l t

form s th e f i r s t

fo rth his

At t h e

im m unity i n h i s a n i ­

th e a g g l u t i n i n s had p r a c t i c a l l y d is a p p e a r e d from th e

blood stream .

T h i s i m m u n i t y was c o n s i d e r e d t o b e m a i n l y

l o c a l b e c a u se of an in c r e a s e d r e s i s t a n c e c e lls th e

of th e

c ast o ff in the

in fe c tio n are th is

to x in .

The d a m a g e d c e l l s

c a t a r r h a l c o n d i t i o n s e t up by th e

s u p p o s e d l y r e p l a c e d b y new o n e s p o s s e s s e d o f

acq u ired in creased re s is ta n c e

lo o k e d upon t h i s the

of the

i n t e s t i n a l m u c o u s m em b ran e t o t h e p a s s a g e o f

i n f e c t i n g organism s o r of i t s

w hich a re

on t h e p a r t

in itia l

B esredka

c a t a r r h a l sta g e as e s s e n t i a l to

estab lish m en t of a s a tis f a c to r y

a d m in is tr a tio n of b i l e

p ro p erty .

l o c a l im m unity.

By t h e

j u s t p r i o r to th e fe e d in g of the

a n t i g e n * h e f e l t h e was a b l e t o b r e a k down t h e i n t e s t i n a l b arrier.

H i s e x p l a n a t i o n was t h a t t h e b i l e

had a desquam at

i n g a c t i o n on t h e i n t e s t i n a l m u c o s a , d e n u d i n g t h e r e c e p t i v e c e l l s w hich w ere th e n a c c e s s i b l e The f u n c t i o n o f b i l e

to the m icro -o rg an ism s.

i n t h e p ro b le m o f o r a l imm uni­

z a tio n has been s tu d ie d by o th e r w o rk ers. showed t h a t b i l e a lk a lin e

sid e

s h i f t e d th e a c id -b a s e b alance

i n t h e duodenum an d t h a t

in tra -in te stin a l

A rnold

(1928)

to w ard th e

t h i s wa s t h e b e s t

r e a c t i o n f o r the p assag e o f b a c t e r i a and

d isso lv e d p ro te in through th e w alls F i n d e r a n d Simons

of the i n t e s t i n a l t r a c t

(1932) p r e s e n t e d a c o n c e p t t h a t

d i f f e r e d from t h e m e c h a n i c a l d e s q u a m a tin g m echanism s u g ­ g e s t e d by B e s re d k a .

They c o n s i d e r e d th e m echanism t o be a

p h y sio lo g ical p ro cess. of b i l e

is

T h e y f o u n d t h a t when a s o l u t i o n

s u d d e n l y b r o u g h t i n c o n t a c t w i t h t h e empty r e s t ­

in g stom ach d u r in g th e p o s t d i g e s t i v e p e r i o d ,

th e norm al

7 acid se c re tio n is

dim inished*

D uring t h i s

p e r i o d o f dim ­

i n i s h e d s e c r e t i o n a n a n t i g e n m ay b e p a s s e d u n c h a n g e d t h r o u g h the g a s t r ic sh ift

lumen i n t o

th e duodenum.

W ith the r e s u l t a n t

of th e a c id -b a s e b alan ce to the a lk a li n e

passage

of th e a n tig e n through th e w a ll o f the

sid e,

the

in te stin a l

t r a c t w ould be e n h a n c e d . The m ore r e c e n t w o r k h a s p r e s e n t e d c o n f l i c t i n g e v i ­ dence as to th e r e l a t i v e v accin atio n .

e ffic ie n cy

S e v e r a l w o r k e r s h a v e a d v o c a te d o r a l im m uniz­

in g p r o c e d u r e s and have f e l t of p ro d u c in g a g g l u t i n i n s 1937; F r a e n k e l, cates

o f o r a l and su b cu tan eo u s

th at i t

(Sim ons,

i s an e f f i c i e n t m ethod

1 9 3 1 ; Moor a n d B ro w n ,

1 9 3 8 ; Grimm a n d S h o r t ,

1938).

of su b cu tan eo u s i n j e c t i o n have u t i l i z e d

ferent te s ts a b ility

to d eterm in e th e

The a d v o ­ several d i f ­

c o m p a r a tiv e im m unizing

o f t h e v a r i o u s modes o f a n t i g e n a d m i n i s t r a t i o n .

Moor a n d W a l l a c e

(1941) i n v e s t i g a t e d p r o t e c t i v e a n t i b o d i e s

p r o d u c e d by t h e

o r a l a d m in is tr a tio n of ty p h o id v accin e and

concluded t h a t

o r a l v a c c i n a t i o n c o u ld n o t be recommended a s

a p r a c t i c a l im m unizing p r o c e d u r e . plem ent f i x a t i o n

O t h e r w o r k e r s u s e d com­

t e s t s and blood a g g l u t i n a t i o n t i t e r s

and

concluded th a t w h ile a n tib o d ie s

c o u ld be p ro d u c e d by t h e

o ra l a d m in is tra tio n of a n tig e n ,

i t was n o t p o s s i b l e

t a i n th e h igh t i t e r s of i n j e c t i o n

(T uft,

to ob­

t h a t w ere p r o d u c e d by o t h e r m ethods Y agle,

K e n n e d y a n d C um mings ,

and R oger,

1932; E lle d g e ,

1 9 4 3 ; Naumer a n d K e r b ,

1943).

Mos t o f t h e w o r k d o n e o n o r a l a d m i n i s t r a t i o n o f a n t i g e n h a s b e e n d o n e on t h e S a l m o n e l l a a n d S h i g e l l a g r o u p o f organism s. p ro tec tiv e istratio n

K o ss

(1934) has r e p o r t e d , how ever,

an tib o d ies

c o u ld be p ro d u ce d by t h e o r a l ad m in ­

o f Type I p n e u m o c o c c u s to' r a t s .

h e was a l s o a b l e

th at

He f o u n d t h a t

to produce th e s e p r o te c tiv e a n tib o d ie s

in

man. The a b i l i t y

of an o r a l l y a d m in is te re d a n tig e n to

produce a g g l u t i n a t i n g a n tib o d ie s e stab lish ed .

The b u l k o f t h e e v i d e n c e ,

t h a t the t i t e r s

a g g lu tin a tin g a n tib o d ies in persons

p athogens, c u tan eo u s

how ever,

in d icates

p r o d u c e d a r e n o t a s h i g h a s w hen s u b c u t a n e ­

ous v a c c i n a t i o n i s u t i l i z e d .

and a ls o

in th e b lo o d serum i s w e ll

suggests

The r e c e n t r e p o r t

in the feces

of fin d in g

o f im m unized a n im a ls

in fe c te d w ith c e r ta in of th e e n te r ic t h a t a co m parison b etw een o r a l and s u b ­

i n o c u l a t i o n o f a n t i g e n b e made t o d e t e r m i n e t h e i r

e fficien cy

in producing f e c a l a n tib o d ie s .

The p r e s e n c e o f a g g l u t i n i n s s c r i b e d by D avies

(1922).

i n f e c e s was f i r s t d e ­

T h is w ork was o v e r l o o k e d u n t i l

r a t h e r r e c e n t l y when s e v e r a l R u s s i a n i n v e s t i g a t o r s u t i l i z e d the p resen c e nosis

of fe c a l a n tib o d ies

of b a c illa r y d y sentery

Skochko,

fo r th e la b o ra to ry d ia g ­

(P r e d p e c h e n s k y a n d Moroz,

1942; Y am polsky, G o r f u n k e l an d A r o n ia ,

1944).

I t was s u g g e s t e d i n t h e r e c e n t w o r k o f B u r r o w s , E l l i o t and Havens

(1947) t h a t t h e

immune m e c h a n i s m

1940

resp o n sib le

for. th e

in itia tio n

o f r e c o v e r y from i n f e c t i o n s

c o n f i n e d e s s e n t i a l l y t o t h e lumen an d m ucosa o f th e i n ­ testin al- tr a c t,

v iz .,

c h o l e r a , b a c i l l a r y d y s e n te r y and

c e r t a i n S a l m o n e l l a i n f e c t i o n s , may i n a l a r g e p a r t b e d u e to f e c a l a n tib o d ie s .

They h a v e shown t h a t a n t i b o d y i s

e x c r e t e d ' i n f e c e s by- b o t h i n f e c t e d a n d i m m u n i z e d a n i m a l s a n d im m unized v o l u n t e e r s ; m ental in f e c tio n shown t h a t t h e

t h a t , im m unity t o e x p e r i ­

is a sso c ia te d w ith i t s

appearance

o f serum a n t i b o d y , serum a n ti b o d y i s appears d e sp ite

also

presence.

of c o p ro a n tib o d y p re c e d e s t h a t

reaches a peak t i t e r still

I t was

and d e c l i n e s w h ile

r i s i n g and th e n co m p letely d i s ­

the p e rs is te n c e

o f serum a n tib o d y .

They

s u g g e s t e d t h a t t h e i n d e p e n d e n t b e h a v i o r o f serum and f e c a l a n t i b o d y m i g h t be e x p l a i n e d o n t h e a s s u m p t i o n t h a t t h e la te r rep resen ts p o rte d to th e

i n t r a c e l l u l a r an tib o d y ,

lumen o f t h e b o w e l by l y m p h o c y t e s .

The f a c t t h a t the appearance and Banvard

fe c a l a n tib o d ies

of serum a n t i b o d i e s

(1947)

th at

su g g ested to H arriso n of fe c a l a n tib o d ies

in acu te

and in c h ro n ic d i a r r h e a l d i s e a s e s .

en teric

in fectio n s

F e c a l and serum t i t e r s

w e r e r u n on t h e p a t i e n t s w i t h e n t e r i c in fectio n s

were fo u n d t o p r e c e d e

the a p p ea ra n c e

may b e u s e d a s a d i a g n o s t i c a i d

activ e

p o ssib ly tr a n s ­

in fec tio n s.,

and c o n v a le sc e n t c a s e s .

w ere d e m o n s tra te d a s e a r ly , as th e t h i r d

both

Fecal ag g lu tin in s

day of the d is e a s e

an d d i s a p p e a r e d so o n a f t e r s t o o l c u l t u r e s became n e g a t i v e

10

a n d a f t e r r e c o v e r y d e f i n i t e l y was e s t a b l i s h e d . how ever,

th a t the

sig n ific an t t i t e r a te d .

They f o u n d ,

serum a n t i b o d i e s d id n o t a p p e a r to a u n t i l a b o u t -t h e tim e r e c o v e r y was i n i t i ­

These serum t i t e r s

t h e n r o s e t o a h i g h l e v e l and

rem ained a t a h i g h l e v e l f o r a c o n s i d e r a b le p e r i o d . Goodlow, R i t t e n b e r g , th e c o p ro an tib o d y resp o n se

and S i l l i k e r

(1948)

stu d ied

i n hu m an s t o TAB v a c c i n a t i o n .

T h e r e w a s no o b v i o u s r e l a t i o n s h i p b e t w e e n f e c a l a n t i b o d y a p p e a r a n c e an d t h e t im e a n d number o f a n t i g e n a s h a d b e e n r e p o r t e d by B u rro w s, ch o lera v ib rio .

et a l,

in jectio n s

(1947) w ith th e

They s u g g e s t e d t h a t t h e a p p e a r a n c e o f

c o p r o a n t i b o d i e s was p o s s i b l y d u e t o some m e c h a n i s m o f s p i l l - o v e r from t h e b lo o d s tr e a m i n s t e a d o f a l o c a l fo rm a ­ tio n is

o f the an tib o d y in th e

su b sta n tia ted

in te stin a l tra c t.

in p a r t by the o b s e rv a tio n s

G o rfu n k e l and A ronina

T h e ir view o f Y am polsky,

( 1 9 4 4 ) who h a d e m p h a s i z e d t h e h i g h

c o r r e l a t i o n betw een th e p re s e n c e of a g g l u t i n i n s and e r y t h r o ­ c y te s d e m o n s tra b le on m ic r o s c o p ic e x a m in a tio n o f t h e ' f e c e s . In th is stra in

experim ent,

of E sc h e ric h ia

an a n t i g e n p r e p a r e d from a

c o l i was a d m i n i s t e r e d s u b c u t a n e o u s l y

and o r a l l y , w ith and w ith o u t th e use o f b i l e . was made o f t h e v a r i o u s a b ility

th is

im m un izatio n r o u t e s a s to t h e i r

to p ro d u c e serum and f e c a l a g g l u t i n i n s

hom ologous o r g a n i s m .

a g a in st th e

The m a t e r i a l s a n d p r o c e d u r e s u s e d i n

s tu d y a re d e s c r ib e d and th e r e s u l t s

se n te d and d is c u s s e d .

A com parison

o btained are p r e ­

CHAPTER

III

MATERIALS AND PROCEDURES P rep aratio n

of A ntigen f o r I n j e c t i o n .

was p r e p a r e d f r o m a s t r a i n

of E.

c o li

Tiie a n t i g e n

o b ta in e d from th e

U n i v e r s i t y o f K entucky A g r i c u l t u r a l E x p e r im e n ta l S t a t i o n . The o r g a n i s m was i d e n t i f i e d a s t h e

z u e r i c h s t r a i n and

possesses

th e s o m a tic a n t i g e n s IV, V and X II a n d t h e

flag e lla r

an tig en

a n tig e n ic a lly to

z^ q .

The o r g a n i s m i s

clo sely re la te d

S alm o n ella p a r a ty p h i B and i s

rea d ily

a g g l u t i n a t e d w i t h Group B S a l m o n e l l a t y p i n g s e r a . Several te s t

tubes

o f n u t r i e n t b r o t h were i n o c u l a t e d

w i t h a sm ooth c u l t u r e

of th e

5 7 ° C. f o r 2 4 h o u r s •

To p r e p a r e a n a n t i s e r u m c o n t a i n i n g

only a g g lu tin in s to

in ac tiv a te

h e a tin g the

organism and in c u b a te d a t

fo r h eat stab le

the H a n tig e n s.

a n tig en s

T h i s was a c c o m p l i s h e d by

24 h o u r b r o t h c u l t u r e s

b o i l i n g w a t e r b a t h f o r tw o h o u r s . been h e a te d ,

i t was n e c e s s a r y

o f th e organism s A fte r th e

in a

c u ltu re s had

e n o u g h 1 0 ^ f o r m a l i n s o l u t i o n wa s a d d e d t o

a 0 .3 % s o l u t io n f o r the purpose U sing s t e r i l e v accin e b o t t l e

tech n iq u e,

give

of p reserv in g th e an tig en .

t h e b r o t h was p o u r e d i n t o

ste rile

and s to p p e re d w ith ru b b e r caps t h a t had been

b o ile d f o r f i f t e e n m in u tes. To c h e c k t h e amount of t h e

ste rility

of the a n ti g e n , a sm a ll

v a c c i n e was w i t h d r a w n f r o m t h e v a c c i n e b o t t l e

12

w ith a s t e r i l e

n eed le and sy rin g e and in o c u la te d in to t h i o -

g ly c o lla te b ro th .

T h e r e was no g r o w t h s o t h e v a c c i n e wa s

co nsidered s t e r i l e . Pr e p a r a t i o n o n e-liter

flask s

of A ntigen f o r O ral A d m in is tr a tio n .

Two

th a t had been s la n te d w ith n u t r ie n t ag ar

w ere s t r e a k e d w i t h a sm ooth c u l t u r e

of E.

c o li.

These

f l a s k s w e r e i n v e r t e d a n d i n c u b a t e d a t 3 7 ° C. f o r 24 h o u r s . A s m a ll amount o f s t e r i l e

p h y s i o l o g i c a l s a l i n e was a d d e d

a n d t h e g r o w t h f r o m t h e m e d i a was s c r a p e d o f f w i t h s t e r i l e g lass

stirrin g

rods.

th en poured o ff in to s t a i n made o f t h e p o sitiv e

T his h eav y s u s p e n s io n o f E. ste rile

c en trifu g e

c o l i was

t u b e s a n d a Gram

s u s p e n s i o n a s a c h e c k a g a i n s t a n y Gram-

co n tam in ates.

I t wa s d e s i r a b l e t o p r e p a r e a n a n t i g e n c o n t a i n i n g only h e a t s ta b le

a n tig en s.

the su sp en sio n of E. b a t h f o r tw o h o u r s

to

The c e n t r i f u g e

c o l i were p l a c e d in ac tiv a te

tu b es

c o n tain in g

in a b o i li n g w ater

the H a n tig e n .

To d e t e r m i n e t h e n u m b e r o f o r g a n i s m s i n t h e s u s ­ p e n s i o n , M a c F a r l a n d ’s n e p h a l o m e t e r b a r i u m s u l f a t e

stan d ard s

were u t i l i z e d .

of th e

su sp en sio n , lu tio n s

i t was n e c e s s a r y t o p r e p a r e a s e r i e s

of th e

stan d ard s.

Due t o t h e e x t r e m e l y h e a v y n a t u r e

su sp en sio n in

order to

I t was f o u n d t h a t t h e

of d i ­

compare them w i t h th e

o r i g i n a l s u s p e n s i o n wa s

10 t i m e s a s t u r b i d a s t h e No. 3 b a r i u m s u l f a t e

stan d ard .

T h i s r e p r e s e n t s a c o u n t o f a p p r o x i m a t e l y 12 b i l l i o n

organism s

13 per m illilite r . pension,

W ith 25 m i l l i l i t e r s

of the o r ig in a l s u s ­

i t was c a l c u l a t e d t h a t t h e r e w ere a p p r o x i m a t e l y

300 b i l l i o n

organism s i n th e

su sp en sio n .

T h i s s u s p e n s i o n was t h e n c e n t r i f u g e d a t 2 0 0 0 RPM f o r 30 m i n u t e s .

The s u p e r n a t a n t f l u i d was c a r e f u l l y p o u r e d o f f

and th e c e n t r i f u g e

t u b e was t h e n p l a c e d i n a d e s s i c a t o r

o v e r c a l c i u m c h l o r i d e a n d u n d e r vacuum f o r 48 h o u r s . end of t h i s d ried

p erio d ,

th e

jar

At th e

o rg a n is m s were fo u n d i n a th o r o u g h ly

sta te . The d r i e d

o r g a n i s m s w e r e t h e n a d d e d t o 15 g r a m s o f

d e x t r o s e an d t h o r o u g h l y m ixed w i t h a m o r t a r and p e s t l e . T h i s m i x t u r e was c o n s i d e r e d t o b i l l i o n o rg a n is m s p e r gram .

c o n t a i n a p p r o x i m a t e l y 20

The d r i e d E .

m i x t u r e was t h e n p l a c e d i n g e l a t i n

c a p s u le s w hich w ould h o ld

a p p r o x i m a t e l y 0 . 2 5 grams o f t h e m i x t u r e , about 4 to

6 b illio n

c o l i and d e x tr o s e

g iv in g a count of

organism s p e r c a p s u le .

These c a p s u l e s w ere t h e n e n t e r i c

c o ated to in s u re

th e a n t i g e n w ould n o t be a f f e c t e d by t h e stom ach g a s t r i c ju ices.

T his e n t e r i c

c o a t i n g w ould a llo w th e c a p s u le s to

p a ss th ro u g h th e stom ach and i n t o th e before

t h e y w o u ld be d i s s o l v e d ,

stru c tio n

in te stin a l trac t

th u s e lim in a tin g the d e ­

o f a l a r g e p o r t i o n o f t h e a n t i g e n by th e g a s t r i c

secretio n s.

The c a p s u l e s w e r e s t o r e d a t r e f r i g e r a t o r

tem p eratu re. jjjvgPa-ration o f B i l e f o r O r a l A d m i n i s t r a t i o n .

D ried

14 o x -b ile

( D i f c o ) was p l a c e d i n g e l a t i n

c a p s u l e s w h ic h would

h o l d a p p r o x i m a t e l y 0 . 2 5 t o 0 . 3 grams o f t h e b i l e .

The

r a b b its b ein g te s te d w ith o r a lly a d m in istere d a n tig e n w ith b ile ,

were g iv e n t h r e e

o f -these c a p s u l e s

a t a tim e , w hich

was t h e e q u i v a l e n t o f 10 m l . o f l i q u i d b i l e . I m m u n i z a t i o n Pr o c e d u r e s .

T h i r t y - s i x y o u n g New

Z ealan d r a b b i t s w ere d iv id e d i n t o f o u r g ro u p s ,

t h r e e groups

were im m unized by t h e v a r i o u s r o u t e s a n d t h e f o u r t h s e r v e d as a c o n tr o l group. 1.

S ubcutaneous r o u t e .

of ten ra b b its . of 0 .5 m l., A fo u rth

the t h i r d p o ssib le

They r e c e i v e d a s e r i e s

1 .0 m l.,

and 1 .5 m l. a t

in je ctio n .

T his

seven day i n t e r v a l s .

i n j e c t i o n was g i v e n f o r t h e

O ral ro u te p lu s b i l e .

day p r i o r to

T his gro u p c o n s i s t e d o f

t h a t r e c e i v e d on e e n t e r i c - c o a t e d

c o ll an tig en th ree

tim es a t

the a d m in is tr a tio n

capsule

of the a n ti g e n ,

the

of b i l e .

On t h e f o l l o w i n g d a y ,

n e c e s s a ry to ta k e g r e a t

rab b its

of

th e y w ere g iv e n s i m i l a r

Two h o u r s f o l l o w i n g t h i s b i l e

a n i m a l s w e r e f e d one c a p s u l e

The

cap su les of d rie d

w h i c h w a s t h e a p p r o x i m a t e e q u i v a l e n t o f 10 m l .

liq u id b ile .

of

seven day i n t e r v a l s .

r e c e i v e d no f o o d a n d w e r e g i v e n t h r e e

doses

in jectio n s

s tim u la tio n of f e c a l an tib o d y e x c r e tio n .

ten ra b b its

b ile

of th ree

i n j e c t i o n o f 1 . 5 m l . was g i v e n 12 d a y s f o l l o w i n g

2.

E.

T h i s g r o u p wa s c o m p o s e d

of a n tig en each.

feeding, It

the

was

care to in su re t h a t e n te r ic - c o a te d

15 c a p s u l e was s w a llo w e d i n t a c t . 3.

O ral r o u te .

rec eiv e d the a n tig e n

The t e n r a b b i t s

capsules th re e

tim es a t

of th is

group

s e v e n day

in te rv a ls• P r e p a r a tio n of A ntigen f o r th e A g g lu tin a tio n T e s t s . S e v e r a l o ne l i t e r

b o t t l e s were s l a n t e d w i t h n u t r i e n t a g a r

a n d a l l o w e d t o s t a n d i n a n u p r i g h t p o s i t i o n f o r tw o d a y s t o e l i m i n a t e any m o is tu r e from th e s u r f a c e The b o t t l e s

of the m edia.

were s t r e a k e d w i t h a sm ooth c u l t u r e E.

a n d i n c u b a t e d a t 3 7 0 ° C. f o r 2 4 h o u r s .

co li

A fter in cu b atio n

t h e g r o w t h i n e a c h b o t t l e was s u s p e n d e d i n a s m a l l a m o u n t of s a l i n e w hich c o n ta in e d 0 .5 $ p h e n o l.

A Gram s t a i n wa s

p r e p a r e d from th e s u s p e n s io n s to

th e absence

Gram p o s i t i v e

in su re

c o n ta m in a tin g organism s.

The s u s p e n s i o n s

w ere m e a su re d and p o o le d i n an E rle n m e y e r f l a s k . f l a s k was b e i n g s l o w l y r o t a t e d ,

The a l c o h o l - t r e a t e d

poured i n to

b o ttle s

A p o rtio n

and th e se d im e n t d is c a r d e d .

b r o t h to be su re the

The t u r b i d i t y

of th is

s u s p e n s i o n wa s s t e r i l e .

s u s p e n s i o n was a d j u s t e d t o

t h a t of a barium s u l f a t e

s t a n d a r d No. 3 , a n d i t s

a lc o h o l c o n te n t to a p p ro x im ately 30$. sisted

s u s p e n s i o n was

o f t h e s u s p e n s i o n was i n o c u l a t e d i n t o t h i o -

g ly co llate

10 t i m e s

Viftiile t h e

54$ b y volum e o f 95$ e t h y l

a l c o h o l wa3 a d d e d . ste rile

of

of a s a lt

so lu tio n

The d i l u e n t c o n ­

c o n t a i n i n g 0 . 5 $ o f p h e n o l com­

b i n e d w i t h 54$ o f 95$ a l c o h o l .

16 For the a g g lu tin a tio n t e s t s , trated

1 p a rt of th e concen­

s u s p e n s i o n wa s c o m b i n e d w i t h 9 p a r t s

of sa lin e

to

o b ta in a su s p e n s io n e q u iv a le n t in t u r b i d i t y to a barium su lfa te

s t a n d a r d No. 3 . To t e s t

an tig en ,

the a g g l u t i n a b i l i t y

s e ria l d ilu tio n s

o f 1;

2 ,56 0.

of E.

tin a te d to a t i t e r

of the f in e g ra n u la r type th a t

sa tisfac to ry

The s e r u m was

A ntigen p re p a re d

c o l i b e i n g u s e d was f o u n d t o b e a g g l u ­

o f 1; 2 ,560 a l s o .

so m atic a g g l u t i n a t i o n .

zu erich

o f 1 ; 2 , 5 6 0 w he n S a l m o n e l l a p a r a ­

t y p h i B was u s e d a s t h e a g g l u t i n o g e n . from th e s t r a i n

c o li

o f Group B S a l m o n e l l a t y p i n g s e r a

were p r e p a r e d to a d i l u t i o n sh ow n t o h a v e a t i t e r

of the E.

is

The a g g l u t i n a t i o n was

ch aracteristic

of

The a n t i g e n was c o n s i d e r e d t o b e

fo r fu rth e r ag g lu tin atio n t e s ts .

CHAPTER IV EXPERIMENTAL B lood A g g l u t i n a t i o n T i t e r s . tra tio n

of th e

an tig en ,

a ll

P rio r to the a d m in is­

o f t h e r a b h i t s were b l e d from

th e m a r g i n a l e a r v e i n and th e serum s o b t a i n e d were u s e d for

s e t t i n g up tube a g g l u t i n a t i o n

run to d eterm in e th e p resen c e a g a in s t E.

c o li

zu erich . were d e te r m in e d i n t h e

A n i n e t u b e a g g l u t i n a t i o n t e s t was p e r f o r m e d

on t h e se ru m s i n w h ic h d i l u t i o n s 1:160,

1:320,

The t e s t s w e r e

of any n a t u r a l a g g lu tin in s

The a g g l u t i n a t i o n t i t e r s u s u a l m anner.

te sts.

1:640,

1 :1 ,2 8 0 ,

of 1:10,

1:20,

1:40,

1:80,

and 1 : 2 ,5 6 0 were p r e p a r e d .

Upon a d d i t i o n o f t h e a n t i g e n ,

d ilu tio n s

1:160 ,

1 :2 ,5 6 0 and 1 :5 ,1 2 0 w ere

1:320,

o b tain ed .

1:640,

1 :1 ,2 8 0 ,

The t u b e s w e r e i n c u b a t e d a t

and th en r e f r i g e r a t e d

o v ernig h t.

th e a id o f th e concave s u r f a c e is

1:20,

1:40,

1:80,

4 7 ° C. f o r tw o h o u r s

The t e s t s w e r e r e a d w i t h

of a m icroscope m ir r o r as

o f t e n d o n e i n t h e r e a d i n g o f Ka hn t e s t s . Pour days f o llo w in g th e i n i t i a l

th e su b c u tan e o u s and o r a l v a c c in e to a n im a ls w ere b l e d and a g g l u t i n a t i o n ings

and a g g l u t i n a t i o n t e s t s

a d m in is tra tio n of

the r a b b i t s ,

the

t e s t perform ed.

B leed­

were t h e n p e rf o r m e d e v e r y f o u r

days f o r th e n e x t s i x w eeks. g Q c a l Aggl u t i n a t i o n T i t e r s .

In the

late

aftern o o n

18 r a b b i t s were p l a c e d i n i n d i v i d u a l c ag es t h a t h a d b een c a r e ­ fu lly

c le a n e d of any r a b b i t f e c e s .

in th e s e cages m o rning.

The r a b b i t s w e r e l e f t

o v e rn ig h t and r e tu r n e d to t h e i r

In. a lm o st a l l

cases,

t h e r e was e n o u g h f e c e s d e ­

p o s ite d d uring t h i s p e rio d to p re p a re The m e t h o d o f p r e p a r i n g

cages in the

the f e c a l e x t r a c t s .

the f e c a l e x tr a c t i s

d e s c r ib e d by

E a r r I s on (1 9 4 7 ). The f e c e s b o ttle s

c o l l e c t e d was p l a c e d i n I n d i v i d u a l

th a t had been p ro p erly

m en s w e r e r e a d i l y

l a b e le d so t h a t

Id en tifie d .

To e a c h b o t t l e

the s p e c i ­ a p p ro x im ately

4 v o l u m e s o f 0 , 5 % f o r m a l i z e d s a l i n e was a d d e d p e r v o l u m e of fec es. it

Due t o t h e

firm n atu re

of the f e c a l p e l l e t s ,

wa s n e c e s s a r y t o c r u s h t h e p e l l e t s w i t h g l a s s

rods to in su re proper e x tr a c tio n .

The m i x t u r e was t h e n

f u r t h e r d i s p e r s e d by sh a k in g th e b o t t l e s a m echanical

stirrin g

f o r 3 0 m i n u t e s on

s h a k e r t h a t p r o d u c e d a p p r o x i m a t e l y 250 o s c i l ­

l a t i o n s p e r m in u te w ith a l|r in c h s t r o k e .

The b o t t l e s

were p l a c e d In th e r e f r i g e r a t o r f o r 3-4 h o u rs f o r e x t r a c t i o n . At t h e end o f t h i s

p erio d ,

a p p ro x im a te ly 7 -1 0 m l.

m i x t u r e was p l a c e d i n a c e n t r i f u g e BPM f o r one h o u r .

A rela tiv e ly

off

flo ated

on t h e s u r f a c e .

in to funnels

circum vent t h i s

tu b e and spun a t 3 ,0 0 0

c l e a r s u p e r n a t a n t wa s o b ­

ta in e d except fo r larg e p a r tic le s th at

of the

of c e llu lo se m ateria l

The s u p e r n a t a n t wa s p o u r e d

lin e d w ith a f in e grade f i l t e r p a p er to facto r.

I n some c a s e s i t w a s n e c e s s a r y

19 to re c e n trifu g e

to o b ta in a s a t i s f a c t o r y e x t r a c t .

e x t r a c t was c o n s i d e r e d a 1 : 4 d i l u t i o n

of th e

T his

feces.

The f e c a l e x t r a c t was t h e n u s e d f o r t u b e a g g l u t i n a ­ tio n te s ts

in th e u s u a l m anner.

A fiv e

tube a g g lu t i n a t io n

t e s t wa s p e r f o r m e d u s i n g u n d i l u t e d f e c a l e x t r a c t . d i l u t i o n s w i t h t h e ad d ed a n t i g e n were 1:8* 1 :6 4 , and 1 :1 2 8 .

The a g g l u t i n a t i o n

1:16,

P in al 1:32,

t e s t s w ere in c u b a te d

a t 4 7 ° G. o v e r n i g h t a n d t h e n r e f r i g e r a t e d f o r 8 - 1 0 h o u r s b e fo re b ein g re a d .

The t e s t s

were r e a d w i t h t h e a i d o f t h e

concave s id e o f a m ic ro sc o p e m i r r o r . A f e c a l a g g l u t i n i n t i t e r was p e r f o r m e d p r i o r t o th e a d m in is tr a tio n o f th e a n tig e n to th e an im als.

The t e s t

wa s r e p e a t e d 2 d a y s f o l l o v i r i n g t h e a d m i n i s t r a t i o n o f t h e a n tig e n and e v ery o th e r day u n t i l the end o f th e e x p e rim e n t.

CHAPTER

V

RESULTS Serum A g g l u t i n i n R e s p o n s e . E. c o l i a n tig e n by th e v a rio u s su p erio rity

of s u b c u ta n e o u s

A d m in is tra tio n of the

ro u tes dem o n strated the

in je ctio n

over the

two o r a l

m ethods a t t e m p t e d as m ea su re d b y serum t i t e r . tio n

o f t h e m e a n o f t h e maximum t i t e r s

rab b its

in each group,

is

1:80 to 1 :1 ,2 8 0 . the

subcutaneous

i n maximum t i t e r s

of 1:160,

tite r

a n d o ne a t i t e r

t h e s e maximum t i t e r s

r a n g i n g from

were d i s t r i b u t e d

three a t i t e r

of 1:320,

in

of 1 :8 0 , four a

of 1 :1 ,2 8 0 .

The m e a n o f

was fo u n d t o be 1 : 5 0 4 .

Peak t i t e r s

w e r e r e a c h e d tw o t o t h r e e w e e k s f o l l o w i n g t h e jec tio n

1.

in je ctio n

one r a b b i t r e a c h e d a t i t e r

a n d one a t i t e r of 1:640,

to

The maximum t i t e r s

fo llo w in g m anner:

r e a c h e d by t h e

g r a p h i c a l l y sh o w n i n F i g u r e

The a g g l u t i n i n r e s p o n s e of the a n tig e n r e s u l t e d

D eterm ina­

of the a n tig e n ,

in itia l

in ­

t h e y w e r e on t h e d e c l i n e b y t h e

f o u r t h week a t w h i c h t i m e a f o u r t h i n j e c t i o n was g i v e n . T h is f o u r t h i n j e c t i o n was g i v e n f o r t h e p u r p o s e o f s t i m u ­ latin g , It

i f p o ssib le ,

resu lte d

the e x c re tio n of f e c a l a n tib o d ie s .

in ra p id in cre ase in t i t e r ,

ceeding th e p re v io u s h ig h e s t t i t e r s The r e s u l t s

of subcutaneous

i n m any c a s e s e x ­

t h a t had been o b ta in e d .

in je c tio n of the E.

a n t i g e n a r e sh o w n i n F i g u r e 2 .

c o li-zu erich

21

,280 Mean of

640 320

Maximum S e ru m

1 60

T iters Reached

80 40 20

O ral p lu s b i l e

O ral

Subcutaneous

FIGURE 1 THE AGGLUTININ RESPONSE OF RABBITS RECEIVING E . COLI ANTIGEN BY VARIOUS ROUTES

25 60 1 28 0 640 Serum 320 Agglu16 0 tin in T iter

80 40 20 0

mull nu n m rrm i

0

in 11

15 19 25 27 * 31 Time i n d a y s a f t e r f i r s t i n j e c t i o n FIGURE 2

35

SERUM AGGLUTININ TITERS WITH HOMOLOGOUS ANTIGEN OF RABBITS IMMUNIZED SUBCUTANEOUSLY WITH E . COLI (ZUERICH) * In jected

39

23 The r e s p o n s e t o o r a l l y a d m i n i s t e r e d a n t i g e n r e ­ s u l t e d in c o n s id e ra b ly low er t i t e r s th e subcutaneous

in je ctio n s.

b eing reached th an w ith

F i g u r e 3 sh o w s t h e

a g g lu t i n in resp o n se o f th e group r e c e iv in g th e o rally

in c o n ju n ctio n w ith b i l e .

by any of th e r a b b i t s

in th is

ex p erim en t.

Two r a b b i t s

o n o n l y o ne o c c a s i o n ,

g r o u p was 1 : 8 0 ,

th is

reached tite r

One p r o d u c e d no

a t any tim e d u rin g th e

gave p o s i t i v e

ag g lu tin atio n

th e re m a in in g w ere p o s i t i v e

to four d if f e r e n t o ccasio n s. tite rs

an tig en

The maximum t i t e r

was a t t a i n e d b y o n l y o n e o f t h e a n i m a l s . d e m o n s tra b le serum a g g l u t i n i n s

serum

tests

on two

The m ean o f t h e maximum

r e a c h e d was f o u n d t o b e 15 3 2 ,

t h e maximum r e s p o n s e

f o r t h e g r o u p was s e e n t o o c c u r 23 d a y s f o l l o w i n g t h e in itia l

in g e s tio n of the a n tig e n . The r e s u l t s

the use

of b i l e ,

sid erab le

of o ra lly ad m in istered an tig en ,

a r e sh o w n i n F i g u r e 4 .

v a r i a t i o n i n t h e maximum t i t e r

r a b b i t fo llo w e d a norm al p a t t e r n sponse b u t rea ch e d a t i t e r

w ithout

T h e r e was c o n ­ o b tained.

One

of serum a g g l u t i n i n r e ­

o f 1*640 w h ic h was c o n s i d e r ­

a b ly h ig h e r th a n t h a t re a c h e d by any o f th e o t h e r r a b b i t s in th is tite rs

group. for th is

f o r th e group. f o r th e group i s response

D e t e r m i n a t i o n o f t h e m ean o f t h e maximum group i s

n o t a r e p r e s e n t a t i v e maximum t i t e r

The m e a n i s

fo u n d t o be I s 125,

th e m edian

l s 6 0 a n d i s m ore t y p i c a l o f t h e a g g l u t i n i n

of th e group.

A nother r a b b i t in t h i s

group,

160

P inal Serum

80 40

-

20

-

D ilu tio n

0

\ 1 1r i n u n 1ruf i v11 u t li ii rm r r r r m i m m m i T m m 0 3 7 11 15 19 *

&

m m m i 11 It tJ.I H 111ill i l i n m i n i PL ~ 23 27 31 35

45-

Time i n d a y s a f t e r i n i t i a l a d m i n i s t r a t i o n FIGURE 3 SERUM AGGLUTININ TITERS WITH HOMOLOGOUS ANTIGEN OP RABBITS IMMUNIZED ORALLY WITH BILE AND E . COLI (ZUERICH) ANTIGEN # A ntigen a d m in is te re d

to

640

-

32 0

-

160

-

80

-

40

—

20

-

f

0 v

r

r n n u i n u t r i n m n I Mmi i m [ l i i i mi n i i n i ' Mil U7TTTT 3 7 11 15 19 23 27 *!£ *${■ Time i n d a y s a f t e r i n i t i a l a d m i n i s t r a t i o n

mm Ituinim 31 35

FIGURE 4 SERUM AGGLUTININ TITERS WITH HOMOLOGOUS ANTIGEN OF RABBITS IMMUNIZED ORALLY. WITH E . COLI ANTIGEN

to

Oi

#

A ntigen a d m in is te re d

26

No . 3 6 , o b t a i n e d a t i t e r

o f 1;80 th r e e days fo llo w in g

i n i t i a l a d m in istra tio n of a n tig en , sev en th day.

l i v e r and lu n g s .

m a t e r i a l from th e

It

r e v e a l e d m any s m a l l l e s i o n s

A Gram s t a i n was p r e p a r e d w i t h

l e s i o n s a n d o b s e r v a t i o n r e v e a l e d many

s m a l l Gram n e g a t i v e r o d s a n d m an y p u s c e l l s . th e

1 ;160 by the

The a n i m a l d i e d o n t h e n i n t h d a y a t w h i c h t i m e

a n a u t o p s y wa s p e r f o r m e d . in the

r is in g to

the

I t was f e l t

r a p i d a g g l u t i n i n r e s p o n s e was p r o b a b l y d u e t o a n

an am n estic

re a c tio n or to a c ro s s-a g g lu tin a tio n .

I n th e c o n tr o l group, p o sitiv e

a g g lu tin atio n te s ts

on t h e t h i r t y - f i r s t 12 2 0 , t h e

bein g rec o rd ed .

day o f th e

o t h e r on t h e

These w ere th e

t h e r e w e re two i n s t a n c e s

of

One o c c u r r e d

experim ent a t a t i t e r

t h i r t y - n i n t h day a t a t i t e r

of

o f 1240.

o n l y two i n s t a n c e s o f a g g l u t i n a t i o n b e i n g

n o te d by any of th e c o n t r o l r a b b i t s . F ecal A g g lu tin in R esponse.

The d e t e c t i o n o f f e c a l

a n t i b o d i e s wa s e x t r e m e l y i r r e g u l a r a n d r a r e . 650 a g g l u t i n a t i o n t e s t s were o n ly s i x t e s t s sponses.

p e r f o r m e d on f e c a l e x t r a c t s ,

t h a t gave p o s i t i v e

Of t h e r a b b i t s

Out o f a b o u t

ag g lu tin atio n r e ­

show ing f e c a l a g g l u t i n i n s ,

r e c e i v e d t h e a n t i g e n s u b c u t a n e o u s l y , two o r a l l y o r a lly w ith b i l e .

As sh o w n i n T a b l e I ,

e n t c o r r e l a t i o n betw een th e tim e

th ere

th ere

th ree

a n d o ne

i s no a p p a r ­

of fe c a l a g g lu tin in

s p o n s e a n d t h e t i m e o f maximum s e r u m a g g l u t i n i n

re­

tite r.

F e c a l a g g l u t i n i n s were s e e n t o o c c u r b o th p r i o r t o and a f t e r

27

TABLE

I

FECAL AGGLUTININ RESPONSE TO ORALLY AND SUBCUTANEOUSLY ADMINISTERED E . COLI (ZUERICH) ANTIGEN

R a b b i t No. a n d mode o f ad m in istratio n

D ate o f fecal a g g lu tin in appearance

Fecal a g g lu tin in tite r

D ate o f maximum serum tite r

Maximum serum tite r reached

No • 27

In j.

8 /9 /4 8

1 :8

8 /4 /4 8

1 :1 ,2 8 0

No. 22

Inj .

8 /1 3 /4 8

1:8

8 /8 /4 8

1:80

No.

19

Inj .

8 /2 3 /4 8

1:16

8/12 /4 8

1:320

No.

14

O ral

8 /1 3 /4 8

1:8

8/16 /4 8

1:80

No. 36

O ral

8 /3 /4 8

1:32

8 /4 /4 8

1:160

8 /2 7 /4 8

1:16

8 /12/48

1:40

No.

29 O r a l p lus b i le

28 maximum s e r u m t i t e r s

were o b t a i n e d .

There a l s o

i s no c o r r e ­

l a t i o n betw een f e c a l a g g l u t i n i n a p p e a ra n c e and th e serum tite r

peaks,

an im als w ith h ig h t i t e r s

dency to produce f e c a l a n tib o d ie s

h a v i n g n o m o re t e n ­

th a n the

low er t i t e r e d

an im als• I t may b e n o t e d i n T a b l e I , reach ed th e h ig h e s t f e c a l t i t e r been m entioned in the p re v io u s r a th e r rap id response to

t h a t r a b b i t No. 36

noted.

T his r a b b i t h a s

s e c tio n as re g a rd in g i t s

the o r a l l y a d m in iste re d a n tig e n .

The a n i m a l d i e d f r o m a n i n f e c t i o n a n d t h i s In d icate

may p o s s i b l y

t h a t f e c a l a n t i b o d i e s a r e more r e a d i l y e x c r e t e d

d u rin g an i n f e c t i o u s

process

z a t i o n m ethods a r e a tt e m p t e d .

t h a n when a r t i f i c i a l

im m uni­

CHAPTER VI DISCUSSION The s u p e r i o r i t y

of subcu tan eo u sly a d m in iste re d

a n tig e n over o r a l l y a d m in iste re d a n tig e n in producing serum a g g l u t i n i n s

i s w e ll d em o n strated in t h i s

C o nsiderably h ig h er t i t e r s in je ctio n s,

a lso i t

were o b t a i n e d w i t h s u b c u ta n e o u s

was f o u n d t h a t s e r u m a g g l u t i n i n s a p ­

p e a re d j u s t as r a p i d l y upon su b c u tan e o u s o r a l v a c c i n e was g i v e n . tin in s

A lthough th e

is not an a b s o lu te

of an in d iv id u a l,

experim ent.

it

is

I n j e c t i o n a s when

l e v e l of serum a g g lu

guide to th e d e g re e

an i n d i c a t o r t h a t

is

o f im m unity e a s i l y demon­

stra te d . The r e s p o n s e

to o r a l l y

a d m i n i s t e r e d a n t i g e n was

c o n tr a r y t o what had been a n t i c i p a t e d . in c o n ju n c tio n w ith E. c o li a n tig e n ,

When b i l e

was u s e d

t h e r e s p o n s e was

s o m e w h a t l o w e r t h a n when t h e a n t i g e n was a d m i n i s t e r e d b y itse lf,

serum a g g l u t i n i n

Many o f t h e r a b b i t s

tite rs

b e in g m arkedly lo w e r.

t h a t r e c e iv e d the b i l e

were n o t e d t o

have d ia r r h e a fo llo w in g the a d m in is tr a tio n of th e b i l e . T h is w ould p r o b a b l y i n d i c a t e

an i n c r e a s e

in i n t e s t i n a l

m o tility ,

some e x t e n t

th e tim e

thus d e c re a s in g to

g e n was a c c e s s i b l e m ucosa.

It

fo r ab so rp tio n in to

is p o ssib le

was g i v e n w h i c h r e s u l t e d

th a t too la rg e in th is

the

the a n t i

In testin al

an amount o f b i l e

co n d itio n .

30 It

is h ig h ly p ro b ab le th a t

a b e t t e r serum a g g l u ­

t i n i n r e s p o n s e w ould have b e e n o b t a i n e d i f d a i l y f e e d i n g s o f t h e a n t i g e n h a d b e e n g i v e n f o r a p e r i o d o f two w e e k s o r so.

The a n t i g e n d o s e u s e d i n t h i s

v o c a te d by p rev io u s w orkers tio n .

(H o ffstad t, e t a l, W hile i t

tite rs

in th e

experim ent i s field

one a d ­

o f o r a l im m uniza­

1929; B urke and B arnes.,

is a n tic ip a te d

1926, e t c . )

th a t hig h er a g g lu tin in

w o u ld ha v e b e e n o b t a i n e d b y g r e a t e r and more f r e ­

quent a d m in istra tio n s

of the a n tig e n ,

use p re v io u s ly te s te d tech n iq u es

i t was d e s i r a b l e t o

in order th a t

com parisons

c o u ld be m ade. The a d m i n i s t r a t i o n

of an tig en

in e n te ric

coated

c a p s u l e s was f o u n d t o b e a n e f f i c i e n t m e t h o d o f g i v i n g a n t i ­ gen by th e

o ral ro u te.

e lim in ates

the p o s s i b i l i t y

by t h e

g a stric

The u s e

of capsules

in t h i s manner

of the a n tig e n b e in g d e s tr o y e d

se cre tio n s.

The r a r e a n d i r r e g u l a r a p p e a r a n c e t i n i n s m ak e i t

of f e c a l a g g lu ­

im p o s s ib le t o d e te rm in e w hich r o u t e

gen a d m in is tr a tio n

is

th e most e f f i c i e n t

of a n t i ­

in i l l i c i t i n g

th is

response.

of the

The c o m p l e t e l a c k

o f c o r r e l a t i o n betw een th e

serum a g g l u t i n i n s

and th e f e c a l a g g l u t i n i n re sp o n se

produced by the p o ssib ilities

as

One p o s s i b i l i t y

subcutaneous

in je ctio n s

to the source is

th a t th ere

of th e

lev el

i n d i c a t e s tw o

fec al an tib o d ies.

i s a s e p a r a t e m echanism f o r

51 the p ro d u c tio n o f th e s e a g g lu t i n in s * and E l l i o t ,

1947).

ag g lu tin in p resen ts sponse i s

due t o

t h e lum en o f t h e

the

(B u rro w s , Havens

The i r r e g u l a r a p p e a r a n c e o f f e c a l the

o th er p o s s ib ility th a t th is

re­

s p i l l - o v e r of serum a g g l u t i n i n i n to

in te stin e

R itte n b e rg and S i l l i k e r ,

from th e b lo o d s tr e a m .

1949).

If

(Goodlow,

s e p a r a t e m echanism s

were r e s p o n s i b l e f o r t h e p r o d u c t i o n o f serum an d f e c a l a g g lu tin in s, tin in s

a m ore r e g u l a r a p p e a r a n c e

appear in

w eight t o

such an e r r a t i c m anner g iv e s g r e a t e r

the p ro p o s a l t h a t

an a c c id e n ta l e n tra n c e in to

the

of the f e c a l a g g lu ­

th is m an ifesta tio n

or s p ill- o v e r

in te stin al tra c t.

i s d ue t o

o f serum a g g l u t i n i n s

CHAPTER SUMMARY AND 1.

V II CONCLUSIONS

R a b b i t s have b e e n im m unized w i t h a n a n t i g e n

p r e p a r e d from E s c h e r i c h i a

co li

(zu erich s tr a in ) .

a n t i g e n was a d m i n i s t e r e d s u b c u t a n e o u s l y , coated cap su les,

and o r a l l y w ith b i l e .

o rally

The in e n te r ic -

Serum and f e c a l

a g g lu tin in t i t e r s

were d e te r m in e d a t r e g u l a r i n t e r v a l s .

2.

in h ig h er t i t e r

Serum a g g l u t i n i n s

by su b cu tan eo u s I n j e c t i o n s

were p r o d u c e d

t h a n b y t h e tw o m e t h o d s o f o r a l

a d m i n i s t r a t i o n t h a t were t e s t e d . 3.

The a d m i n i s t r a t i o n o f a n t i g e n i n e n t e r i c - c o a t e d

c a p s u le s p ro d u ce s serum a g g l u t i n i n s o ral ad m in istratio n

of b ile

of serum a g g l u t i n i n s ,

t o a low t i t e r .

The

d id n o t enhance the p ro d u c tio n

in f a c t,

a b e t t e r serum a g g l u t i n i n

r e s p o n s e was f o u n d when t h e a n t i g e n w a s a d m i n i s t e r e d w i t h o u t the b i l e . 4.

The I r r e g u l a r a p p e a r a n c e

su b sta n tia tes

of f e c a l a g g lu tin in s

a previous p ro p o sal th a t

s p i l l - o v e r from th e b lo o d s tre a m i n t o

th ey a re a r e s u l t

of

the i n t e s t i n a l t r a c t .

BIBLIOGRAPHY

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