Su333L1D 3TUDIES 03 Yd: QJALTIv A”TI3QTS
(‘1‘ 1115

F fir;ffilr. I 3"; 0111‘4‘n
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ifiJfifl EJ'ESRJJJLRIJLS
By
A TKESIS
Submitted to the School of Graduate Studies of‘ m‘21333

State College of Agriculture and Applied Science
in partial fulfillment of ha requiraaonts
for the dagrae of

DOCTOH 09 PE LJSOPHY

Department of Bacteriology and Public Haalth

195%

JHE’QH:

. .
4 ; “~‘LS

DEDICATED TO

Joana, Pasela, Patricia, and Penny

a) 5‘ f”) .5“
L513: 73‘)! 4

”“84
“Mu

fan

‘rH‘I-

‘I. U...

.12" 5 Y ‘ 7“. " ’1 ! rug?” ':‘
A'J.>X.I7)3 5 L-JJ’J: ‘LJJI ‘ a

Sincere appreciation is extended to Dr. Kalter 3. fleck
unfior whose guidance this thesis was carried out.

Gretefulness is expressed to Dr. E. J. Stsfseth, Dr.
Leroy Hallsann, and Dr. R. A. Runnolls for their helpful
criticisms and valuable suggestions.

The author expresses his gretitufie to Dr. Joe“ Stockton
for valuable assistance in instrumentation.

Gratitude is esoressed to Dr. Ralph D. Earner for sug-
gestions and all during the pathological studies.

Tr. to are extended to the School of Grsiuete Studios,
s eh gen State College, for financial aid in the form of a
Graduate Assistantship for the past three years. Appre-
ciation is also extended to Dr. W. H. Ferg"son for providing
original cultures and for many suggestions during the conduct
of the study. Gratitude is expressed to ths.Dopertment of
Agricultural Chemistry for assistance in conducting chemical
tests.

In sdaition, the author would like to mention the
following persons for having some part in the successful
completion of the work; Kiss Lesa §eu, Hrs. Botty'uilllass,

Dr. Lloyd Shell, Dr. Frank There, and Hr. Lloyd Elli.

ft.“

“‘0‘

(fl

3‘ IzsfiouILLIJ’IL
candidate for the degree of

WWfix-o a“ 0-”. n‘u, w b‘
IV&U U3 fiLuu ”JIJY

Final o -ssinotion, January 99, 195H, 2: 00 P.H., Bacteriology
Conference Room,u ui tner Hell

f

'isssrtation: ‘ele ted “ uiies on t? -e Somatic Antigens of
*°~t~";*‘*~ sol; 0111 Be and 0% 85 Isolated
as tHs Igpure .oljsaooniridos

Outline of Studies'

major subject: Bacteriology and Public Health
Hiner subject: irisel Pat: zology

Biographical Items
Born, Hersh 11’ 191H’1.:atgrtoun, South Dgi iota

"n‘orvr~ lusts Stu3ios, Albion College, Adrien College,
i933~Hl

Grsruato Htulies, 14Univors ity of? shigsn,19HP-H3,s'eyne
Univers ity, 19H1n “ioni an Strs to College, 1951-53
xionoo: Instrzoter, Hotroit College of Pharmacy,

19H6~H8g Instructor, Boise Junior Jo legs,
19H8 ~50; Supervisor, Slinie 31 Labor story,
Port Iiuron General Hospit l, 1959; Graduate
SSiSu'lriu, EJ.‘ .111 "‘3 ”til 1&3 331.53 9 1.9 )1’5'33
VstorieLry Pitholovist, Biolo; 133i Lo3ore-
toriss, C :9 Detrisk, “srylari, 1993 to
date; “0430? Ladiesl “LoiniSLrstive Corps,
UH itoi its es fir:1y,Unitod .t.LL Reserve
40r08,11)%H-H3, I‘auo Natioan Unara,
1o:o

Hemsor of Aloha firepoe ”moss, Societ 5' of userieon Bacteri~
ologists, HioHiJs. french.

F4

I i

TABLE OF CONTESTS

Page

INTRODUCTIOH .......................................... 1
REVIEW 0? LITERATURE .................................. 8
Infant Castro-Enteritis ............................ 8
Pathology .......................................... 23
MATERIALS AND METHODS ................................. 26
Cultures ........................................... 26
Fractionation of Cultures .......................... 27
Rabbits ............................................ 30
Preparation of Specific Antisera ................... 3O
Alsever's Solution ................................. 31

Sensitization of Erythrocytes from Various Species
0f Animals With Polysaccharide 00.009000000000060 32

Hemagglutination Test .............................. 32
Hemolysis Test ..................................... 33
Agglutination Test ................................. 3H
Precipitin Test .................................... 35
1;} 1m and In M Tests of Polysaccharide nu... 35
EXPERIMENTAL RESULTS .................................. 3'7
Biochemical Tests ..................................
mm Studies
Lnj11§zg Studies ...................................

115:;

PathOIOgy 0.0.0.0....00.000000000000000...0.0.000... S3

v‘r
Dada

'1)

3"“
8—4

DISCUSSION OF RESULTS .................................
Isolation of Polysaccharides .......................
Purity of the Polysaccharide Fractions .............
Injection of Polysaccharide Fractions ..............
Serological Reactions ..............................

SUMMARY AND COKCLUSIOHS ...............................

APPENDIX ..............................................
Tables .............................................

Graphs 000.000.000.000...00.00.00.00000000000000.000

BIBLIOGIIJKE)EIY .OOOOOOCOOOOODOOOOIOOOOOOOOOOOOOOOOOOOOOOO

Page
‘57
57
57
60
65
71
7h
7 5
96

101+

~42?
1;!

to:
do.

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'9'“ f‘ A

11."; LwEMST 1033'

Infant gastro~enteritis has been of interest to various
investigators for many years. A multiplicity of suspected
viral and bacterial agents has been cited as responsible for
many deaths among newborn and infants. Humorous agents have
been incriminated, sons of which have attracted the attention
of workers on both sides of the Atlantic.

Several years ago the question arose as to whether two
special serotypcs or Egghgzichin gal; could be responsible
for the disease. These types were often found to be the
dominating organism in sporadic and epidemic infant gastro-
enteritis. The two serotypes in question have been design
netcd as 055 and Gill accorfiing to Keuffcsn (1950). Investi-
gations in the past regareing the pethogenicity of many
organisms have been questioned. Serious consiaeration has
been given, therefore, to the toxic substances obtained from
bacterial cells by chemical fractionation.netnods.

The polysaccharides of 3. 9211. were obtained in 19:23 as
a "resieue antigen“ by Zinsser and Parker. Their final
product gave positive carbohydrate ans negative protein
tests. It also precipitated homologous antisera, but was ins
capable of inducing protective antibodies in rabbits. Soith
(1927) confirmed this information.

mt
col

dis

ac‘

Boevin and Hesrobeanu (1937) prepared a glucolipid
antigen fromufl..gg11. The bacteria were extracted in the
cold with trichlorccetic acid and the residue rescued. After
dialyzetion to remove the acid, the carbohydrate was precipi-
tated with alcohol. This substance showed immunolog'cal
activity 1.3 mg and in 213122.

Wong (1933) isolated the polysaccharide of 2. 2211 and
obtained fractions, first by acid hydrolysis and than by
alkaline hydrolysis. The procedure consisted of a hydrolysis
with two per cent acetic acid. The final sxtrection.was
completed in alcohol. After drying the precipitate appeared
as a fine white powder. A complete ch93 cal study revealed
a substance which could be hyirolyzed by mineral acids to
simple heroes sugars. The two fractions differed only in
minor details.

One of the early studies concerning the physiological
effect of bacterial fractions upon the blood of rabbits was
described by Dclafiela‘gtmal. (193%). A close parallel was
shown to exist between fractions which were antigenic and the
production of a hyperglyceuicvtoric state in.rcbbits. all
fractions were assumed to contain rolysaocharides but no un-
altered protein. The products were produced by tryptic di-
gestion followed by alcohol precioitation. The greater the
toxicity or the fraction, as demonstrated by injection into

mice, the better immunizing agent it became when the some

animals were given subsequent injections of the fraction.
Eben rabbits sore inounizsd with the some antigen, further
injections failed to profluce the chemical changes in th'
blood foznfl to mcur in tho non-133330 oni.ssl.

A octerisl was is slated by Dennis (1939) frora fa:

“-rn1? ‘w “-6,

l'

          

infusion broth c' turos of ¢*_ by precip ta-
tion'with alcohol. The as toriel we 9 not characterized cheni-
cally as e polys min mrile, thou; h it proved to be non-protein
in nature. The fraction produced leuoopenis in rabbits. The
effect was more pronounced in reiucing the nuobcrs of poly-
morphnuc cor lcri :ocytes a, though it res a; p;::ont that some of
thoa.survivod the effects of the toxin. The lot“'cytcs sore
not markedly affected in the insane animals, while in the non-
immune serious leusopenic states were observed. Those re-
actions suggested the possibility that the leuKOpenic of
typhoid, which is accompanied by depletion of the myclopoiotic
ele:sonts of bone marrow, might be due to t‘rxis fro ction. In
sidition, the lock of inflammatory cellular infiltration in
the vicinity of typhoid bacilli get e further suos tantiction.
while working with the same organism as Dennis (on..gin.
1939) Horace (lQhO) isolated a substance from fi..tzahoa;
which later (florgsn, 19hl) proved to be toxic for rabbits.
The in m; destruction of leukocytes was used as a measure
of toxicity. The toxic fraction was obtained by alcohol pre-

cipitation from an aqueous solution of the disintegrated

organisms and purified by dialyzation in nellonm nae 3333.
Th resultant toxic material was traatad by tho 333333 of
Savag (1?33) to renova the protein. A light gray colloidal
solution'was obtained when the driad material was r3¢

333333333 in distillad water. 0033333 13.3133 33v3 tha

follov»i.3 results:

   

’31 333.3 ”3-33 NM {‘4’ $335 '

3 h

2 1!-

1 5 50
0.5 k as
0.2 h 0

In rabbits, doses of O.h~to 0.5'36 per kg injected intra-
venously caused prostration'with dySpnCa and marked diarrhea,
terrinatlng in den 3h.

Saith (1939) discovarea that, after five or six in-
jactions of a typhcid filtrate into rabbits at a tima when
the 331331 showed a very high titer of antibodies in the
serum, thara vauld fDIIQW’a typical fall in the white blsod
cell count. morgan (a;..gii. 1930) encountered similar
result3 khan L W“ 31333 aanals r333 23d 32m 1rply to injaction
with a similar fractloa. This change canstituted a 3131-

nution in the nunbar of nautrOphilas. An int3r33ting point

to be observed hero is that aniaals possessing a high an nti-
body titor to the toxic fraction were not protected from
further lyoio or noutrophiloo Upon subsequent injections of
homologous antigen.

The 1333033313 offset of bacterial fractions was summar-
ized by Olitzki (1941) in four stato3ont3: (1) A gonoral
loukoponio consisting of a decrease in both nontrOpnilos and
lymphocytes; (2) LymphOpenic~loukoponia duo to a sharp do-
oroaso in tho lymphocytes, ossooifi ed Ath an inorooso in the
neutroghilos; (3) Poly33313oois 333 to 3 snarp rise in
noutroghiles, associatei with a mild dooroaso in lvo§tooytosg
(a) Gonoral leukocytosio due to a rise in all typos. It was
dotorminod that a loukooonio resistance, which was due to the
activity of noutrophilos to resist lysio by a toxic fraction,
could be developed by active i3annization.with tho fr r3 @103.
This was not noted in all bacterial fractions tested.

A fraction was prepared by Favorite (1932) that,uyon in-
jootion into human adult voluntoor3,o mused chills, favor
and muscular acho, followed by 3 1333333313 which was almost
ant rely due to a loss of noutrop hilos. A return to normal
was noted followed by a loskocytosis. Blood chenistry values
showed no change in total protein, 'roa nitrogen, oroatinino,
chlorides or glucose. 31.;* :h titers of 33; utinating 3n pro-
cipitating antibodies woro found to be present. As was the
3333 in the studios of: iorgan (o2. cit. 1991), titers of

circulating antibody did not seem to to rolntod to the level

0“

of tolsrsm me exhibited by trze enixnl then im sated tith the
toxic antigen.

The most recent tora L33 carried out by Lays, g; 31.
(1923). A polysaccharifio subs so was isolated from an un-
typ ed strain of 2. aggi‘thic h pres Aibly 313 not contain tno

"P” as .tigon of :13-.3cn (19 l). nesctions in rabbits to

tLis 33t3r11l were invest :sted ll: h intsrosting results.

t;
'3
O
*5
P)
If
\4
(J
5)
(‘3'
*1
‘ 1
‘ fi
3
O
O

1.3313 3(1) 11) {13.13 Hstrritasd 13?.LJ 30 )1431311". ' ‘ 4' _
cytcs whicn tore previously scncitizoJ using a polysaccharide
of Q. 32;; as the antigen.

fieter.3§.al. (1952), althongn not wor Eng 3 th a poly-
ssscnnride fraction of,§. so? , sh 331 that somatic anti3cns
present in 31313 culture 30113 can.be chorboJ on the eryth~

”a n

rocytes of many spa “1 es 0. animals. In a later study, “ tor
23,31. (1972) demonstrated spc cific “33333 at nation and
henolysis of erythrocytes previously sensitized 31th the
somatic antigens of 053 and 0111. In a subsequent invosti~
gstion,? fieter Qgflgl. (1952) innibited tne “03-1 cation of
erythrocytes by 333 ing humqn or animal ears or by the
adiition of 333 yolk or various fractions of rat liver.

The seriousness of the proble n of op Jc;‘.r;ic gastro-
enteritis, vhers OS 3 and 0111 so 3 the szooscted causative
agents, led he author to cons iner certain fractions of those
organisms which might ber reSpor sible for tncir to: :icr section

in the host. To arrive st 3 closror unfierstonfiing of the

active principle involved, and ta obsnrve tha effacts of that
principle upon various organs of the host, this stufly ma

"£10? taken.

REVISE 0F LITSRETURE

 

In reporting his famous work in 1886, Escherich describ-
ed a now bacterium which was found to be present in the ins
tostinol tract of ovary manual. 33 hold the Opinion tact
diarrhoal discuss was due to c rcilstribution of intestinal
forms uni not to a Specific pathogen. Investigations since
have discovcrod strains of fi.‘ggli known.to incite disccso of
diorrhocl nature when.prcscnt in tho iutcstincl tract of
infants and expcriucntclly in adulto. (Lancet, leading
article, 1932)

Since tho condition of diarrhoa in infants can be pro-
duced by many agents and clinical conditions, a clarifi-
cation as to the various causes is necessary. Crowley at 31.
(19%1) summed it up vary well by outlining six major catc‘
gorics into which any case could be placed. Tho first ina
eludes those cases duo to an outbreak of 5313039110319 or
dysentery. Such infections are easily idcntifiod and cause
little troublo for the bacteriologist. The second group is
characterized by a high mortality rate, a rapid Spread among
infants with.locc of weight, anorexia, toxemia, diarrhea,

vomiting, and dehydration. Adults arc not offoctcd.

The third is one in.which one may see diarrhea but usually

no vomiting. A fourth in which adults as well as children
are affected seems to be common, and many times cannot be
traced to any specific agent. The fifth type may be referred
to as the influenzal, whore mothers of infants and the
hospital staff are common victims. The last group is charac-
terized by both adults and newborn showing stonatitis in con-
junction with diarrhea. This group would suggest a viral
etiology (Light and Hodcs, 19H3).

Infantile diarrhea, more accurately termed gastro-enter-
itis, has been recognized for many years as one of the im-
portant causes of death in infants. Because of epidcaic
which have occurred in many countries of the world, particu-
larly associated with hospital wards where crowded conditions
prevailed, much interest has been aroused regarding the cause.
It was not until 19%5 that coliforn organisms were seriously
considered as causative agents. All work incriminating such
agents is not yet complete. There are still many workers
who take a dim view of the situation, even though the evi-
dence is overwhelming. '

The literature is abundant with references alluding to
the causes of infant diarrhea. As early as 1392 Jonson.gt
,al. in Denmark reproduced a disease with diarrheal symptoms
in calves. He further infected calves by feeding cultures
of E. 2911 isolated from diseased calves. It was believed

that races of E. coil existed which were pathogenic for young

1.4:.
iii
4‘0 3,
‘4. U: . .. [fl

10

calves, even though strain 3 of suzh or3anisas tare present
in all noraal aniaals.

Lovell (1937) confirmed these findin3s and produced a
connereial polyvelent antiserum against 2.323li. The sane
technique was applied to human infants by Hamburger (1920)
who claimed success with antisera prerared a3ainst,§. coll.
His work was not confirmed by subsequ nt investigations.
Shortly a ter Eaaburger's declaration, idem (1927) described
eoliforn bacilli which he believed to be related to diarrhcal
disease in calves. Referring to these strains as “Dyspepsia-
Keli", he proffered a theo y of ”elinentary intoxication" and
described coliform organisms as related to diarrheal disease.

Following the above work, several e nperinenters prefer~
entially selected the filtrate of broth cultures for areal-
nation. Filtratee from cultures isolated in connection a th
cases of scouring calves were studied by Saith at 31. (1927)
and were’found to be tonic for calves one month old when
given intravenously. This was not true when the sane fil-
tratee were injected intraperitoneally into guinea pigs, even
when the dose, tor is for calves, was increased many times.
Feeding large numbers of bacteria to guinea pigs and calves
had no aooarer t effect.

Smith 23 a1. (1937) furtr er discovered th at than certain
strains of g. call from the ileum of calves suffering from
diarrhea or secure were grown upon aa3 ar plates, nutat ions

occrr‘ed in which capsular substance was 109 t, virulence was

ll

reduced, agglutinsbility was increased, as use susceptibility
to phagocytosis by leukocytes. So to (192?) else studied the
interrelations between bacterial toxins and capsular substance
compared with normal protective factors in the guinea pig.

finch confusion existed as to how each strain or different
organise belonging to the coliform group could be specifically
identified. A tisely clarification appeared when Stuart 3t 31.
(1938) laid the grout were for subsequent separations of the
colifore from other groups of bacteria. Accordirsly, or3cn¢
isms of the some genus c uld have vary as serologicsl re-
actions end at the some ties be bioohosicelly insecureblo.

Still attempting to discover the ccusstive agent of
disrrseel disease in infants, Crowley at 31. (19hl) cresised
the flora of 57 cereal infants. Their results revealed 80
per cent ngtggggilxagflhizijgs, 10 per cent colifore bacteria,
h per cent streptococci, and h per cent staphylococci.

In comparison, 3% infants ill with s dierrhesl disease
in two different outbreaks showed lectobecilli, 30 to 50 per
cent, coliforu, 31 to 55 per cent, with streptococci end
staphylococci exhibiting little change. An 3..32;1 etiology
was not suspected in hose outbreaks, but it seems signifi-
cant that the colifore organises increased at the expense of
other species present in normal stools.

in increasing number of workers began to observe out-
breaks of gsstro-esteritis in infects, arriving at little by

cy of a definite cause. Lecbeke a; 31. (19%3) on the eve

12

“the discovery of Q,;g_1 as the agont associate& with
gastro-0333ri his in inlants, observed an 3313*313 in'whieh
there occurred 22 mild 331 23 severe cases wifih threw 333t33.
no 33033 was founab it £0333 13393333 1. its b3by£ *lss mice
proved fatal to a much greater degree than did feces from
norm ml 1nf3nts. The disaaso appo3rod selective for infants
above and below normal birth wei3ht.

In 19%k, khan K333£333n 59333 tila 1033 t133 of o.oe3u-:3

the work of Stu3rt 92‘333. (1333) he brought for th his pub 1-

cat Mon3ro ardivg he sorolo:y of tha coliforu group. Hero
definite 1131t ations were placed upon the V3rlows str3lns .
accorfling to the occurrence of 3031313 "0", the flagella: "H"
and capsular or ”onwaloge K", antigens. This particular work
3330p 0531313 t39 pres ant stufly as it proved the existence of
many separate strains of‘i..g;;;. T113 numbar of strair s or
"0" groups of Kauffuann.haa been oxtenflad by Knipschildt
(1935), Vahlna (1935), and‘wramby (1948) to includa at least
123 sorolo 3ically dist inct strains. Ora: 10v (1951) 3331333
strains of 3,.9931 belonging to Dogroup, 25 isolated from
cases of infant diarrhaa. He correlated strains found at
uncropsy in nawborn calves with those fan 3 in i3f3nts 1:1th
gastro~enter1tis,a anl inaioated tzzat t3 oy were probably
identical. flavor before had any coliform.organism present in
animals boon snapeatoi of be 393t303 0313 for hu3ana on an

epidamio seals.

13

At the time when.much of the cviionce in favor of a
bacterial etiology was being advanced, many investigators
began to think in torus of a virus-caused diarrhea. It was
only natural, in the absence of a Gorinite agent, to cxolain
the epiéoaic in terms of some'virus. Lyon.and Folsoo.(19%l)
correlated outbreaks of epidemic diarrhea among newborn
babiee with the simultanoous occurrence of virus influenzao
among adults.

Light and noies (19h3) were the first to isolate a
filtereble agent from diarrheal stools of infants and to
produce a disease in young calves by nasal alainistretion of
the virus.

Other atteoots to produce diarrhea experimentally with
a virus were made by Buddingh and Dofid (19%“). An attempt
was made to produce diarrhea in.rabbits by feeding 8&8?0n31038
of experimentally infected cornea from other rabbits having
clir cal eye infections. After an incubation poriod of sovea
days the animals doveloyed characteristic diorrhoel stools.
when portions of these stools were fed to normal rabbits, no
symptoms were observed. The'viral agent which.wos supposed
to be the cause of the corneal infection'uae also credited
with producing both atoaatitis and diarrhea in infants.
Swabs, taken from the stool of diarrheal infants, proiuced a
disoaso of the cornoa'when,rabbits were exocrimcntally in-

fected.

 

 

I. “hall P

Eloir1n -*.QL. (119’15), lo invest i31tin3 on 93.13. czmi of
diarrhea among stuflonts, fai 19 d to corver a filtereblo agent

but LL re £110 to tr:1n$.: t the disease to adult hu:«n.volun-

(u?

oars by 211L L13 1331 iz‘aloe List of filtered nose-phor3n3c-
al Lashings or flit MP 1 stools fr-on patients 3E L113 the

irfieo Further, ti11y 1:0ro un1ble to subst1nt ate the work
of L 3211333312.. 93"". 19113).

Sovitt (lie?) doubted the virus nature of th agent
studied by Li3ht‘3; * .‘qugii. (l?13) as it could not alvay»
be inactivated by boilin3P - rfive minutes.

93 the other hand, Goricn‘:;.;;. (l7‘h ) isolatoa a

filtorzble agent from the fcc J

02

aa~ thro1t we shitgs in an
outbreak of Joctrc-ontoritis among infants and achieved the
trons:_ssion of the disease in efiult volunteers by the oral
rcut-. Hewover, inoculation of this filtrrte into ez1ryou
1tel o, "s by time-3 dii"?:1?o1t rout-33 £11113 3. to 5.101“ 91:31:“: zoo

of virus growth vhcn tested 5 foofling th :9 infioctoi eggs
to sdzlt vol rs.

It ca1oot be d-onied t. at in many of the studies who;e
.3. c ? h1s aozu the surge :tel agent ,
viral agent has not been sicqustely in1esti32tod. It might
even be possible tent a virus is associated r1
strai1 of bactzrii. 13: 211033, 3113 ot31er causes of d
rhea have been sought.

For example, Keitell (1930) in the course of a study

concerning an epiiemic of diarrhea a fee tine both afiults and

infants, discovcrod that in many cases the infants‘ sora
gave increased titers to cold agglutinina and streptococcus
H. 6., both finding 330sting to hi3 a virus infection.
He did not attempt to isolate a viral agent.

Tho ghiqolig and figlggsolla gonora, along with many other
bacteria, have been suspected as tho roSponsiblo agents in
infant gastro-ontcritis. Anong those are the paracolon group
(i-iushin, 19%), the gonna {3,32,12,33 (i-iushin, 19:30), the gomis
3'11»?- « , (2311921111, 1932), “Atom £111.31: (1.111.). and
W m: r '1 W (11111.).

Grannpositivo bacteria have played a minor role in the
causation of diarrhea in conpnrison with grnnsnogntivo. Such
organisms that have attracted some attention have been tho
staphylococci (Kortyn 19kg, Crowloy at 31. 1?h1, and Draper
.£&.al. 19h1), and the streptococci, certain strains or which
were shown by 6119 (19%“) to produce enzymes changing tyro-
sine to tyranino. This substar~o when rod to young rats
proiuccd diarrhca. Tho cultures woro bota nomolytic group D
stroptococci, and were found to exist as too predominating
organisns in the stools of infants in cortaio outbreaks in
Cambriigo, England. Sinilar studios involving tyronino wore
carried out by Dock (19%?) and Bock gt 31. (19%7)*who failed
to obtain ovidonoo of toxicity with tyrooinc rod to hunan
volunteers m 9,3.

Cortcin strains of E. gali have more roooutly been shown

to be associated with Sporadic and epidemic gastrowcntoritis

16

’3

infants. nnrly work carried out in 0 cat Britain and the
Scnniinnvinn countrics has been volunincus. Kany can be
cited, such as Bccuwkcs Q; 31. (19kg), Bray (ITTSD, Bray g;
g]... (19'4-3), Cathie g; £33.. (1931), 3rcw1cy g; 3],. (193a),
Christianscn g; 31, (IQHS), Silas , gal. (1 }+}), Giles gfihgl.
(lflk3), hclzc1.g§.a1. (19h9), fnann (1950), Kirby £5,31-
(1950),“: . agnnsscnugs‘al. (1930), Payne g§.g1. (1931), no crs
.g§.a1. (1951), chitt (IQRS), Stcvcnscn (1952), Taylor (1971),
anfl others.

The t'c prciozinating coliforn scrctyrr as found asso-
iatcd with epidemics of gastro-entcritis have been nnncd by
many investigators and prcccnt a confusing noncnclcturc. The
irct referred to an typo 1 was called 233§Q§1n§,Q211, var.

swignzm by Bra? (1%?) . 2311274112 @211. type c.1223; by
01109 9,; a. (19%), typc D l+33 by Taylor (1951), W
.ggli, B. G. T. by Rogcrs czhnl. (1951), and 0111 En, 0111 Bu
Hz, and 0111 BR H12 by Kauffnnnn (1950). The second t;rpc re-
ferred to as typo 2 has bean named 23;§gzig;.ggli typc.§g§g
by Giles _§.51. (lfihfi), and 02 5'35, 055 Bg’Hé by ,~.auff3nnn
(1950).

The initial discovery of a specific ccrctype of Q..ggli
(to be referred to in this thesis as 0111 or 0111 En) asso-
ciatod with infant gastrc-cntcritis was made by Bray (lfihfi).
A group of organisms was considered which was referred to as
W 39.11, var. Mn 11’2"! . A strain, M933, was found

to be reaponsible for an cpidcnic of summer diarrhea in

17

infants. T23 at rain.voc not tan d in too stools of normal
infants to any significor -t degree, and was rocovcrod from,22
out of'%b cases of 32220r diarrhea. A cooinol odor was noted
upon culture on artificial 2321a, a pxono.ocon.wiic2 has boon
noticod by others.

Applying the technique of slide agglutination, Gray and

22 (1)+8 ) dis covcrcd p sitivc cultzres of t2c special
sorotypo, #938 (0111), to be procont in 87.5 por cent of tho
cases studied. Only four per cont of the control group were
foun i to harbor the or 32213::. 2 coocifio antiserum p oporcd
against the strain of Bray, 2033, was used in all tests.
Biochozicol ocporotion of t2is strain from other coli was
considered i2proctical, if not 1220331212. Tho agglutination
studies comprised a gr up of to co". co 6123.103 ed as gastro-
enteritis. Tho moon ago of t2o infants was 2123 months.
Twenty-oig2t of tho cases were oovoro and a dcfi2ito olor moo
ascribci to the fo cos. It is 12% me 131 gto note t2ot hora,
as in othor cpido2ics apparently duo to the same organism
row gross lesions wore noted at post mortoo.

In an outbroox of infantile gootro~ontcritio in Aberdeen,
Scotland, Giles and Euros r(19b3 ) rocoraoa that tho majori-
ty of tho cocoa otudiod was unrclatod and that no 002202
courco of infection could to oofinitoly cstcblishod. Two

ma outbroaks occurred in‘which it'wao possible to trace tho
infoction from patient to patient. or the 159 cocoa, 65 were

considered to to of diototio origin, 93 app M1 32 to belong to

13

t2o pr "1?? infective gr 2;, 223 t2c regaining 92 shovel
clearly to be a common strain of 2. gall. The latter were
serologically honogcneouo.

Confir2ing the work of Bray 9;. git. (l— 32, Bee 2223
gt 2].. (1929) studied the role of specific strains of __,. 519,11
is epiiemic infest gastro-onteritis. A year later Payne and
Cook (1930) studied the inciiccco of e scecisl serotype of
.E..ag;i from a group of infants over a one year period. (22
investigation of gastro-entcritis in so orphans' hone.) 22o
clinical condition of the babies was unflor constant obser-
vation. .flgctggigg.zzli Olll Bh,wcs isolated fron.h0 of 337
rectal swabs taken. The organism was often present in pure
culture on the swab plate. All the strsins conformed bio-
chonicclly end scrolccically to those of Bray g . 211. (1927).
Ten out of 15 infants in one group shovel the strain in their
stool but exhibited no clinical symptoms.

Smith £1.21. (1950) associated t2o supposed causative
e3 st of an epidemic with the assignoted 055 BS'Hs by
2223:2222 91. git. (1950). This type had replaced the 0111
variety present 12.22 earlier epidemic in t2e some geographi-
cal arcs. Titers of antisere against 035 appeared to be very
low or negative when tested by bacterial agglutination. By
the use of more recent tests those sore would, in all probe-
bil ty, have shown higher titers. (7eter sow Elia (1952) has
shown that low titers of antibodies in the blood of human

volunteers infected with 055 (experisontclly) could not be

13

dat33tcd by the usa of tha ba3t3r131 3'ut133tian t3st, but
were saffiaiantly hi3h ltd 33333 t”o na;33313t nation 0? sheep
3 rythm 03“t33 that had been praviousl' sensitized with tha
boiled culture filtrate of“ " ..22110 53.) 305 were treated
with chlorozycot a 31th 3333 results. T33 fact that treatad

Jirlran those who had r3331ved antibiotic) showed 33ight
13331303 over untraated is of law st in v10: 0! the presant
knowledge of growth stimuh .‘on 333 to 13303ted antibiotics.
Suffi 3lont nanbers of 1~£3nt3 ware tr33t31 to 3333 the ot-
snrvation V3113.

“artharing th 333 :33 far intactians dua to.§.‘22;1

3 11'33 (3111).) 3033?‘Q§.31. (1933) attezptafi to 31333ver

nitis, rdning ti~ and otitis 33313. cultural studies were

"“131 out to determine vh3thcr tbcsa 5303131 sarotypos re-
sorhlad californ bacteria aacording to thair r333tlon3 on
various 33313. The presence of D M33 in the Upper reSpira-
tory tract of infants was Investigatad. In this regard, the
or333133 was £0333 1n fiva infants with non~3pidenio diar-
rhea. ‘11 tho strains 13313tei wore 3103 h3.i3a 11y coliform
amd‘wcra agglutinztol by "pa-31:13 33t13333 .rcpared against
type D #33.

t ch3uld be mantionad that this sarotypo Va a mat foumd
in 33 a iiitional infants exhibiting diarrheal symptoms.

Strains of ;._gg;1 isolated fro:3 cases of per1t3 nitis,

pyelitis, meningitis, otitis media, and septicemia were not

of this Special type. An infant, upon being fed a culture

of D #33, exparianced diarrhea and waight loss. Lzaosure to
the patient's own atrain of L. 33;; 331153 to procluce synptozs.

In.drawing 50:3 important conclusions, Later 33H31.
(1951) recorded four points: (1) Two sarotypas o. 3, 32;;
were £0113 to "r01249 33033310 c1333 of 1“fxnt diar?h a.

(2) Contact with these special types 1313 to symptcss of
gastro-enteritis. (3} Thare is a carrier stata in infants as
tha organism hai been razovara1 from the 3.333? raggiratory
tract from whieh they caulfl ““0’101 an air~bor ne infection.
(3} Lhasa states may b3 closely assaciatei with api3031¢ diarc
rhaal disease 0? infants.

In Lngland, Taylor (1951) mafia 32W Siva invesfiigatians
into outbreaits of infan gastra-enteritis. marina a single
53333, are healthy b1 33 excrsting Oifi’uas aflmittad to 3
residential nursery, and scan an 03tbmr1L of gastro-enter tie
occurrad in.which no beta ty 3e crj1ni. m (055 )‘WES isslatefl
from all of five 3133 im ants. The organism'"as also 150*
lated from six of save a bslbias Law'i“' an 11331161 stnol

033.32v, anl frag two of five infants with 35203331 stools
133111101 that

but with no incranse in frequencv. Taylar

'1

ca
his and other epidemics were flue to a Special type of L.

3.3.“:-
In the 5339 yfiar thr.-1a g; A. (1951) r0; orted on a
stuiy of two yer rs 3uration in*mLizh tLey rocavere 3333

(0111) in 26% cases of infa nt diarrhea

?ery interesting 333 signifi03nt results were published
(1951) and €03.2rs Q; 31. (1351) when they PGflOftGd
on tr a 133-33 3 e of cross contaainat -on'nith ir ant diarrhea
when the patiant s xere canfined in a cubiclcd ward. hit 333
founfi to be cont33innted with ooliform organic3s due to
routine ctivitios in the ward. All the articles ass 31.:ted
vith tha 13333t3'ware also c03t33333t33 13.3 1133 aafinLLL

a”
3:33

(‘22

isaase was 5:03 to: mead r3;ifily fro3.cuaic -3 to ubiclo.
The trc 1t333t cf floor apps with five per cent phenol failad
to prevent air contamira Mi 3 of clean cuuiclw It was ob-
served tilat a 513313 33:31313 00311 b0 c03pizLely cu 3L3 Linatod
within 13 hours after admission of an infant harboring a type
strain of Q. ggli, and that this organism was able to remain
viable in $33 duat of the roan for 27 days. ibis work
certainl' cnphzs sod tha close assaaiation of both ty;es of
.2.‘gg;1‘u Lh outbreaks of infant diarrhea, osyecially in
hoggitals whore intimate OGnLac bJLLL patiozzLa is unavoid‘
8b100

After atujies on the spreazl of 53 h or 5331533 Li itnin a
hosyital ia.£nglaud, Rogers g;,fi;, (ihiio) furthar revealed
obsefvat ions mafia on tha rapidity with which the sa syocial
tsfpos of c liform arganisas could be traced fro3 033 heapital,
known to harbor the organiL3s, to a 5000i 3, than to a txird,
and still a fourth, each succeeding ona previausly known to
ba free of the strains.. In an utt ompt to pr vent further

Spread of the disaaso, Lha auahors r~L couweuL Lu bacterial

GX331333103 of 330013 £303 311 infants entering 2 h0331t3l
for treatment of a diarr heal can mien, vomiting, or both.
In addit_on, the stools of each nauborn infant 333313 be ex-
33*nel bacteriologically.

In the Uni “ml States, Fergus 333 and June (1972) ware
int 2resto-d 1n the gossibllit y of 33 11% lufactien with 0111 34
333” t.3 o3~a3-3.3‘vn33 13333331. 3313 velunteers 333~
aelactefl for tha 32313. 033 group was 533 3333133 £33,3r3 of

the 03323132; 3 secon-l :33 39 served as a cantral. uhen

330,330,030 032331313 01 3230 hare 1333 ate} (3333:: 8111),

9"”

C)"

Q g Q ~. .1, -‘ ‘ k- . q :n. - ..
sydnvuaa 31311;? +0 )33 “3332.31 {and 33133313; more 23 31.

’uantitibs 1333 than th- above 33:2. 3? of 33233313 f3’l3d to
3121332: uranton The 303tr01 o:;3nlsn,f3:l to 3
3333333 3333 of voluntaers, was 3 333313 of Q. 33‘ isolated
$3.1) 1.1.33.3) «.33 may

#3 ‘u-«l

fron ti 3) stool o- 3 333:231 13';‘;nt133vn not

, 9... 3.. 3 ~, ~ . .. .. 3‘ - 7'. s “ .5...

szvclal hype 01 30113333 03322134. “33; o- 333 3233313 in-
!“ an. A». A 1-..- ...... t- . 0.... 5.1..

533t1~3 332 333312 of 01H 3% 333233 ~33133 3133 - : ,33t

¢. ,3 an .. .. 4 . .. ,... a. .. 3...!
spr3ln. 139 331 331 3322313313. 333 tL3t 334133 a: 333t0

.3

r33132:““ to n3tt 31 infection'witiz 0111 ?+ strain 33‘2..33;1.

09-.”
Lulles 0333233133 tn) £23113; 3? the 0,3’3 333313 to
33233 :31.nt3 333 3: v3 3133 3333'33333t2333 3333 ecafitly by

3333 £3.31. (in 33333). "333 3-3 work h.3 3333 333113 293,

anv I."

thnra 3 little doubt that flnlings 5131;: to 33333 of
F3? :11" .03}. m :21- 30 £1.20 (195°) Will have 1360'} 21:22:13.

20033 303: by 30:1133 3 .31. 331 Steven? 3 (1772) has

3.331 333"t :r 4233*3 to 333 1333 of 33 0111 333077M10103y

of infant gastrauonteritis. 303133.2i.£l- (1932), ovar a
period 3 seven months, observed 55 33333 of diarrhea in‘which
80 per cent showed a spe 31fi3 colifor2. 823 hundred and
forty-six 12f22m not suffaring from diarrhoal 3133230 show3d
the org mis2s 0111 to be prasoat in h.7 p12 r cont of the cases.
Einety-tiw 233 332 Its in tize 3223 hospital were shown not to
contain the org niam'by bacter 21 cultures. The reported
mortality 32323 inf.1nt3'wa 3 only 10 per cent, tha low figure
bein3 croait ed to the us 3 of chloroqycctin in treatnfint dur-

ing tho 33129213.

 

fiistopothologio findings -2 the 3253 of infants known to

33 infected with 0111 or 033 have boon.varied. The most
onstant findi23 has been 3.2113 fatty 31-.3233 in the liver of
infected infants.

Giles giugl. (1938), in an epidemic of infant gastro~
enteritis, fou1d that 223m 392 33333 harboring 3 301202
strain of.fl..ggl; the liver was tho only organ to show'con-
sistently 3023 eviden3e of damage. 322n333 in this or 332
varied from mild fatty c 3233 to a sovero necrosis. The

peripheral portion of the liver 1031113 was most a f33t3d.
T313 spleen and kidney 3 33r3 often fou1d 302312 tad. Only a
£03 32303 appeared to have definite naphritis. Broneho-pnau-

mania was noted in a few cases but was oonsidarad terminal.

The presence of noningo-cncephalitis in infants s=”fer-
ing with a similar condition'ues reported by Christensen 3;
.gl. (19h6). Giles at gl. (1949) in a similar investigation
found no such lesions in a large masher of cases.

fistula (19h3) carried out extensive studies noon infants
deed of a diarrnonl disease in usieh 0111 and 033 were the
suspected agents. The pathological findings closely parallel-
ed tnose found by others mentioned above. “he intestines
manifested little change with no ulcers of the mucosa and
only slight lymphedenoyethy of the mosenteric lymph glands.
Only the liver consistently showed pathological changes.
Host marked were fatty chance, congestion, and jaundice, in
that order. the fatty changes appeared peripheral in the
liver lobulo. There was usually evidence of early prolifer-
ation of the cells forming the bile cannlieuli. The thynus
gland was smaller in every case, as were the supra-rennls in
some cases. A spleennmegaly was observed in sose. finphysemn
of the lung with atelectasia was noted in four cases. Th so
general findings only indicate a toxemia and wasting co.-
dition and are in no way specific for any given disease enti-
ty. These find n33 are not uncommon to many wasting diseases
of children.

in examination of infants deed free a dierrheal disease
was made by Kirby.gifln1. (1950). In four of the patients no
gross changes were observed in the intestines, other than a

congestion of the mucosa. Hesenteric lymph nodes were normal

or only slightly enlarged. there plasma and protein hy~
drolysetes were given as treatment, little fatty change was
noted in the liver. Changes in the brain were limited to
mild congestion of the pie-oroohnoid vessels and edema of the
meninges. There were no lesions to compare with those found
by Christensen on. m. (19%).

Few other investigations have been node in the cose of
known coldenics of 0111 or 055 intent gastro-entoritis. In
on investigation.in_which.the author took part, findings
similar to those cited above were observed. Only 0- case

out of 13 showed any appreciable pathological manifestations.

. h‘flf‘f} 5' ('fi r1? 3, ‘51-?“ s- C"
fiztiu;&I.-$&IAJ -'5.-6.D ~:J;«IOJ9

he strains of §.,gggi,used in this study were obtained
through the kindness of Dr. H. w. Ferguson, Division of
Laboratories, Hickigan Departoent of Health, Lansing, Michi-
gan. The strains were identified on 0111 3k and 035 35,
numbered 5373 and 13027, recpcctively. Several tronsfzre
were made from the original cultures to brain heart infusion
agar (Difco Laboratories) to insure the purity of the cultures.
The organisms were transferred weekly and observed throughout
the course of the study for roughness of colony g wth.
Originally, both strains were isolated free the feces of
human infants ill with geotro-enteritis.
Biocheo-cel tests were carried out in duplicate to check
he cultures for typical reactions. It was found that they
fermented maltose, sorbitol, duloitol, dextrin, rheoinoeo,
erobinose, monitol, lactose, and xyloee. to foreontation of
sucrose or inniin was observed. Indol was not formed, gale-
tin was not liquefied, and citrate was not utilized. They
were Voges-Froetouor negative and motrvl-red positive. After
biochemical examination, the organisms were agglutinated by
means of group Specific entieora. All cultures were observed

to be pure.

 

The haste; -3 for fract ionation'we r3 'raun in nut: 133:
broth at pH 7. Att33 pts "220 made to 5r 3 ho 023331335 in
a svntlmsia 30-11233, but; the llaits-l growth obtainol 3:33 2132‘.
33333333 for the purpose of tho study. 333y workors, under-
taking 31. :ilzr studios, haven sad 33:1333 typss of 3rlin3ry

33113 31th su 32333. $33 Qz‘gl. (1930) utilized try '3: io

di'est agar satisfactorily. NO .1233}. (1933) usad an

63

oriinnry agar 333133.

To grow tho cultures for fractionation t3 metnois were
used. T33 rdif ferod only in tha amount of medium contained
in a single flask and tha type of sizksr employed. In the
firstm ethod 500 ml Ehrlenmnyar flasks were 3333 in'whish was
placed 250 ml of nutrient broth. fter seading, the flasks
were six 1333 oontin:ously for 2% hours, in 33,133 ubator, at
37 3. The constant agitation'was assomplishad by 3.3 ans of a
Dorell shaker. fiethod two involved six liter flasks that
containad three litsrs of 33313 333k. These were shaken-on a
platformntype 3333133 at 37 3 for 2% hours. hero growth por
301333 of media resulted from grau.h in the larger flasm

Tho r3 pid removal of the growth from the medium rexuirea

ho use of the Sharples centrifuge. 333333 of approxiaataly
12,000 rpm.vere required to 3333 the sogaration. Only a
small pfirsentage of tiia grozth.was lost in the first centri-

fugation and this could b3 reclaimed D? a second cemtri-

3‘)

fugatlon. Tue org. alums 'oro 301 wtofl £303 the rotor with
a Spatula and s spomfiod in distilled water at a ratio of
about 033 to 133 parts vat or. Tho water suflpoyolon of b33-
tor a 333 glaood in a water bath at )0 S for ono-half hour.
After r:33t1r3 t ‘.3 cells tiw 3t1333 “y contrlm mica, they
V333 outrs-otoJ thro3 tlgos with ethyl ether, and finally air

. ‘ 1" ‘.‘ 9‘13}, . Q _, u. r fi 5. *1_\- .h , \‘ g -
driol. D~n un3 9-33 Cuduififll ro3olaros throughout no 3:333,

":1

ho b33m r13 3:033 lzeptf “.03 as much contamination as nossiblo

(:3

by tha u 30 of 31333 313 Stare and careful oovorin3. The
driad baot3r1= were placed in a mortar and ground thoroughly
V th sand until 31330333313 33.3333.-on $31103 to revs.
intact calls. T.13 period variaa from b9 tab to batch.
Tho disintagratad coll fragments were extracted three
times with distilled water. The supornatant liquid was
savod each time and the sodiaont 613333333. It was 3533333
at this point that the materials sought were water soluble
profiucts. This provei to be the Cass. Hays £31. (193 3).
The water extract containing tho inguro water-soluble
polysaccharido fraction was shak3n.uith a mlxturo of chlor03
form and 33y1 alcohol after the method f SeVag‘fifi‘fllc (1333).
The ratio of chloroform to alcohol was 0.1 part to 0.25 part,
respootivoly. The total mixture was shakon for a period of
“5 to 60 minutes, by hand, in a sopar3t’ry funnel. The
mixture was centrifuged in 50 ml portions at 3,000 rpm in.an
Intornoflonol Centrifuge , type PR-l, for 10 nimatos. A

separation of tho chloroform from the water layer was noted.

At the interface a gclaticous-cppcqring membrane was formefl.
This layor contained intact protein materials and was
discarded. Tho formation of such a lcycr indicating the
presence of protein substances is, according to Sevcg a; a1.
(22¢,Qiio 1933), a test for protein, scncitivc to coo port in
#0,000. The aqueous fraction was treated in the above mannor
six times after which no gelatinous mombrano was noted at the
chloroformwvctcr interface. The chloroform was found in tho
bottom of the tube. Finally, tho absence of the gelatinous
layer indicated that all of tho protein possible had boon to-
movcd from tho mixture. A negative Biurct reaction (labia I,
column 2), indicating the absence of the peptide linkage,
substantiated the observation that little intact protoin.rc~
coined.

Tho water layer, froa the cbovo extractions with chloro-
form, was precipitated with two volumes of ethyl alcohol, 95
per cent, which contained a trace of coflium acetate. A fine
white precipitate appeared and was recovered by centrifu-
gction. This prccipitato was discolvcd in.distilled water.
(A ratio of about 100 parts water to can of precipitate.)
Prccipitaticn of the pclycacohorido material was carried out
for a total or five tiocc. Any cocimcot appearing in the
centrifuged water solutions was discarflod each time.

The final procipitate was extracted three times with

alcohol, three times with other, and dried by evaporation.

3c

The final mtcricl appeared as a light brown powder. The
polysacchariie was weighed accurately, and portions for
tmiodiato use were dissolved in warm saline solution. In
cold saline solution it formed a collciicloliize sucponsion
which dial not settle out upon shading. Other polysaccimridcs
have been reported as being colloidal, muraly those of Horgcn

(19%) who isolated them from a culture of $1. "3-. o. .

‘v-Ihitc albino rabbits, weighing from two to three kg were
use-i. e11 enizials bod s norml temperature prior to use in
any carporment. Blood samples for total and cii'i‘cmntiol
lomzocyte and erythrocyte counts, blood glucose and hosie-

globio determinations were taken from the marginal cor vein.

 

Living and hoot-lulled vaccines were prcpcrcd in saline
solution (0.85 per cent sooium chloride, pH 7.0) from 32‘... 5221.1.
serotypes 0111 :31, and 033 35. wherever the votes "saline
solution" appear tin-oughout the study, the above Specifiw
cations apply unless otherwise state-:1. Rabbits were injected
intravenously with gradually incrcaciog doses of vaccine, on
alternate days, for periods of six to twelve days. All

animus were rested for at least one week prior to bleeding.

31

Blood was tnsen from the heart and the so on separated from
the clot. All such sera were checked by bacterial tube e3-
glntinntion for titers and were phenolized (0.1 per cent) for

l
a.

preservation. foo various sntisore were node free tzo blood
of rabbits issunizsd with either boiled (100 3 for one hour)
or living suspensions of organisms. Sons sera were proenred
by injecting a mixture of boiled orgenisns (prepared as
above) and the spec fie polysaccharido antigen. Sore node
from rabbits receiving only boi’ed orgsr es contained only
0 antibodies. (Boiling of 0111 or 053 s‘reins destroys t;
3 antigen, so that no 3 antibodies result fro; injection.)
Sore made from rabbits receiving the living culture contained
both 0 and 3 antibodies. These cultures were not hosted;
horefore, the B entigen.wes not destroyed, and upon in-’
Jeetion proouoed B antibodies.) Sore prepared from rabbits
receiving only pelysecohnriio antigen contained both 0 and
B antibodies. (The polyseoohande antigen was not made from
boiled organises.) is the cultures utilized were found to be

non-motile, no consideration was made rogsrding H antibodies.

5,, ant-I52. : SQ1H+IAfi

ill cr'tnrooytes were collected and stored in Alsever's
solution. The solution.wos node by dissolving 2.05 per cent
dextrose, 0.8 per cent citrate, and O.h2 per cent soiinn

chloride in distilled water. (llsever‘gt‘gl. 19%1) The

erythrocytes were washed in saline solution several times by
oentrifu3otion, and re spenfiod in Alsever's solution. uaoh~
ed cells were stored in Alsovor's solution at h Cu r.til used.

cells showing homolysis were discarded.

' ”-**.ioo.o£...z:s QIZQLV“££Q~§
52.12;} '36: 905‘1313 a.“ l“ :1 owing/5n mfl4m

 

Packed erythrocytes were added to a known polysacchariao-
saline solution to make a five per cent susoonsion of coils.
Tho tube containing the mix .turo of erythrocytes and polyu
saooharido was placed in a mtsr both at 37c and incubated
for one hour with frequont agitation. it the end of this
period, the cells were thrown.down.by oontrifugotion at 2,333
rpm, and the supernatant fluia removods The sensitizes
poo5ed cells were t? on‘ws bed three times with saline solution.
Finally, the sonoitisod washed erythrocytes were diluted to
approxiootoly one and one~half per cent w th saline solution
and used promptly in homagglutinstion and hooolysis experi-
ments. A final dilution of polysaccharido ens to 1,000 was
found optiool for sensitizing erythrocytes.

“-- 1%.};qu

 

Erythrocytes sore sensitized with the desired poly~

saccharile or oultxre filtrate. Those were both boilod and

33

unhostoi.) Soriol dilutions of sntiooro to be tgotad vor=
3335 in soil no solution. W~ou3j~*'vo hun1redths m; saline-
serum mixture was placed in each tube (Kano). Contm3 1 tubes
inolufisfl (a) no scrum ofid (b) noroal rabbit soruo. ?innlly,

£1 tlJO roooivai 0.? 3 ml of oonoitixod srythrooytoo onfl was
oiokon tho: ou*”lv. Tho taboo were thou olo3od in a twat
both at 37 C for two hours. Tho rosotion was road at 30
minutes, one hour, and two hours.

uf “or the final tooling, tF13 tu13oo ware cout3iia*ol at

approxioatoly 2030 rpa and again road. A poo i ivo toot was
ind oatod bye adios at tiao bto to: of tho tubs, broader than
tho control, anJ hsvi n3 a sorrotsd eigo. $bo disc wouli not

slip fro: posit‘ ion'uho 3 tbs tuba was tilts}.
425255353153

Frooh guinoo pi; sorun (pooled from the blood of sovorol
sniaals) was asoi as a source of con: lea 5t. Before sash
test tho-3 armlooont was titrotod ith norms six-sop 2.13:1 2111.513
erythrocytss to ootoot tho presence of lysino for norosl
shoop and human blood coils. Eho lo“oot dilution of ooL3 lo-
mont which showed no homolysis was solootod for use. accouoo
the tosts conductod were of o prolininory rgturo, it was not
oo.sid MT 1 L390: tant to sM 333r3138J13 co;i: lomont in to?
of 1131435 honolytio as for no? £1 -hoop or*‘“”03"“" .

Sono diffm sin ho; olytio ti era'wos notoi, duo origarily

to differences in samples of complonont. aerial éilutions of

~tod woro mafia in tho flasirod concentration 0.

U!
0
*3
U
“A
u
0'
(J
t?
‘1
u

complement (0.23 ml was added to each tube. Kahn). The
control tubes oontainad (1) hoatod soru3 but no guinea pig
couploagnt; (2) guiron pig complogont clone. Twenty-fivo
hunfiroflths.$l of a 039 per cent snaponsion of sonsltlzod
sheep or human erythrocytos was alflod to each tube. The
tuboa ware shaken and placed in a 37 C :ator bath for one
hour, after whioh a final reafling was taken. The honolytl:
titar was road as tho highost dilution of earn; in which
hauolysls oaourrod. $ho honolysis of nornal sheep or human
erythrocvtos in th- presence of specific antlsora was investio
gated previous to the beginning of each test. Ko hamolysie,
due to sgooifio antisera alone, was noted in dilutions of
antisoro of one to one hundroa or greater.

35N13*4nn§45, rnn+
..- .‘.w v 1.5%:- _.‘.~."‘.x

 

All bacterial tufio agglut nations more carried out

‘

333033133 to the technique for the Elia host for kn

‘vvn'fir‘ 9:.

   

(filial, 1896). Slide aafilutinations ware performefi by
placing a small amount of the culture, ailutod in a $3113
soluilon, on a clean glass 51130 and mixing in a drop of
Speaifio antiserua. Agglut nation was obsorvad by infilrcot
light as a clumping of tho calls within a period of ono to

two minu 35. Reactions occurring after two minutes were not

consiflorod significant.

mm 7M"

Polycacchariécs for testing wore made up in saline
solution to tho cesircd conccntrction. (fl 1-1.000 flilution
gave good results.)

To perform the test, small prccipitin tubes (Durham
tubes) were filled to about coo-third with tho specific anti-
serum to be tasted. The polycacchariflc solution.uao layered
carefully moon the serum so that no mixing of the two liquids
occurred. Tho procipitin.tubcs wore placed in a cpocial rack,
so constructed that it was not accessory to rccovc the tubes
in order for readings to be made. All prooipitin tubes were
incubatefl at 37 C for 18 hours and than read. A definite
cloudy layer at tho cctiocrum-polysaccharido intcrfccc indi-

cted a positive toot. A calincacntiocrun control, run at

tho some tics as the tcot sazplc, was included.

g; g;;; and I3 VétggETcsts ofwgoizcaccficridc

 

1;; gm tests were made in rabbits by intravenous 1n-
Jocticn of known acountc of tho cpccific polysaccharid
suspendod in calico solution. ‘Blood samples taken for study
worc collcctcd in paraffinplincd taboo containing sufficient
heparin to prevent coagulation of tho blood. The tubes were
ctoppcrod with parcffin~coatod corks. The blood was collect-

ed from the cut marginal car voin.ao it flowed trcoly into the

tube, coda mtly mizzod to pro MV9.3 coo 3min tion.

,La;2i§gg tests for the lysis of polymorpicoc 1m 92: euk
cytes were conducted coir? freshly collected cord blood from
human newborn infants.

ques for the cello ctieu of blood were oreoeretl as above,
and delivered to tine oostct.r loal section of the hospital. The
blood was collected in wax-lined, heparinized tubes free the
cut umbilical cords and placed inacdiotely in a 37 0 water
bath. despise of cord blood were run in two different hospi-
tals. Five ml of blood was taken Eros each of several new-
born infants. Three cooplete cell counts were made on each
sample; the totol counts were evoruged. Differential counts
were also carried ext 1:1 triplicate and avers god.

Two mg of polysaccharide in two cc of saline solution was
placed in sec h five ml of blood sample, mixed sell by gentle
rotation of the tube, and then placed in a ester bath at
37 3 for one hour. At the completion of the incubation
period, triplicate sour ts (as above) were made and t ‘re final
average counts of all samples obtained. The results shown in
Table VI are on average of the several separate blood samples

investigated in the experiment.

77"” ‘1 " “f" c "s five-ff? ”an
“I..." “In. .44 4 an; a ifuautéidx...’

V5.4, Q’X‘WI" rim: ”Facial-

An adequate chocicel characterization of the final
polyeecohoride was not carried out due to look of the noces~
Bury equipment and the detailed cheoicel examinations roe
quired. Few bacterial polysaccharides with minor exceptions
have been adequately studied chemically. flowever, basic
chemical tests indicated that the material was carbohydrate
in nature with many nuclei containing nitrogen in their
ctmcture. lismmrth at a]... (19%) found that. in. an 0 antigen
preparation.£roc‘§.‘tzgnogn there was 20 per cent of a solu-
ble nitrogenous constituent present. This type of material
would not be removed by ordinary de-proteinizetion methods.
Rays at $1. (1950), using the cote method as was used io.thie
study, did not recort the per cent oitrozon present in their
pclycecchcride product.

From the results of he chemical examinations shown
(Table I), it can be reasonably concluded that there is a
carbohydrate nucleus present. Intact protein is not present
as indicated by the absence of the gelatinous layer, figzgg
‘et‘al..gn.‘git. (1938), and repeated negative Binret re~

actions. Ritrogen-containing compounds, probably peptide in

nature, are pr-ooot to an opyrooioblo extant. Tho poroon a;
of nitrogon present in o=oh preparation (as noted in ?ablo I,
column 3) is high.vhon.co:parod with polysaoohoriao fractions
of others such as'fiong‘gt,gl. (1933). Howavor, those workers
did not fractionoto a strain of E, ggli‘knoun to possess the
D antigon. It shouia bo brought to mind that tho presence

of vari us ohoniooi constituents in poiyoaoohorido fractions
doponfls greatly upon the method of isolation, and no results
can be compared on on equal basis unless tho sage strain of
organism is fractionotod and the Safia method of oz rootion

is employofl. Tho chemical subst;noos that can be isolated
from a givon organism, evon'by a single method of isolation,
aro numerous and should not be cougarod with those isolated
by other motooao. Thoreforo, the froztions discussed in.this
study are not oonporod chemically with othor g, gall fractions
obtained by similar or different oothofls.

Phenolic subsuonoos were found to be present as infli-
ootod by a positive Kilion’o reaction. All organic compounds
which contain tho hydroxy phonyi group give the Eillon.re~
action. The toot is gen-raliy oonzidorod ono for the amino
acid tyrosine. Doaoxyribonuoloio acid was found present to
a significant degree (Disoho toot). PhOSphorous anfl sulphur
were also strongly positive. The presence of phOSphorous~
containing nuclei, oooowiiov to Raistriok gt a;,(i:ii.),

seems to pro: ado a certain dogree of toxioity for anisals.

They stressed the inpartcnce of phosphorous and sulphur in
the biologically active molecule.

Reference to Table I, column 7, will show that the
substances were hydrolizod with sulfuric acid and, upon
neutralization, yielded reducing sugars. Those sugars were
not characterized as to their specific nature. Further cheni~
col analysis of such mater 313 should be undertaken to give
more exact information rognriing their structure. Until such
is tho case, ono can only characterize tn-so materials as
incurs polysacc’ocidos.

Until more workers undertake the isolation of poly»
socchnrido fractions fros the 2. Q211_knoun to possess the B
antigens, it is the Opinion of the author tnct the preconcm
of those antigens, which render the bacterial cells 0 in~
agglutinnble, is of treucndous importance both from the cheni-
ccl and inunnolog col point of View. As no one to date has
isolated the D antigens, it m jht be well to consiler than
as c cnocicnl entity, which, with careful cheniccl extraction

ctnods, could be isolated. There is no indication at this
point to consider than as being ont rely of a polysaccharide

nature.

T_ 1.74." chm-15":
‘ V. «A v '3 3-. v. 33.. 5 ;-|~' 3

”3‘15 . "‘
«1.1.1332; Of 9013333331311 16 OZ} MILDMI

Intr3V3m one ina3fi 133 of $0.3 mg per 33 body weight of
tho pol"scc hariflc in 331133 solution proved lethal in 333
of five rabbits. Table 111 cor. toino c rocori of the looko-
cytc count, cry hrocyte c3unt, 31334 513.3333, 333 tam taro
aft3r injection, of two r3 3bits. Rabbi 333bor 2 io incluaod
in t3 above fire. It c3n.bo econ tr 3t a £033 ofn .3.33 per
Lgofpo‘”oooc.1riio proved lethal to rabbit nufiber 2; I”? 113
rabbit nuaber 3 survivod a dose of 0.b mg nor 3;. 1:; r3. )3lt
anchor 2 a mar1zoil reaction icvolopoi 31th 13 two to three
hours and was cnnrcc 1323 by crootr3tion on; éiorrhoa. T33
porio.or31 circulo ion'w3 63: "co so] 33 evifionced by a pal!»
noon and bluoir g of the ears. 333th occurred 22 hours after
the onset of tho symptoms. 2113 heart 31331 was subooauontly
colloctod from four other rabbits t.3t had been injected with
a let: 23.1 close of the {calamari-:13 and 313331333. There
existed a morkod leukoponic and 33 elovotei blood glucose.
(2123 normal blood glucose for rabbits is from 90 to 11? mg
per 130 m1 of blood. (Joys.g§u° 53)) The temperature
rose only slightly in each rabbit. T133335 rare t3333 of

various organs& dter docth and fl? ed in 10 per cec fornolin.

#1

As much as 15.0 mg of tho polysaccharido adoinistorod
ngzflga to normal rabbits failed to elicit any noticeable
response. Similarly, baby mice fed in the sums manner showed
no obvious reactions.

Injoctions into nonriozuno rabbits of 0.“ m3 of poly-
saccharido per kg body woight did not prove lethal to any

rabbits injected.

Changes in tho Soils and Glucose Lovol of Vonous
Blood of normal and Imauno Rabbits Following
Introvonous Injection of Polysaoohasids

when O.h mg of poly: oohoriao pot kg body weight was in.
Jootod intravenously into normal rabbits, marked symptons
occurrod. After a two-hour period, samples of blood showed a
leukoponia accompanied by on increasod blood glucose. iho
leukoponia was characterized by a disappearance of poly-
morohnuoloar coils from tho blood. About 2% hours after in»
Jootion the rabbits developed a leukocytosis, ohoraotorizod
by an increase of tho polyaorphnucloor cells. In 72 hours
the blood picture was essentially normal. (Rabbits 1 and 3
found in‘rablos II and III.) (Soo figures 1 and 3).

Two hours after injection of polysooonosido tho blood
glucose increased in prooortion to tho size of the insootod
does of toxic material (see figure 2).

Tests wore mafia to determine whothor a rabbit .oving

antibodies against tho coll vaccine or tho polysaccharido

1.0116 ro1ot in a m1onor sioilar to tho nonpinjootod animal.
Rabbit number k (I ablo IV) 113 inaoctofi int ravo.'usly with
one ml.dosos contain_m1tso o3 oooh of tizo pol"saoofi11;1i&o
fro; 0111 on every othar day for a total of nine timos.
Those injootions wore givon in pr1p1r1tion for tho toot,
res~lt s of vole h are giV1n in.Toblo 1?. A chock on tho blood
so hours aftor oaoh injection stored the nu "111 look openia
and increased gluooso. Aftor t‘.1o abovo sorios of injections
with 391331-: urijs from 0111, t213 1111111 1:13 rascal for a
porioa of throo 13:13. intiboiiss against tho polysao oharido
injoztod sore tostod for and Ao2onotratod by to otoriol ag-
glutin1tion (lablo XIV). It is shown that th1 palysmohorm=
antiserum oontninoi O and D 1:1tibol ios, as living. , intafi
bootm 111 soils wore sbélutino.oi.

A sooonfl sorias of injootion 3 of 0111 go ysoooh1ri£o
was om ngivon to rabbit number k, tho res: to of which are
s1zmorizzd in T1blo I? (so: £i3uro R). Go tho first day,

hy

after 1131 2tion, a characteristic loakoponio r19

“3

1113. Approximate y 35 par cor t of tho leukocytes had dis-
appeared, #2 p 3r cont of which were ooutroohilos. A 131110-

oytosis was noted on the day after tho initia l infi otion-

Two h«:rs aftor ingootion 01 tho second day an ov-o mo rs

marked leuim onia has produced. in 85 per cent reduction
occurred in tho total number of 191‘21oytos,s mooiztod with

a 92 per cont reduction in noutrophilos. mxlts on the

The
thiro day moro : oilar to thooo ahovo. ?oir13o1 tion'uas

#3

given.on the fourth day; haucvar, a 2111 132103 tc.is 213
found. 02 the fifth any an increase in th' total leukocyte
count was obccrvcc two h3"rs 1*twingcction. This 2-13 113
due to an incrcacc in the yaphocytc lcvol, as an actual de-
crease in tho numbers of ncutr philcc occurrc1. Th3 r1nbit ,
(number k) on the sixth day, 0239 15312.prcccntcdahi:
leukocytc 032 t but was not injected. A tolcr1nc3 to thc in-
accti ens bczc n to be sh01n1 by tzzc scv3nth 115, as the gcly-
sacchaviflc produccc only slight 0311:1309 in t11c tctal lcukocytc
Gaunt, rcprccer .tcd by an 013 it pcr cc? t rciwl tion. ?29 loss,
th.2g. 12111, was mcdc up entirely by ncu 2321133. 32121-
ncticn on the eighth day revealed 1 normal blficd pi.turc. A
blcofl glucose catarminntion on the ccvcn h 113,1nd two hcurs
after injection, Izowever, shc'.:cd the usual increase. fio
app; cut tolerance had been 9331b ishcd for this 23321..111.

The s12c 9" :.,11 as 1297c was rcctcd for a p3 3101 of
eight techs and given a 2.0% ~~ 3016 intravccncsly of thc 3129
polysaccharidc. Fithin two hours an excjinction of tha blood
rcvoalcd a leukoyenia and an increased blood glucose. that»
ever tol1r1nce the 121111 had previausly’vasn 010“"cr in
evidcnae.

A rabbit (22251: 5, Table V) which had becn previously
given a coil vaccine prcpc: od fr02: 32. CQLL 0111, van injected
intravcnnucly with 0111 poly.ccchc-ific (0.2 23 per E3 body
waight), turac and one-hal f1wcc‘:s after the 103 t intr.-1 11015

infiecticn of cell vvccinc. The blood seru2 tron this 121211

13.1.}.

at“ 1.133 the p03 303333 an 121333.11. 1:131:1311 tit-3r of 0:13 to {312-113.
T110 respozma of 1:113 rabbit to 3113 11133331011 13 1233111113531} 121
Table V. 2130 and 010-»31311’ hours after ’61 10 11130:: 1.1031 .2: 1311--
150333113 13:15 establisi‘md, characterized by the disappearance

" polymorpimuclear calls. In 3331:1331, the lyrsphocytea were
markedly reduced. The total leukocytes 111er reduced 21:10:11:
70 per cent; the lmnpiwcytes, 5'3 31- r 3931.; the polyaomh-
311310;:3 cells, 35 per cent. 1110 1111103 133:- :‘3333 .1... cussed
simply. 112:0? a {1013:1011 of L1 11011333, :1 :11 lzmiaaytssls
02115201 1.11.1111 30:13 3.101 increased 33103.1 glucose. 1.3331111 81
E’hzbmr porlod :1 «3011111113 10.13; 0:33 3.03.1.3 03.13.3133, 11330.1;1111110d
by a 110.3: .13? 1131.11.11 blood 3.330%}. 1 301111113 .0 134.3. 11221 to
113.321 11:13 30.113331} 1:; '22 11:1: ’3 after-.3 the 11130 33.3.- :1 0’? 3:10
13-31;, 3.11. 31:21:13. fianocyta estimtions resulted in no definite

11.5

0333111310113. 11-333-031.11 Value. 1:330 1101'. 31331131330313;

 

Lysis 01? Lani; :0331'1133 .3113; by 0111 Pmlrsacclw. 1:36

The (:3 311311.13 03:01.3"913 by Dennis 9,3 m. (1939) 3.1:} hays
a: 3d,. (1333) 3:111 use) .3 130.013 .1131.) 1:333 ability of 3:110 poly-
34113021313133 from 02.11 to 3.333 101313371203 :3; 313.223. Freshly

calla-33:13:! car-:1 731001 of five harm). inf-ants at birth 133:3 mimd

”5

with polysaccharide in saline solution. After a two-hour
period of incubation at 37 C, the results found in Table VI
were obtained.

A #2 per cent reduction in the total numbers of leuko-
cytes occurred compared to the nunber present in the control
sample. The neutrophiles were reduced 83 per cent, while the
lyrphocytes showed a 35 per cent apparent increase.

The pclynorphnuclear cells observed in blood films made
at the termination of the incubation period were interesting.
These cells were found in various stages of dissolution.

Some appeared greatly ballooned and others appeared broken,
with a pouring out of the granular material. Other cells
possessed only a nucleus; the cytoplasm had passed out through
the fractured cell w. . The cepious amount of debris found
was accounted for by the fracture of the leukocytes, in this
case neutrOphiles. Lymphocytes appeared essentially normal;
only a few showed any alteration in morphology. The erythro-

cytes were intact in all samples observed.

Agglutination and Precipitation Tests

Following the preparation of antisera, made by injecting
a rabbit with the polysaccharide from 0111 or 055, it was
necessary to deternine if the antisera contained both the
somatic O and the "envelope" B antibodies. As a pure 3 anti-

serum has never been prepared, it was thought that possibly

#6

one made by injections of polysaccharide would be free of 0
antibodies. To investigate this possibility an experiment
was made in.which an antiserum was prepared by injecting a
mixture of 0111 polysac chariie and organises, that had been

previously boiled (0111 or genie as), into a ratbit, while a
seco.d rabbit received on ly the polysac cEL rile. If he pol;'-
saccharidc were pure B anti3en, only 3 antibodies would be
producei. By the sc.e rule, f the polysaccharid a were pure
B anti3on, nixir’ it with a boiled culture would supply an
anti3en complete with both 0 and B anti3ens. Such an antigen
when injec ted into a rabbit telld res alt in an antiserun
having both 0 and“ c antibod -es. The results of such an
experiment can be scan in? able VII.

is is evident, the boiled organisms were agglutinated
in a l to 2553 dilution by the polysaccharidooccll vaccine
entice 3, tr e unheated to l to 320 dilut -on. Antisera pro:
pared against the polysaccharide alone agglutinated the
boiled or3anisns in a l to 6&0 dilution. had the polysac-
charido antiseruc contained o.ly3 u33.; lutinins, it would not
have a331utinated the boiled or anisns, whim Inessessod only
0 anti3en u. The B anti3en on the bacteria had been destroyed
by boiling for one hour. (Kauffnan, 1950) It ea c, horef o: e,
concl adei that the polysaccharide was made up of both 0 and B
anti3ens.

Having established that tn polysacchariie was as do up

of both 0 and 3 ant i3ens, whach.upon injection would produce

1+7

0 and B e3glutinio s, a pr ooipitction recoticn.wcs inn sti-
.ct~3 tor iotcr:cinc if the 03 anti3 n of he poiysccchnrile

would precipitate cell vaccine onticcrc possessing 03 preoipi~

‘I
i:

s, as well as those hesin3 only 0 precipitins. The

ac sump otion'tcs na3e tin t the injection of boiled or'"r;iens
into rcccits for; ed procieitins specific for the 0 antigen.

3 pr e p itin reaction took place between tiie cell vaccine OB
antic ran and then polysr 33m ride GB antigen (fable XII). flow-
over, uncxgeoted results were obtained'wnen ti -e polgsncooiriio
03 antigen failed to precipitate the cell vaccine 0 cnti~
serum. The possibility was therefore presented that in-
jection of boiled or3cnisnc into a rabbit does not produce 0
pr recipit ins. It 313 1t also oo can “in ed t'm .t only the B
cnti3on present in the polyeecchctiae is able to elicit the
pro: 1; itction reoc tion. Since only one anincl was used to
produce tiie ontisoruc, it 2133 1t be possible the the oniool

used was not capable f groduoing prec-pitins.

3enc33lutinntion and Hemolysie Expericonts

Kcny'workors in the past have shown that erythrocytes
are capable of eésorbina various antigen. which render them
c33lu innble by Specific serum ontibofiy. Reactions of this
tygo have been reported by Keogh 3*,31, (19%7), are wchcnko
g; 31. (19%?), fiill‘flc"oon.2gHQL. (17%3), Hose 3’33}. (193 0),
Pic her (1930), z"‘ie‘srer 3;,QL. (1951), and fieter Q; 31. (1932).

Polys 3333331 38 -303 v.? 333 3933133 of 333t3334 Lav
an afisoraai by the red blaod cells of many species of
313. T:1 p31 - 33333r11o antigxo 33 of 331 untynai 333313 of
.3.‘;2;1 have been 33331133 3y the erwuu33ovuus of 3333313 33

Lorentz.” 33 19; 2. by 1:1;73 «1-1; a... 33333me m. 3.23;. (1:? 7)

"J
3

33333333 the polysaccharides of V323 33 bacteria 0:133 Lanna
type 0 crytlzocytes.
To det 33133'3333333 thn polysacch3rilss fr33 ;. 33;;
0111 and O§§ could b3 33333333 onto the r33 cells 0? V13isus
ape 3333 of 331.,13, the £31133: 5 3333213333 333 3333333333.
The erythr33 ' 03 of a can, rasbit, 303 ,333, 53333, and
32133331 .323 L33333 and 3333131331 Lith tho p313333charifla
£333 0111. 233 results are pros 3333 in T331 VIII 11L33I1
31733 tLa r3.mt133 for th 33313333 33.3..33;;G 1; howavar,
tbs test 333 repeated for th: acrotypo 055‘L_th 513133 r3*
alts. 3 like reaction'was carried out by 33333 a; 31.
(1932)'us 133 the antigens present in culture filtrates from
th 0111 and O 55 th at had been bailad for 033 hour. It can
be obsarvofl that the various blood cells adsorbed the poly-
sacchariflo 3331533 and 3333 353133133tzdb ;* 3333133333 anti-
sera to about tha 3333 titer. 1ntis<zra cor 3113113 0 333 03
an 3133 133 3333 333311? 3ff33tiva 1:1 p333 113133 3313;311t33p

ation.

1+?

1&3 03*3 tic n of $vo 33t13ens S 311311903313

In orfier to detormlna whether r03 31333 cells are 0333313
of aasorbim tha 3nt13ans of poly 13 M" 11103 0111 and 0%
513ultancously,1e follo1M113 experiment was 333313 1 on t.
53019 red cells were modified by (a) 1333?Jation.w1th the poly-
5333313 33 from 0111, (3) 13333311 3";‘ th the yslysacchariaa
£233 0"? and (c) incubatian'vith Ln; polysaccharides from
1111 331 033 51311333301213. $33 ratnlts f tha 3133311333
are presental in Table It.

331 31333 33115 tyeatél 33 i1 3 333 ha 33va U3? * lu 31~
nated by their h11313331sant13331. 30113 that had 13103303

‘ .I t"" I ‘I‘ C & ' -
'3 3333 33311311;.cfl by 3131

L
a
g

cabin: that each cell had 33303331 bath anti; ans. Heyeated
caper 113nts gave 3131113 33311t3. arm?"3:, 1331 0:3113 prev
paraa as in a and b were 21233 t131LL;-, 1313331u91331101

" 44" I, a. ‘9‘" 9% ' I “4'" ‘ 1": ' I1 r
3133 - 513310 13.- 3133 (313 - 33 C111 03 0337 013131 o .1?

J.

t “1 3“’atk 33133 0: t3: blscfl 33113. "3;”1 ina‘ion of

W ‘UU"

v_1sit1:efl as in 0 pr 13333 a 99331331 reactisn main“

“‘-

O

Q
4

1.4

..:..~ :_ .. 11-,- .1, ,, a '3 .-"— Vb..i.,.., -

01313: -3141033. F113» n"? 9V133219 0; tag 31131‘133313 ad-
1. _ _ ,_ e .3 , “a ,- 1. . ‘ ,

0-3»-o1fan313313 :13 311331 1133 5133111231 33113, as in

a at b, Hero 11:33 vita 313333.31 39
tha 333 3133313 335111333. 3313 1133131 1331131311133'333
Obsarvafl, 11111131.3 that only 333 33“"1*133d 33113 3336 in,

V3 1‘70 '3 c

r'
20

The Snbsoquont Troatnsnt of Sheep Rod Blood
Cells with Polysnoohnrids

If tr 9 polysao onnriae antigens con id be adsorbed onto
red cells independently and sinultnnoously, the question
arose as to whether tl1prosommo of one polysaccharifio on
the red 0911 would sxolufle the ads rhtion of another. 1
blocking effect night be present to pr svsnt subs sqzont aa-
sorbtion of tho polysaccharido antigens. To in .’:o mi3sto tnis
question, repented s:porl:1 onts 1:3rs‘unnort Ezen in.which wasn-
ed snacp rod cells were first treated wit11 the polysaccharido
from 0111 followed by tho polysaccharias from 055 and yigg
‘23233. The reactions srs shown in Table X. tlnost ifiontionl
titers were produced by the various reactions. 30 bloc: fins
of honnggl1tination was noted in any to: otion. Blood cells
treated with one antigen dii not honagglutinsts with the
heterologous antiserum. Titers were sinilnr to those obtnin-

ed when snoop rod blood cells were treated simultaneously.

Hanngglutinntion and H0301¥619 by
ndiition of Complement

Fisher nni ’~o*n in 1930 rooortcl that erythrocytes

{-45

oonsitizoi by banter nl noti~o“s‘wo171‘unflo go lysis in the
to: non of oonrl1zont. To notornins whothor this 33..

_ 11-1 0351:1321 113115.111: finnenstmtod using 1:213 antigens of £2.

,QQLL (“0‘"“nochn 1333 ) 0111 and 055, the following exporinont

was undertaken.

Snoop and h‘x 33 e""*fi.o“"‘es JOPG no-1sitizol 0y the poly-
saccharide antigens from 0111 and 05?. To cnrry o'.1t a test,
“sitiz ed eryu1rocytes (having adsorbed $308310 polysac

charide) were added to tubes containing two-fold serial
dilutions of E1e honolog one antic arun. Con: e:3o.. made from
pooled sore of several guinea pigs was adfied in a final di-
lution.of l to NO to each tube contn ning sonslt 33' orythro
cytes and specific ontizor“3. fhc tubes were gently nknkan
to insure nixinga z‘é tnon 1:0re incubated in.n tutor 3333 nt
37 “ far one hou.. Lvsis occunred int 3303 001 t33.113 snoop
orythrooytes but failed to tnke nlnce in tubes canto: n13:
human red cells. Lvsis of sheep or hunnn crythrooy'es did
not econ: in tho absence of cocolnficnt. Vnon conplonont wns
nixei with 5939111203 erythrocvtos 0.. man or sheen in the

Q

absence of antiserum, con;lc:ont dilution of l to 30 nLc

q a A y. . ' g-x .
33m rdifl not canoe lysis of tha orythroegtes. A final ni-
lutir of l to #0 of conjlonent was scloetoé, the:3?orc, as

3. antisonn on p.loyod in the

C?

ontL 313 for homo lysis tes
homolysic tests were heated at 56 o for 30 rinntes in or lo:
to destroy complonont normally present in 31333.

Table XI indicates that nonolvtic titers were M13133
thn. corresponfl 13 he :zgfilltin tion titers. Katinarn used
in thooo experiments vor= 3133331 '30; cell vaccine used as

ff . o 9 43" J. ‘
antivezs. 331alycis exporizents tore also c011312tod :13;

-133139 antigens.

anti"n*r or

\u' ‘1‘ I...

Q
‘ 1
13
*1
f)
u.

a

A.

c *0 «. ‘o ._ ,. o. ._‘J
Sen lyois ti toss in.tneo~ ens ovoro conoi.cont j lobe; tun.

\51
f0

when cell vaccine antisera were employed. The hemolysis test
appeared to be more sensitive than the henagglutination re-
action. It is interesting to point out that no lysis of
human blood cells took place. Durim the test human cells
were washed in the sane manner as sheep cells, but apparently
small amounts of contaminating scrum renained to block the
reaction. fleter gt 31. 1932) have shown that 5.33113 zzounts
ofs erun present with human red cells in the sensitizing
medium can prevent the adsorbtion of an‘igens onto the erythro-
cytes. Henagglutination reactions, on the other hand, were
not affected. R3pea te d tests yielded the 5339 resalts. It
was also noted that treatment with more than on polysac-
charidc antigen did not prevent the lysis of sheep red cells

in the presence of homologous antiso um and complement.
The Effect of IE catL Jpon Polysac charides

rater gt‘al..gg. git. (1952) showed that in order to
made an antigen from 0111 or 055 active (so that it would
sensitize erythrocytes for henagglutination), boiling for one
hour was necessary (Table XV). It has been shown in this
thesis that boiling of polysaccharide-salm esolutions was
not necessary in order to render them active for red cell

sonsit is ntioz in experiment was indcrta E: on to determine

:5:

Who her boiling of pol; saccharide antigcnS'would prevent them

from being adsorbed by erythrocytes and‘whether they would be

able to not specifically in hensgglutinstion reactions if
adsorbed. Table XIII shows the effect of host upon the el-
sorbtion of_the polysaccharide from 057. {coagglutinction
titers of erythrocytes sensitized with he boiled polysac-
charide antigens from Ofifi'were nearly equal to those of the
unboiled. However, it can also be observed that when a
culture filtrate was used for erythrocyte sensitization, it
was necessary to boil it in order for the antigens to be ad-
sorbed by the erythrocytes. A test using unheated filtrate
showed a titer of l to 100 dilution. It can be observed from
Table XIII that the polysaccharide antigens produced better
sensitization of erythrocytes than the boiled filtrate.

Eetholesz

Five rabbits, all of which.were normal half-grown.enimsls,
were given.doses of polysaccharide in.excess of the amount
known.to be tolerated. Eight-tenths mg per kg body weight
proved fatal to each rabbit injected. The snissls were
prostrate within.s few hours following the injection. The
animals were necrOpsied soon after death and sections of the
various organs taken for histological examicetion. These were

fixed in 10 per cent formalin.

Gross Examination

The lesions found upon cross erasination of each anlnal
were those suggestive of a rather severe toxemia. The sninols
were bleeding free the nostrils. Green.wstery feces covered
the hind parts of three of the rabbits.

The lymph noJes, particularly those of the bronchial
area, showed a marked degree of hyperenia. The spleen.was
also very hyperenic but otherwise generally normal in appear-
ance. Petecnisl hemorrhages were seen in the thymus gland of
three animals.

3 mild hyporenis was noted in the intestines upon being
Opened which suggested the presence of catarrhnl enteritis.

The heart in all rabbits was flabby and lighter than
normal in color.

The condition of the lungs varied from nearly normal to
sons in which areas of emphysema could be detected. The
condition.was more pronounced in the basal lobes.

Examinetion of several livers revealed snail focal areas
of necrosis well distributed over the organ. The organ
appeared generally lighter in color than the normal.

Ens kidneys, with the exception of a slightly aaharent

capsule, were grossly normal.

nicroscopic Examination

trans 52125. e« r of these were hypcrenic and some show-
ed minute hemorrhages.

The spleen was congested but no other changes

 

were observed.

. Sections of intestine chosen a hyperu

ntc" re
secretory epitheliunuwith an increase in the number of goblet
cells. masses of musin'were present covering the columnar
epithelial cells. A definite increase in the size of the
intestinal caoillerics was seen.

.Qizgg. HicroscOpic criminetion showed the result of a
toxic reaction. Areas of necrosis were scetterci throughout
tn. section, and were associated with the inner third of the
lobulcs, chiefly in the region of the central veins. There
was coagulation, cessation uni some liquefaction necrosis
present in the liver cord cells. The portal canals were
congested but the areas around themuwere free from necrosis.
The sinusoids, when not disrupted by necrosis, were filled
with erythrocytes. Fatty changes were present in the hepatic
epithelium of lobules where little .ccrosis had taken place.
Thronbi were found in many of the central veins. Other veins
showed normal erythrocytes in the luncn.with an occasional

one containing fibrin.

‘4‘
Q,\

filiaazfip Coagulation necrosis was visible in the proxi-
mal and distal convoluted tubules. Some gloneruli were
hyalininnd. finalei, where recogn gable in the tubules, dis-
played pycnosis, knryolysis and knryorrhexis. nany convo-
luted tubules contained debris and occasionally albuzlnnus
casts.

‘ngg;. The lungs in threa cases showed emphysena. This
confiition could have been tarninal. Hanorrnngo was present
in areas surrounding many of the bronchioles, which contained
numerous erythrocytes. Th3 capillarias or the ung contained
pink staining material having tha appearance of hyalin.

There was no inflammatory cellular infiltration of the alveoli.

.flgfiaz. Vacuolntnd nrana among the bundles of fibnrs
indicated fatty change. Thane were not extensive. Limited
necrosis was seen.in small focal areas and where present the
bundla fibers snowed loss of striation. Onnnsionnl hemorrhage
was present to a mild degree in the bunflles of fibars. In
such arena the fibers appeared to have undergone myolyaia.

”nain no inflammatory cellular infiltration was seam.

FLU

DISCUSSIG? O? RSSULTS

 

It was recognized at the beginning of the study that a
method of isolation must be utilized that would produce as
little degradation or the fractions desired as possible.
nony methods used in the past have involved strong acids or
alkalis or extreme temperatures. The method chosen for this
study was mild and the polysaccharides obtained were not
subjected to acids, alkalis or high teopcrsturcs. For the
disruption of the bacteria, the grinding method or Lsngner
and Forrester (1939) was used. flood results were obtained,
Judged on the basis of nicroscopic examination of the
fractured cells.

Inasouch as the polysaccharides from 0111 and 055 reacted
chemically and biologically so nearly alike, discussion of
the polysaccharides will apply to both, unless otherwise

stated.

_§yrity of the Polggcccnoride Fractions

Various agents have bcen used to extract the O antigens

of the E, 9911. Among those are, to mention a few, trickier-

acetic acid, trypsin, glycine, diethylene glycol, phenol, and
gucnldien, followed in most cases by alcohol precipitation.
These preparations have not been demonstrated to be pure
polysaccharides, but mixtures of many chemical groups such as
D-glucosesine, phospholipins, glucuronic soifls, polypeptiaes,
phothutides and some sulphur-containing nuclei. All of
them, however, have been positive to the fiolisoh reaction,
although this would not necessarily prove that they were
carbohydrate in the strict sense of the word but merely would
ind cate the presence of the carbohydrate recicel. In the
fielisch reaction, the carbohydrate radical remains intact as
an aliehyde group present in.a new grouping known as e
furfurel. Raistrick at al. (193%) stated that there is no
Justification to assume that bacterial fractions obtained by
present methods are pure polysaccharides. In edeition to
what has already been said in discussing the biochemical
aspects of the fractions, a few points are to be made re»
zeroing their purity. Few polysaccharides have been isolated
that can be termed pure carbohydrate. Tel gt a1. (1943)
stated in cescribing antigens isolated from gsiggllgflgzsgg-
geriqe‘thnt the leukopenie present, in tests conducted, was
produced by a conJugctcd protein and en'undegrsded poly-
saccharide fraction, both substances having a phosphorous
group attachea. conceal or this group rendered the fractions
inactive. The fractions of 0111 and 055 have been shown to

possess this group.

59

33133-132; g; 31. (1931+) 1313321113321 3 £r33ticn frog: 3.33,,-
.13:1§3 333‘ which cantainofi t3 3 33331313 polym 33‘:133113

in an "antigenically" pure form, thou3h m3ny 3311t or 33; 33333

3313..

   

profiein prod eta were present. It 333 cnphasim A1 that the
fractions wrn 3 corta 1r1y not pure 3333333333 3h3313 311g, but
3333 3033133331 t3 b3 phosphatida as U011 as polysazcharide.
Their exact structure 13 still a matt3r of coujacture.

Delafiald 33 12.1- (19310 claimed a fraction that was a
p31y333313153, containim no unnlterad protein. £333V3r,
31t3red prstein prolucts 33:3 in evidence.

Ha 3.23.31. (19% 0) found ta‘o £333 31333 from a, 33‘ to
be of a 331y333323r133 n3t‘“3 blt 333 33331r: maayo othx 33331-
331 nuclei. Tha fractions from 0111 and 0;? 33:33r3 33331«
33113 31th t- 333 f0“ 31 by ther 13333313 tors in 3036 r3 3933ts
in that (1) they contain r3333 133 33 33 3 1n 33311y 2333333313
amounts, (2)t thAy do not 03:31u.ur~33.Adci protein, (3) they

3 a ., 3. . ‘.. ,A .. .. 1 . L. 1:
3333113 3; 3.33333, uni (+) 333; 333131 33n33131ng

3333333

'13

3313A“ .3: and p3 spheroua with (E) 3313r3 t3 asgznts of nitrogen
in.V3313:3 nonpprotein 3rr3333z3nt3. 335333 A th and Stacey
(1939) 323:31 that an 333133n of £- EJQQQQQ 3333.1A31 50 to

63 per 3333 polysaccharifle, 16 per cent 133313313 polypeptide,

3 to % Aer 3333 of 3 3313513 nit rogonoxm 303 s 1t~3nt, and 3
to #3 3r cent of 3 113015 comnnnart. D-3lv333331,3 33-

33:1t313321 as a 1:31t.

63

A preparation by fiorgon a; a1. (19%1) proved to to a
polysacchariflo-protain cooplox which was both toxic for rabbits

and also antigonic.

Inlootion ogfifiglysggghariflo ?r22tions

Reference to Tablas II and III indicates that the most
marked reaction of rabbits to intravenous injection of poly-
saccharido 0111 or 055 was a rapid rofiuotion of the louko-
oytos of the venous blood. The granulocytos are affected to
tho groatoot extent. A quick rise in the blood glucose level
was also marked.

Loukoponio effects or various bacterial frootions, some
of which have boon polysaccharido in.noturo, wero first report-

3 by Kolmor (1891), Kanthaok (1892), Loowit (1392), and

Q

Golochoiior and Jacob (189%). Tho lo‘kopooia in most cases
was followed by a rise in blooa glucose levels. flora recent
work (roforred to elsowhoro) has offered similar findings,
tho present study not excepted.

rot all fractions havo boon alike in their selective
ootipn noon certain colls of tho blood. Those reported in
'his study woro.moro pronounced in resuciog the numbers of
polymorphnucloar louKooy 93. Such results can probably be
attributed to tho phagocytio action of tho noutrophilos, as
20115 generally considered to be non-phagocytes are not

rofiuood in.a;¢rooiablo numbors.

61

The foot that largo oesos of “31? ..nn ride or on fed to
rabbits and mice did not produce sweeten: seems to indicate
that tic.m:tariil was either (1) not em) orbed from the di-
gestive tract, or (2) inactivated by digestive enzyooo present
there, r (3) not absorbed rapidly enough to reach a t0" c
level in the blood. The possibility of the detoxification of
certain amounts by the liver shoulfi not be overlooked.
italics upon the blood of oniools that ingeoto3 the fractions

re dot ialioot3d.

The r~o1ootivo destruction of the gre.nnlocytee of the
b19032 r33 b2o2 reported b3! Drnnis gg.o“ (193 9), {organ an.
.aiQ.(19LO), ”ovorite on. o'*. (1932), and Hays gfi‘gl.‘gg..213.

(1955. The veriouo oochenio: responsible for such e re-
action have each been vocational The possibility of the
response being due to on adrenalin increase woo coeoiiored.

In two pop2ro by' "roy e‘ ol. (w B) and 3(1913) the re. eults
o? 3goct Lgr roebite eni humans with odronolin.did not pora1~

lol the findings of the proeon study. In both investi-
gations on i? nelioto rolot ivo e33 o2solute rir e in all leuko-
cytes occurred upon injootion of 1-1000 dilution of adrenalin.
Io loulorcnio was observed in any case and the increase in
leukocytes was izmodioto.

Other conoiflorotioes as to the cause of the cellular
rooponeo m the mentioned. Loukoponies produced by the in!
joction of o‘cleic acid derivatives have been reported by

Deon 0* 31. (19?8). These studies indicate a highly specific

stimulatory effect, liiitod to tho 5zonulocytoa of tho bloo3.
Injections pro m‘ocod marked 1. roooos in the total circulatim
neutrOphilos. Eo loukoponio or rofliction in any othor collu-
lar olomont too notod. In anothor study Boon 53 51. (1135)
failed to pvoduoo roootions in rabbits by tho intravenoas in~
Jootioo of 203,900,000 living or5oniono. Only mild trancioot
constitutional and hematologic rcac “is 5 53:3 observe}.

many v " r3 = . attsop 333 to p “oiuc alcukopcaiis
similar to those prodzcol by tho golysocchcri3os of 0111 333

'3'.“ ‘0‘!~ 1 !' ” r‘.' V on I “ -' 4 (“8" ‘ '.)~i‘ . .
G); by UAQ iojccoion o. virio 3 5r ”3-: ococtcncco into

.— f ‘- 4- ?9 ‘- r-. ‘ . r e - '4 - m ‘- ' -~ 5.31 ‘4 -‘- &

rdJJ1u3. 2:09....)1-‘3’ 51.13.15; ttl‘SCG 3-33 "1:33.413 ( "44. ‘2'...- CuflMS"
" J n *' N F! P 1 1~ ‘v. . I'.‘ - ao- ‘ . . v J... .

toroi -323 pro.oics us o55 alouoin. .crci5n procaia -r-3 a

variety of sources, when given intravenously in rc“bits, has
exhibited the common property of otiooliting I'mocoooiasis
in as No 1:12 o mooncr as the nucleic a: L()oci Q; 51.
.22. 2‘ . 1923) a5g2oirod to iooroaso tho oyoloii elements.

The roop n o of rob Hi 3 injects-3 with tho polyocschorido

U3

3
froa 0111 or OS 3(233135 11-?) does not 5 *ulnto results oo-
tainod oy mortars Eith pro tcio 510”tot o3, while injection
of various poly: cachorido fraot 235 35 a comm i3orcblo number
of p2rm no has rov~c ml 3 sioilar roo.c tions. Therefore, it
docs dot appear toot protein ouosta1co co proiioo rooalts
cooporc blo to thoso found upon in 12c tion of polysaccharido
fractioo o intravenously into rabbits. Horoovor, to o h; por-
31"03113 state roportod hero has not been 053 crvod in protein-

inicctod animals. However the rvporg l'ocmio rc mo tod could
v 9 w

63

be due to an adrenalin response, though this is not compati-

ble with the leuKOpenia observed.

~14

Iistoricelly, the first case of a ba3teriel egranulo-*
cytosis in this country was reported by Lovett (192%), in
‘vhich_gggillg§ gzgcvgnegufi was the irfi “ti 3 a3ent. Later,
Ricyer 35,51. (193%) failed to produce an a granulocytosis by
injecting bacterial toxins into rs bbits. A toxin from
‘3. ggzonoQQJ‘"as the only one found to be not ve in this
manner. No doubt, investigations would show that the in-
Mffective3esso these fra.3 tions would be due to the method

f fractionation.

O

Alterations in the leukopenic response have been studied
extensi rel 5'. Tables IV and V shov the results of injecting
pol:em ccheride into cell v0 ccine and polvszzcchcride immune
rabbits. Irieunity to th leifxopc:1ie or hyperglycenie was
not observed. The reSponse of the vaccine i.mnne rabbit
(Table V) indicated some attenuation of the effect of the

polysaccharide. Studies in.vhich no Lnjunit; could be demon,

strnt ed to a leukopw cresponse are those of Favorite gtflgl.
93'.91§ (19”2) lays 2Q m~.22i@“(19%), and Morgan gt
.gl. on. Ell. (19%1), mush‘Morgan gave no indication of the

time between the last irnunizing dose and the challenging
dose. workers claiming to have producede an immunity to
cellular and chemical one res of the blood are Dennis,g§ngl.
22- ..it- (1939), Delafield st 31. ea- sii. (1931+), and
Olitski gt 31. 23. git. (l9hl). Any consideration, however,

6b

of the toxic or lemme response to injection of bacterial
fractions zamst be tempered by the realization tint the toxic
and icnunismg substances present in such fractions cannot be
normal to represent the total toxic and manning potenti-
alities of the intact bacterial cells. rot-sever, injection
of whole organises has been shame to produce little change
in the blood of rabbits (Dean 21 m.- .92. 3132- 1935).

The reduction in the oust-Jere of lemeocytee 1:; 11329 has
been reported by Bennie 51mm. (1939) anti Hays 9,9,. m.
(1930). The destruction of leukocytes in each case took
place at the expense of the granulocytes. A reduction in
granulocytec has been shown to tel-2e place L3 11m in the
cord blood of infants (Table VI). The effect was marl-zed and
the cellular destruction was complete in the cells effected.
An interesting: question arises as to whether infants suffer-
ing from a metro-enteritis due to 0111 or 055' show a lam-io-
penie at any tine wiring the course of the disease. Though
the author has no inforootion in this regard, it is very like--
ly that the absorption of the bacterial credo-toxin is relative-
ly slow so that a leukocytosis dev’GIOpe without a lemzoponic
state. ‘slere 9. Emma ini'ant injectoei with a large doee of the
polysaccncrmo fraction from either 011]. or 0‘55, it is
reasonable to assume that lewzopenia, followed by a leuko-

cytosis, would result.

 

Voluminous material is found in the literature dealing
with hecagglutinction.reactions. ‘Usoally reference is code
to direct bacterial or viral hemogglutinotion. 'Howcvcr, it
has been shown that the antigens of many microorganisms are
ceosble of being adsorbec by red cells, thus rendering them
agglutincble by specific antisere. this tyoe of reaction
has boo. celled indirect bacterial hcnngglutinstion. Keogh
st 33.. (new, niaciebrooe st cl. (19%), says 93. a1. (1950),
Fisher (1950), ficys (1951), Fisher g§,n1; (1951), Hetcr ch
{21- (1932), and others, have demonstrated the phenomenon.

Hater (1932) and Hater at al. (1752) reported a hcocg~
glutination reaction, produced by sflsorbing the soluble
antigens present in te- filtrate froo.Olll or 055 cultures,
onto erythrocytes. These antigens were not descrioed cheni-
colly. fietcr found that adequate sensitization of crythro~
cytcs for the indirect hcengglutinction reaction occurred
only it the culture filtrate was heated at 100 C for one
hour (Table XV). The author stated that the.scchcnism of
rendering the 0 antigen capable of easorption by red cells
might be due to the inactivation of the 3 antigen by boiling.
Two conclusions in this re; rd can be drawn from the results

3 the orcsent study; (1) 3 antigen did not soccer to be inp
activated by boiling as boilec polysaccharice scepensions

secocd to produce reactions equal to the unboiled (Table

XIII); (2) it might be assiged that hes 2330 of the culture or
culture filtrate to "i motivate" B enti;en brings about a

modified fractionation of tile pretei n.£ron the po lyssccboriio,

allot:ln3 the polym xxnride fractions to be adsorbed onto
erythrocytes. It mi ht be of 1.-u m~t to test boilclc ulture

fi trates of the two serogroups for the presence of polysac-
cits-rides.

flLe 0 inog3lutinability 0:73. *" s~t roles that gosscss
the 3 antigen (Kouffosnn, lOfiO) has been recognized for some
t J9 (3:;ble IV). This reaction, thought to be due to the
pro: an cc of s 3 enti3on on the surf so or? the or3nnisn,
results in interference tith.0 agglutination end erythrocyte
so :1sitgation.' ts"’sann p1 revel the absence of a cabsule,
since no swelling occurred then the ol3.nleott‘s trait;d titb
a Specific antiserum. sati3ons present in the si' sero-
3roups of a, 33;; containin3 the 3 ant °en'tere referrad to
as "envelope" enti3ons. The sore-3roups Gill and 033 possess
the 33 and t3.e B; antigens, respectively. The sore-groups
mentioned can also be fO’nJ to oos sees en.H antigen, in which
case they are motile.

Var one serologioal reoc tions tinlch have be en reoortei
by Eater 33,‘31§. (1952) have been possible flue to the ad-
sorption of the 0 antigen from boiled filtrates on the
surface of the erythrocytes. fioeevcr, in the present experi-

nts tbor "as good im ioution that similar reec tions were

due to the B anti;en.es tell as the 0 antigen. tater also

6-7

pointad out that the adsorption of the O antigans frog cultura
filtrates by er ythrocytos was blo wd by tho presence of
solubla 3 antigen in the filtrate. The polysacchariio anti-
ons difl not ex? bit this failure t-o be adsorbed by eryt21ro-
cytx , even tho1r 31 t? oy have been sho*i.*n (Table Vii) to possess
t21e 3; .en in sorologio1lly ac tive form

Ervthrooytes sensitized with the polys1ccharide antigens
sl.ovol con sistantly higher homa331tinatin3 titers t3 an.rci
cells treated in the 5133 manner w th heated cultures or
boated culture filtrates. It should be ezphasia 31 that at no
ti? -o during t? e fractionation process, in the preparation of
tho polysaccharid 9, did tho to spa r1 t1 o rise above 533 0
Culture filtrates, on tha othar hand, o3uirod boilin3 for
one hour in order to destroy the blocking effect (nontionod
abovs) of the D antigen.

Th3 pr esonto of tho 3 anti31n in the polysa.o chariflo
fraction was demonstrated than, mixed with boiled organisss
and injected into a rabbit, thore was produced an 03 anti-
serua. Tho 3 antibodies oould c039 only from.3 antigen
present in the polysacchariie, inasmuch as B antiganywas
destr0391 by boiling in the b1 otarial calls injected. The
aoovo an erun agglutinatod living or; 111538 (Tabla VII).

As pure 3 antisara hgve not been profiucod, the exact
identity of the B antigen is unknown. ‘Whether it exists as
a coating on the surface of the living bacterial 0011 or is

distributed throughout are mattors for further investigation.

It app3ars that a gonwr3l distribut103.of the antigon through-
out tile 333333131 3311 3;)313 33‘ plain‘most satifi $0311? its
praura 3333 11 t: 13 poly3333333133 £333 tions, and also in culture
filterQSC

In all 5033331u£133L13Q.r03“t1033 33 0931333 33r33 di-
lut1on was obsarvad for agglutination. Complate inhibition
of heaaggluti :x3tion'wa 3 not obsarved to be 333 to a 133
303333 13th on of serum. For any 51- en r3 Mi 3333, h3333313-

33331333 3333 using 33 :3 p3333333 againsfi tha vaccine, show-
ed can sistontly higher titers than sera prod 1333 against the
polysaccharide.

The length of t133 :33 orythr cytes 3333 3333333 during
sensitization and tho concentration.of the polysaccharifie
were izportant. The sensitization tiuw 33 r3931 ted by Hays an.
.31;. (1950) appeared Optimum for use in this study. Rater
an. an. (19:72) four! the 33:; :3 1333333 3 far: 3321313133
erythrocytes to be satisfactory, using boiled 331t‘33 fil-
trat33. Joncantrations of pol"333333r1&3 used to 333313133
33y iflccytoa were higher th3n 133319 £333; to 33 0931333 by
3333 Q2. 21;. (1951). Longar pariods for 33nsit1zation than
one hour did not 3; apear to increase tka hemagglutination or
hegolysis titers, 939133133 the 3333 33t13333.

From observations 3333 during this: tu3y, 1t 333 ha
postulated that only a 33311 p03tion of tha total 3333 of a
31333 erythrocyte 13 3333333 3% any 033 time by a polysaco

3333133 adsorbed to it. Indications that this might be the

case are presented in.chlos IX and X5 whore it was decon-
streted that more than one polysaccharide could be asserted,
either simultaneously or subsequently, by erythrocytes. The
facts presented seem to indicate an.edhorence to certain
colloidal principles. ‘whothor the polysaccharides actually
existoe es colloids cannot be stated. Though the total
number of polysaccharides that can be adsorbed by a single
erythrocyte has not been determined, Hays on.‘git. (1951) suc-
ceeded in eflsorbing four at one tics and in demonstrating
their presence as specific eetigons in hoccgglutination re~
actions.

Krevchcnko (19%?), who reported the reaction of polysooo
chorides with erythrocytes, stated that human erythrocytes
apparently cocaine with specific polysaccharido by adsorption.
In exolsining the phenoconon, the formula of Prcundlich'wos
given, in whic. X - Kcn,'where X is the amount oasorbod per
gram of adsorbing substance, o is the concentration in
solution, and K and n are constants. He postulated that if
this rule hold, erythrocytes should be able to adsorb poly-
e'cch:ride and transfer it to other erythrocytes or to a sus-
penfiing medium. A future test of the principle would be of
interest.

Several probloos arise as a result of the investigations
into pnlvsacchcride adsorption; namely, (i) that is the mini-
mum amount of adsorbed polysaccharide accessory for hcoegglu-

tinotion to occur in a high titer antiserum, (2) what is the

70

Optimum amount of polyescchor no that must be adsorbed on
the erythrocyte surface to bring about the highest titer
possible, (3) how much of the erythrocyte surface is occueiod
by the presence or one polysaccharide, (h) is the polyscca
choride bound strongly to the erythrocyte surface, or could
it be eluted so homogglutinction'with specific antiserum
would not be possible?

It would appear that the answer to many of those problems

lies in the fiolis of colloid oner stry and immunology.

71

SUEHAKY $33 CxTSLUSIOHS
The isolation and preperties of the somatic antigens of
”echo .ggli, sore-groups 0111 3k and 0§S 35, are

described. These antigens are ehevn to possess specific poly-

   

saccharide substances, as well as other chemical crouoing
containing sulphur, phoephorus, and nitrogen. Phenolic
groups are present.

Rabbita injected introveneue-y show a marked reaction,
which is chore: orizod by leukopenie and hyperglycemia. The
leukepenia is due essentially to the less of granulocytos
from the blood.

Rabbits ienunized by injection: of the specific polyeeec
charide antigen develoe a tolerance to repeated injections,
but when given a period of rest, and again injected, develop
a leugopenic end hyperglycemia equal to that of a nonoimnune
chisel. Preetration and diarrhea are also noted. Rabbits
legunizod with the cell Vaccine of 0111 BA, when rested, and
then injected with a non~lothel dose of the specific poly-
saccharide, show the characteristic leukeponia and hyper-
glycenia.

The loti’lzocyteo present in the cord blood of mmbern
infants ere lyeed‘ig‘giggg by the pelycaecherifle of 0111 Bh’

The loss of leukocytes is principally one of creyulecytes.

72

Red blood cells of non, pig, chicken, £03, cow, and
sheep are shown to become sensitised by the pelysaccheride
snti3r one one egg ntincted in the presence of heuolo3 one snti~
serum. Preparations contoinim the 3 not gen are effective
in rendering cry hrocytes e33lutinsble by their Specific anti-
sers. A pelysec wheriie 13 King the B antigen.wos not prepared.

Erythrocytes of sheep are shown to adsorb more than one

m13en eitm acultencously or in succession. Ho block cc
of reaction 8, due to the presence of more than one of tee
polysaccharides on the erythrocyte, is noted.

Polysccchnride antigens adsorbed on sheep erythrocytes
are shown.to combine with specific antibody, and, in.the
presence of conclecont, pro-duos new olysis. Titers of homely-
sis reactions are generally hi3ner tion are those of hemog-
clutinetion.reactions.

Cell vaccine cntisere are more effective in precocing
higher titers in the honsgglutinntion reaction than polyssc~
cherid e entiscre. Boiled polysaccharide preparations appear
to have as good sensitizing ability for erythrocytes as on»
heated preparations.

The intravenous injection of polysaccharizle antigen from
0111 Do into rabbits in lethal doses cans ed sin nuts to occur
in.vcrious organs. Among these, the principal effects in the
liver re: (1) fatty chm c309, (2) coagulation and cessation,
and liquefaction necrosis, and (3) thrombi in scny of the

central veins. In the kidney necrosis of the proxincl and

73

distal convoluted tubules occurred. Pctochicl hemorrhages
are found in the thymus glee . A cntorrhcl enteritis is
produced in the intestine. itclectasis and emphysema are
shown to occur in the long.

It appears that fractions such as are described would be
useful in the diagnosis of gastro-entoritie in infants, por-
ticulcrly in hospitals where bacteriological procedures are
not feasible. It is probable that antibodies could be demons
strotod to be present in the blood of infants .iffcring from
gastro~cntcritis of coliform ori3in, by utilisation of the
hemcgglutinstion or hocolysis reactions, in‘wb.oh.s polysac-

charidc proper tion.mi3ht act as the sensitizing antigen.

APPBXDIX

Tables
Graphs

7h

.omou abwpauaamnv a.uo«mmcom an madman mu“: vdmmmm

.wuoa ouoaounamuuhMOmovuou ammo anuaum:

$33.30 523 n3: noflafifiaaa E handgun
.opnunmaondnnmomnAnnaaoama no coauaauou an «sponamegmm

.ucmo «on ma wonmoumme mam mammdm ad
vane» consummam «a unwumz.na ummmn demouuwu HnamHmnMH

n, rlr Ir

1

 

xx}. xx}. xx}. 34‘. x}. ‘5. 3.1». 33. m 5m mm o I xx ‘3.
o; _. .. .¢. ...o41o .o, .u . ,7. y. .5 ._ .Jlo.
. . p. . . n L . . , -. r r. . n .
Qhflbflflm d .If m . d d O in I. ~
mm. Wamé " :583 n 1239..“ u Nnfifimmfia " $3933 " 3.3% . €315

 

who nn¢ flHHO no aifiHfiadjarnmeL
mwa ”“0 «Ham «.3 "H .3. no 0mm

H33 “FNMA...

76

12:31.3 II
.1331. 9":13 0? A 730? ”it 435' ”x. 3311' (3:137? MIL :43 032
TO 11'33 '11"... J=J3 IFJZZCTIJH 12. 13.1
§;,§Q;I 0111 POEYSACSHKRIDE

 

' on-

! ‘ 1 I- ’1'??? ‘*t':»*. ‘ F3; . 30..
Rabbit 2 Tina of 3 Total :Per I :Per 3
l -1 .- -1 . {'H' ‘3 i‘iC J 4“. f3; «30 ,5...“

 

Rab-me #1 before 1113 7,000 55 I..620 33 2,310
00" 333/153

bedy wt 3 hours 2,300 82 1,836 1% 322
6 ham-a 1+,2oo 1.9 2,058 51 2,1»2

Zh-hours 20,000 12 2,h00 88 17,600

us hours 9,800 36 3,528 62 6,276

459 hours 7.500 #0 3.000 5'3 3.975

#3.; .4. _-- _-_ __ AL
.— ._., , w. w , _.._

1Leukocytes to the nearest 100
Qfirythrocytes to tha noarest 100,000 per Gum blood
3Hemoglob1n in grams per cent

1‘-<a‘1.u‘:::c>:::e in grams per cent (blaod)

--IMMWI~W—“-m- “- mm‘wmmv—u—s-mwfirw w.—.«.- -v- uh ans-cu .» -A»

77

31:32,: II (continua-'3.)

 

 

     

 

W:— i3*:*“' ~. ‘vizz'hv— ¢:~—~—rw g4; ‘vauu “a
$302: T: ‘1 i Eryti‘moeytesz i 1333.3 33. 3100" ;T9:,~.gorattu*a

1 70 5.8 x 105 1b.). 102 102.0

to 9:2 5.; x 135 11+.o 210.0 103.!»

o o 13.3 x 1-35 13.6 m mm

o o 13.7 x 10‘5 13.2 132+ 10%.?

2 195 3.1» x 105 12.2 135 102.2

7 523 3.1 x 105 10.1 97 39.1

A“. A .....__ - A— A

73

MBLE III

33590233 0:9 2:372:31. rag-17331723 ( Era-mm; mo
5.23:) mars) cw Irr:::2,,w::;trurss FTJLE‘I‘IDI-i
09' 33;. gear”; 055' POLI$ECZMRIDE

     

._vmhw,w}unpwr.m,- u;m iwm-,
Rabbit : Time of a Total tier

 

IA :Per 3

L 13*. u 3' " ‘~ ' - "tr 0 . a

Rabbit :32 before 1m. 8,800 51+ 5,010 no 3,520

0.8 mg/kg

may m; 2-}hom’a 2,100 65 1,1400 33 700
6 hours 7,700 [*1 3.200 59 !".5730
2"} hour3 .0 Q. .0 an. '“

dead
a 318 £13 .

body wt 2} hours 1,300 82 1,056 1%- 182
6 lwurs moo 2» 1.050 76 353%
25 hagrs 15,200 1% 2,128 86 135072
#8 hours 10,100 #2 h,2%2- 50 53056
72 hours 6,500 68 “1&20 30 1,950

A

1Leukocytes to the nearest 100
zzrythrocytea to the nearest 100,000
3fiemoglobin.1n grams per cent (blocd)

l*aslucoso in grams per cent (blood)

TABLE III (continued)

 

 

 

 

“'T"- R A Erythrocyt@sz : Hb.3: Blood“: Tegporatura
5.3 x 105 lino 10% 133.0

0 o 5.1 x 105 1!. 5:: 2.23.0 105.0

3 231 1w; x 105 13.5: 5-3.1..0 103.0

2 10L. 5.; x 10” 1a.; 115' 3.03.3

8 10!. 5.6 x 105 13.9 139 103.0

0 o 5.3. x 105 13.9 101+ 103.1

0 o 5.2 x 105 13.0 90 102.0

8 808 w. x 10” 11.9 87 99.0

2 3.30 w; x 105 2.1 85 93.5

so

£313?

  

Dose 3 Day film of Countzi'otal Mmmyteszs

r5133 LE

1111 WM .;:1 11.2.1311 I ""1" :3 1:11:11 0111 mm:
H 33:33:. 11.1w i0 HUJJNLLJA :‘ 171}

I.) 1... (213‘ P0

ran-«ma

1410 5.1.»...

 

- -1 1 - W
131: before
2 mg injection. 15,800 1+0 2,720
2 ham-a 2,!100 62 1,968
2111 before
2 m3 injecfiion 13,500 6 1,110
2 hours 3.700 51 1’37?
3rd before '
2 m3 injection 9,700 3’11 3,293
2 hours 1,200 82 90‘.
- kth .. 12,700 65' 8,255
2 1.1;; 51:11 2.3331011 831-00 59 11,956
2 hours 10.700 70 7.1190
-. 51:1. «- 15.100 33 5. 738
7th harem
2 mg injection 9.500 1'12 3.990
2 hours. 8,700 1.5 '0,002
.- 1311. -- 7,100 1.:2 2,982

1T1th of rabbit. mmber b :- 1/63+O
amukocytas par 022122 of blood

gang
5.) LJJ‘.

MID
11.31333

   

i Mllu:..i' .. i. :" A‘V' '“

 

T5333 1"! (cont finned)

 

 

 

 

:35 3,808 ’+ 373
11. 336 it 919
91 16.835 3 51+?
1«3-3 1 5 295 270
(,1. 6,203 1911
10 120 3 933
3:. 1,318 1 127
1+3 3 9 3 30 1 82+
23 2.995 1 1'37
60 9 ,0130 2 303
53 5" 5’10 0 w

51. 11,698 "'

3.2

313133 "323 3:33 2333 :33 1:133 3. 03 I 0111 v.r103
Rut)? U?! :JiJ TO 1’11 11":3U‘JIJ I :JL‘J: 31;)! 0:
3. SOLI 0111 30L333.0333ID31

  
  

Before
injectian

23 hours

11 figure
2% hsurs
#3 haurs

72 hours

'1‘ 3'3 {E V

22‘; ”nu"'JL"huh“«wwwipPolkuorphnuclearL'
:.< LEV,Q$¥ an
6,800 5% 3,572 45 3,128
2,003 76 1,520 22 #30
3,53» 15' 1,275 85 7,225
12,530 32 h,030 8,500
9,000 51 3,590 #7 u,23o
7,200 53 3,176 #2 3,025

_____ .4.
w—v—‘u — — V —. 'W fi

12% opolysaccixarida intravennusly
zLeukocytas to tha nearest 100
33rythrocytes ta tha nearest 100,000
“Kb cm as gram per cont (blood)

531006 3133333 ina

1-3 per 103 ml or blood

f‘
.3“ -

7..“ng V (cantinuafl)

1‘5".- .— v'd» unqu- .3.» gnu-W

 

. . ‘
8 H133"F I Blood; I '1‘ B00t11

   

o o 3.3 x 10’"5 12.5 132 101.1
2 1+0 3.3 x 10‘3 122.5 232 101.0
0 3.1 1 10° 12.2. 183 102.1
0 3.1 x 105 12.1; 158 102.2
2 130 3.2 x 106 122.3 160 102.1
0 0 11.2 x 106 3.5 165' 102.0

DSOflUM $fifl§8fiflhfla MO fiOfluflfldfifi

ouawo non ma vauawuauo
manqnaonunaa and manhoomaahu neoa vacuums any on manhooxaaa Adana“

.flhn as 333 no 38 3»
scan vouoondoo coca» uauuaa oaanamom gun» «a ommnybo on» ”a“ nuanuomn

 

  

 

ax: «m 08.“. a 8m.» Q R comia m
“was .R 9m .3 an 85...." .R on 85:.” c
H . : .., .. a.” a A. ,.... 1.7: V 2‘... , . .. a: ‘ 2 Fa.» . ‘4‘ h... 4H. .1 . . . 3%.“.
sauna . ham. :6» < .a0 a Haney .uouuabm.lonnahqu «duck . gawk
ludddaaauuuuulaluuamuaaaHHMI. . . u .
EMF . 353 8.3

 

«do 53 .m
momm $8th manamoofifiom mg
”393 $8 mafia .8 BESMDS mo Emma mafia a

Hb mqmdk

an)
8

Tm LE VII

AuCLdTIh’l‘IJT 0F BOILID AID UL" in? 31.33 E. COLI 0111
BY In“ 0 ”‘1in7 LA I? {L’TIULA’L

     

 

 

 

‘poiledt dolled ‘W
Organisms Plus 3 0111 a 0111
1/20 I I
we .: .r
1/80 I I
1/160 4‘ I
1/320 I J
1/6h0 { -
1/1280 4‘ -
1/2560 I .
1/5120 - ¢

__ 4“... _._._ A

lsarum.prepared by injecting polysaeaharida of
0111 into rabbits.

TABLE VII (continued)

   

an“

PolysaccEarido : Boiled 8 0111

..........anr L J V V'Cér 5‘, M 1 fl 1W3...—

' 1/20
W

I
,t
1/80 ,l
g
I
,(

   

 

 

1/150
1/320
mm
1/1230 «- -
1/2560 - «-
1/3120 «- .

4.1.1.,Y'V“? 2??”01 “11‘1" ‘1!" AR '.-1.r-1 :1-1'1- ' (:1 '13..an {am-1.1:.“
I: 1.5.1.11; 500‘ 1 1.6.“. u U. Hu' '43..qu .1 :uv'o 51.1.}. 1 H Lu .1.

{Lube-ML! 1—9:” .aqus.) :3 z VUITI—a‘d) LII-215114. UGLI
0111 PG" 33.331111); .1‘?’ 3:32!

 

 

 

‘13 .fix : 1*Jj A:L::§T:TY W vw-_3»: WAT ;‘;:-ffi ::<*»:
a 3 s 8 3
Cow 3 Rabbit 8 Dog : 249.11 8 33139;: 3 Chicken
MAM 1 h a a z z .

 

0111 Antigara
1/100

.1

1/203 ;
1/4100 I
1/890 /
1/1500 I
,K

7

‘5 ‘k. ‘5 ‘k. 3\ ‘*. 1k
‘s ‘k. ‘s ‘k. ‘~ ‘k. 15
‘~ 1~ ‘k.“. ‘k “k. ‘5
‘4 ‘*. ‘~ Y~ ‘k. ‘. 11

S
g,
a;
s
1
a
s
I
8

Serum Control - - «- - ~ -

Saline Solution
contrOI fl . a a u n

033 (Antiserum) . n n q . -

C.)
C?

T 3L3

S '3er 14.)») h: 11.4.1.1:1‘1‘33 0...‘ Til: ELIAE’S «833‘. LARIDB
3|. 5 J
Afiiad ‘ U‘ DE. CULI Olll “f3 055
BE an“... “.3 131.390 0.3.3.33

 

' loLdLL‘b-Lnkibau :13]. Hy... .41. 81011 numLouul 01 4 t-Kl'd blood

 
 

E. 3011 t . .r n' 1-. a. , I'.. d . a fi"?l§i. Iemp
Antisera : 0:21]. a 05.9 a 011.1 {an-.102)
“_I _ I__;J_P0;gs.gzug:;&o: ,000 “a” 30

0111
1/100

1/200
1M0
1/800
1/1600
1/3200
1/0100

     

 

mxxxxxx
.1
xxxxxxx

M2800 . . ..
Op 3 1/100 - / I
1/200 - I I
1M0 - I I
1/300 - .l I
1/1600 ~ I I
1/3200 - I /
yam - ,z ..
1/12300 . - .

" 51:?3luu1JJVAQL} Jim-«11300

311nt1—0ra after Treataent with

E. 0011
antisara I

*3.
Lac

1"!
. .

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“4.— :QLLL J 1

0111

O

p

3

1/100
1/200
1/100
1/800
1/1600
1/3200
1/3100
1 2300

1/100
1/200
Mm
1/800
1/1500
1/3200
1/6100

\‘L‘s‘k‘ki

«2 4’7“???) a,“ «13; h

33;;J~3':1_...i 5.1.1 1.1;... $4.. J. wanna
) 613554;; :H; _: .1 Tilt-1f; 11'013111'550‘ {)53
0:1, :13; {.3 L.) L 33‘... 4 .1.?

13_IJTS

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V311 .JLJOJ walls 05"

3M }53

303) follguedcfllll followed

\K'k'k‘k‘k
I‘ki‘u‘k‘kk‘k

8
I

\‘k‘kk‘k
l‘k‘ls‘k'k‘k‘k

I

90

O'DQ‘ MAI-(I’M M. I in YH“? A “. Ti: ‘5 ' 5-09%. g («a aqr“ mono-5 “gm” ~51,
311...... i. “Ix-JUL)... A “L XL»): :1... u:.LJu...UIaJ \J.‘ ust£§-~\3U.LJ.w

8:;f3'1LluJ his... 11:" In.) lJJLOqu LJJ ».LtI4):; ALL J33::£$

{9:331 £1. JJ’ 31 0111 331:3) OJ").

 
 
  

       

055 Polysaoczarida

8
O
I'Qfi"“’"‘ 19.4.
8
3

«unicoral Hangs Itination of

Spec _f133 0111 Polysacchkrile
for I

Antigen :

   

Huang» 3 A Shaog

-r

“11% = 51mm...—
om 1,600 , 3.200 - .
3111 G’W 12,800 ‘ -

 

055 ~ . 1,500 3,200
0353 - - 1,600 1,600
Controls
Complemenx* . ~ * '
Saline only ~ ‘ * '

1HAM)

91

T 132.3 XI (cozx’cinuoa

 

’ - ' ' " - 3‘32... “3--....W
30210157313 of 0111 Poly- 3 liomlysis o; 055 3’0" «-

 

 

 

saccharilo Sonsitiged a sacch3r1io Sensitizod
3. 2:33:22th 3 x 1 _ v Trytfzrar.“ *1 3
a 8 3
431an ‘1 fine: 2 Emma : 1 {331292
. .25. 600 o g
- " " 5'1. 200

o o a 51,300

‘1' :73L:3 XII

P‘RAJVIPITIII “1:313:13" O? 337171;,353.’ ,PIII'JP UNI) J “LUZ" A
301%} 33??) U“ 5;; =.T131') 0111 “TD 0') "'5' in. CiliLI
U"::afi|~::g 1;), “1:1 221*; l‘JIJLJ :JJ‘L.I~LJ~I

FJTI'I ' T3 1‘ :0}! 01.11

 

 

02 -

A —-L .._ A4 . .-

uv w‘w— w w w vw w—v- v— w—

lantisarum made from living culture

azmtisar‘m made from boiled culture

93

TABLE XIII

smamrmrmrz or mm RED 23mm 3:15.51
mm was 3911.29 arm mas-Iran 3231643
03‘ E. com: canvass m)
Pazxsmcmamus

 

 

 

.‘ .
of: I Polyuo- : Polyno— : Futratsa : Fun-at.
3 char“. 1 chum. I Heated : Haunted
055 1/100 I I I I
1/200 I I I -
Mm I I I -
1/800 I I I «-
1/1600 I I - .
1/3200 I 1‘ . .
1/0000 . - - .
0111
1/100 - ~ ~ .

 

1am typo 0 blood 001.1.
2mg“:- from bmth cultural of orgmm
359:. containing on agglutinnu

TABLE XIV
AGGLUrIvarIaxl a? UKHQATED 0111 339 055

Bo GULI 033133315 BE 5333 Cuxraxxxxu
POL33333333193 33333323135 03

E. CDLI 0111 ffifl) 0‘55

       

having 3 of unheated : of Unheated
t

Imagining : W w 1 9.13 $01.11....
0111

 

03 I a.

0
55 03 - 1‘

v w— "— ‘v—v—Vv— —-—v vv w

lzsgglutination by the 31136 technique

m1r\?1“§ ‘f
1.3;. Julia AV

1

0111“ U 3“»; 1"“? J. Inf-:3

3 53; JTI‘ILTI 1‘? 0‘3" -0” 3') 337'” "7? : 
3

‘J
' Z—Z‘ITE’E'TS

 

sew-m : iwgmuimation t .- tidyim
wi“h

   

of

 

lCulture boiled for one hour at 1003

[J
9

A graph to show tha leukocyte and glucose
resnonsa of a rabbit to the intravenous
injection of 0.k mg of the polysaccharide
from 0111. Rabbit number 1, Table II.

Fimme 1.

 

2. 8 oo no on 9 9. an on ma cu m. o. n o
_ _ _ . fl _ _ 4 _ A d 4 _
Lu .3
If? 4.0.?
L0 11°”
30.5 1.2.0 mm... C»... x. mutooxafll a
33m 5.2.0 00. cum 3002.5 .3211..- < 1m .bo
lllllllllllllll 1/ 1.0. .62
III/I {
I/I/ +~Té§
[I], u
I'll," "'ll" u 1..
1, .1! o!
, .
, .1238.
, .
, .
,. .12 .52
. .
C 18.68
..

 

 

Figure 2. A graph showing the leukocyte response to
injections of the polysaccharidas from.0111
and 055 in two rabbits, numbers 1 and 3,
Tables II and III.

99

d?

mmaoz

on 3 00 an on 0.0

.360 m0 zopomalelu
2.0 no zo_._.ouaz_ll

_.,Q
u—dbn

w

ohbnwnu—

 

00
'N'W'O 83d (EOIX) SBLAOOMOB'I

 

00018 :IO

1.“
.17.!

F1530 3. A graph show :1”: the manhocyto mag-30300 to
injection of 0.3+ mg of polysaooimrido from
0111 0023;231:021 to the polymrpnmclocxr
lemiocyte response to 1:210 5:11:30 injection.
Rabbit number 1., Table II.

lOl

mmaoz

 

 

 

Oh 00 oh no on n0 00. on on on ON a. o. n o
Wfi _ _ q _ _ — _ A A _ — d d x,
\\ II
x A
1 \
[III] \
II, \ I
IIII \ 1
/ \
/ \ I
/ \
/ I._
/ \
/ \ I
// \\ l
/ \ I
/
// \\ I.
// \ I
/ \ ..
/ \
// \x I
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Adam, 3.1927 Eur Trago dor baktoriollon 3031910510 fior
50’onanton all oaontaron intoxikation. Jo. RindorRoilk,

12333.10.

Alsovor, J. B. and Ainslie R. B. 1991 You'mothod for pro opa~
ration of dilute blood plasna and Operation of oo3p10to
transfusion service. E. Y. State J. Red., El, 126.

Avery, 0. T., and Gobol, E. F. 1933 Pnou3ooooous ope gifio
substance. I. J. Rxp. Rod.,.;;, 731-733.

Boo :too, R. 1999 .110 of Specific strain of 0011 bacilli in
o 133:1: gastro-o ‘ ritis of inf:1nts. A.R.R. GiJS)Wrt1
11030;..3131 and $1.0:1. ton 381313.13. liaandsohr. V00? £13110?"
"onoooR3n30,.12, 195-212.

mzarl 5. R. H., and Or sRov, F. 1931 O-grouping of Q. gall
ostralno isola ed from 0-809 of w’h to scours. Rota path. at
3211131'00101. SQNMO’ a, 87’93.

Bray 1903 19010 tion of antigenic ally homozonous strains
01‘ “M11113: 3:111 3.39.11.13.11 from mmr diam: m of

iflffiufvS. Jo E01311. and £38. ’39., :2. 239‘21'P70

RR, and Boavan, T. 3. D. 1948 Slide agglutination of
~*2;133.3111 vor. 1-.'? ‘133; in suaaor diarrhea.
J. P3131. 0111 35.1090,

  

Brood, R. 3., Rurroyz 3. G. 9., anR Kitchens A. P. 19MB
Bor:.'oy's Ranual o. Deter 31notlv0 Bacteria o. :y. Sixth ed.,
11111333 and RilRins 30., Baltimore, R3.

Buddin.3h, G. 1., and Dodd, K. 190% Stou.titis and diarrhea
in.:1nts caused oby a hithorto unrecogniz 0d virus.
J: Fowllat., 9',M-109.

Cathie: I. R. B. and iIaolarlano, J." 0. R'. 19:1 Inoilonoo of
93*.ori' o 0 group 111 in sporadic infoRtila gas tro-

011t§3rit130 grit. fled. Jo, 11, 1'90 2-10019.

Christonoon, 0. and PlorinU-oooron on, K. 1996 Ronlngitis-
and o onzalltis—llRo 0133303 in tbfl oraln of infants with
covoro gastro-eotorltls. .Roto path. 0: microb. $0333.,
2.3. 39 9.901).

U1

'1.)

.1537’, If. 301”};02' 1‘1. 3:7. $3.130” 1 $3., a.“ .C} 11313, G. 30,
lfiwi L‘ ~i #1: OR “GonflQal d13?:q Eda 13.33u0rh1ty hflflfitfilSo

DaoR,: f' X. 19%? Problems and orrora 13 assigning causes of
@131 food poisno .3. Am. J. Pub. Hoalth and the
1:12:1qu 33'11‘51 v.1, fl, 3 10-3

 

‘1’ f‘fivcx-’ CI 230,3118'1‘77‘1', J. I3" 8.33:1 13411.52‘311, £2.
19F771oed1ng tests on 11333 yoluntesrs with entoroooooi
and tyraw no. J. Inf. 313., 23, 131v138.

Dolafield, R. E. 193% Rlooa-au"n“ changes and toxic effects
n“o4“oeé in r: bbits by certain froot103s darivol rm
--'-"""‘k.’?r§1 : “A?“U‘ A“ no Brit. J. “Igptlo 5(1t.1., u, 138‘137.

h.‘

 
 
  

   

    
 

Dennis, I. 3. anfl Sen Izjian, R. 19?;9 Leuoooidal activi y of
t;-'?I101{1 :iitrateo {1.13. to. :Iyg'o. .1.} 21.3?! )0

Dragpor, T.3 and Brown, 6. R. 1995 Staphloooo cal onto ritm
in «21.11.1311. Austral. 139:1. .1., 1., 11394174 .

Dean, C. P.., "orfos, L. 8., tar..n S., and 1305, 0. W8
A stutIy of tho mac E1:.n133 of nus 9199t8 iflduoad leuKOponio
Md le*”ooyt11 states with special roforanoe to tb o rola~
tive roles of liver, spleen and ban :1.marrow. J. Exptl.
“ado, £52, lb?" 4.3?33.

.LW* 2 . 1935 Clinical Implications of Roflarn FRysio-
logic Romatology. Breed Publ. 80.. St. Paul, H.nn.

 

I?» ”An 31')

Rad 5.13,, T. 3.19,?.. Tho status of sorou

u “(L— 1.; ’ i . ;'-., 1‘ f‘
logic ty313 in tho f3311y Rnterobao toriacoao. 13. J. Pub.
1/
l1‘331th’ J, ‘ "35.673.

orioh, T. 133-5 Die DRr maahtorfien Sauclings und Ihzo
Roxie hun.on.13r Physiologie der Vordauung. .ordinand ano,
tattgart.

m

12.33?
C'
U

Favorite, 3. 0., and Rorv~n, ILR.19%2 Injootiou of atoxlo
antigon from R. Lguugsg 1J. Clin. Invest.,‘al, 539-199.

For JasoR! R. R. and June 2. C. 1992 ngori3cnts on food~
in: au alt vo untoors 1 th T11;93’“‘*gigg;; 111 3R,a
coliform or 13133 associated with im ant d arrhea. Rm. J.
Eiyg.,.ii, 129-139.

F19h0?."u¢ 1950 The hemfgjlutination of ngmggb no"-
U.35130 Wt 31. J. -xptl. uiOlo 338 “3Q. R31.,L,‘39-511.

191h- Eur Frags dor funrtionollen £113 diagnos tiR

:‘Z‘Gy h.
131133153 Adrenalin. fitscRF . d. 398.. 13311. 2.01., 3:

;--‘~.

Frey . and Lury, S. 1913 Adrenalin zur funktionellen
Milz diagnostik der Milz: Untersuchungen.an.klinisohen
Material. ZtSChro f. d. 833. ’ Exptlo fled. ’ 2’ 250-258.

Gale, E. F. 19%h Castro-enteritis in infants. Brit. Med. J.,
1, 631-632. ,

Giles, 0., Sangster, 6., and Smith, J. 19%9 Epidemic of
gastro-enteritis of infants in Aberdeen during 19h7. Arch.
D13. or Childo’ 2E, “5'53.

ii, and Sangster, G. 19%8 Outbreak of infantile gastro-
enteritis in Aberdeen: Association of a special type of
Best. 9.911. with infection. J. Hyg., 55;, 1-9.

Gordon, 1., Ingraham H. 8., and Korns, R. F. 19%? Trans-
mission of epidemic gastro-enteritis to human volunteers
by oral administration of fecal filtrates. J. Exptl. Med.,
.fifi, h09-h22.

Hamburger R. 1920 Die Behandlung der Toxikosen des Sauglings
mit coiisemm. Jb. Kinderheilk, 23, 25-32.

Hays, L. 1951 Specific serum agglutination of sheep erythro-
cytes sensitized with bacterial polysaccharides. Austral.
J. Exptl. Med. Sci.,‘g2, 51-61.

ii, and Stanley . F. 1950 The preparation and
prOperties of somatic antigens from c a {£211.
Austral. J. Exptl. Biol. Med. Sci., , 201-211.

Hayworth, N., and Stacey, M.- 19k8 Chemistry of the immuno-
polysaccharides. Am. Rev. Biochem., 12, 97-11%.

Holzel, A., Martyn G. and Apter, L. l9h9 Streptomycin
treatment of infantile diarrhea and vomiting. Brit. Med.

Jo, 2, “M57.

Jensen C. 0. 1892-1893 Ueber die Kalberruhr und deren
Aet ologie. Monatschr. prakt. Tierheilk.,‘22, 1892-1893.

June, R. C., Ferguson, w. w., and Worfel, M. T.
Am. J. Hyg. In press.

Kauffmann, F. 19hh Zur serologie der Coli-gruppe. Acta.
path. at microbiol. Scand.,‘21, 20-h5.

‘52 71,10%9k7 The serology of the coli group. J. Immunol.,
: ' 0

 

107

Kaafrnann F., and Dupont, A. 1930 “Jezorichia strains from
ile epi3onic gas tro~enteritls. Acts path. at micro~
E3101. 4.31. "C‘lnelo’ £12, 5);..-)JI‘F.

19 ”‘5th

-w__ , 1951 Lnterobacteriaceae. Copenhagen:
Junxsgaard.

xeo 4:h, a. v. Ianrtn, E. A., an] Iarburton. 3. l. 19“?
a39;33514£131ns of tlxe JaenOphilus group. 3ature.‘LflQ.
D31‘a334.

‘
#Afl- __.....+_.__ .nau-w-m. 19‘,3
alsorution of Dict3rlll ralysachJr1333 to erythrocytes.
EIthme’ m. 6.17-3;{30

Kietel 11.. C. 1930 Occurrence of cold and streptococcus JG
Iggla in infants tith gastro-enteritis. J. Inf. 915..
L3. $.19:2:..)+.

Kirby,‘w. H. J. 1931 Kanazglutination reaction in strepto-
coccal infections and acute rheu.1tic fever. Free. Loo.
42.359111. 1.1101. and 3.923.. 23’ 5190522.

Kirby, A. 3., $311, 3. G., and Soacklo oy J. 1930 fieonatal
”1511311051 and VOiltl. 5". Lancet, 2’ gall-207.

finipschilit, J. 3. 19+) UnJerso:eloor over Coligrunoons
L3rolo_:ie. Englisiz quJer. Copenhagen: Jrnold Lusch.

Krake, 3.1anJ Pzarkcr, :‘. P. 193% The etiology of granu-
13:.enia Lith.part‘*ulnr reference to tne draggs cont.i“inu
the benzene ring. J. Lab. and Olin. Jel.,4.2, 739-813.

Kravchonko, A. 2., and Sakalov, II. I. l9¥6 Adsor 'ion of
specific bazterial no? -ysac sizariles by hunan rytnrocytos.
21.14... ..:-r0,31'0)101. (437133 1.1101. 1;...‘11‘11'1333101 3..., 12’ lO‘l‘J.

Seen in abgto only, '3. A. a, 742's :, 1N7.

Lealin; article. Lancet, 2, 393-395, 93?.

Lexoel as, P. A., 1;?11ntivan, J. J., aha Orchard 3. u. 1933
35133413 diarrnoa of tim .Q“bOTn: A rower of two out-
b'?08:13. All. J. 311.). zioalvll’ ll, 12: 13-12:?3.

Lizjht, J. 3., ani Loses, 3. L. 1933 Jtndies on epiieJic
hiirrrzea of the newborn: Isolation of a filterxble virus
causing diarrLea in calves Am. J. Pub. Lealth,‘33,
1%}1‘14J20

Lovell, R. 1937 Classification 3? :_g U0 $;;.? ; fron di-
seased calves. J. Path. and 13:13 ., “338;, “.135- -- 39.

108

Lovetté Egogi 192+ £121.31? .JlOCYtic angina. Jvofivo' E539
. ' c...

Lyon, 6. 3., and Folsom, T. G. 1931 Ioidocic diarrhea of the
newborn. Am. J. Dis. CLild.,‘fiL, 327oh33.

Lagnuscon J. II., Lourcll, 6., Friccll anJ Vcrncr
1970 Kuraomycin treatment of in? antilc diarruca or 13 voait-
ins. Brit. Izod. J.,.1, 1398~1%OO.

Itartyn G. 1939 Staphlococci in tho nowborn. Brit. fled. 3.,
”510.71%.

Joycr, O. 0., and Tahowlis, E. u. 193% A ropcrt of failure
to produce granulocytop nic vitiz bacterial toxins.
J. Clin. Invcs.,‘13,h37~hha.

Liddlobrook 0., and Duooc, R. J. l9+3 Specific scrum ago
glutinat on of crythrooytoc sensitized with extracts of
tubercle bacilli. J. xptl. Mod.,.§§, 521-523.

Hodicc, R. 1., Furs coon . L., and Duccy, F. F. l9)2
Epilcoio in Tactile d arr Eco cflsociatod with ggjngzLflmha
col; 111, Bu. J. Lab. era Olin. Mod.,‘32, 1? -2-127.

Jargon. H. R. 1930 Preparation of antigenic material in!
duo ng lcu=30ncnia from _3thgcl 5.3131233 cultured in a
synthetic mouimn. PPOQ. ~06. «X9131. .3101. t’iGdo. E3. 529‘

M3

Morgan., 3. T. J., and Partridge, S. h. 1940 StuJias in
iamlnoohcmictry: II. The fractionation and nature of anti—
genie matcriall isolated from‘aggg. .dxficagzziga (SL133).
Biochom. J.,.3¢, 169~191.

J. l9+1 Icmunolo:ica1 craportics of an.anti-

waic material isolated tron 323gugl.3.txnhggg J. Io-
AUHOIQ , a, 1 31-130.

Huchin, R. 1990 The bactcriology of infectious diarrhea.
3.3853. J. 3.113 trill... 2,161.1.“320

J. 1939 Studies on arccolon bacilli. Austral. J.
“Lichanj -’ {1113110630 5501.. 22, 43-01352.

Lctor, 3.,1+ owls .435!, C. 3. 1930 6~hqgictig.g211 coro~
typo D #33: 0c currencc in intestinal and ro .piratory
tract: Cultural characteristics, pctho :onicitsr , cons itivi-
t? 130 antibiotics. (1‘00. {203. #123031. 2721,431.. and 3.3410, 25,,
531*‘1‘370

 

109

3998?, 3. “005’ U. R. Simmer? C. 5.7., and a‘lurflock, £1. :1.
13.71 91:11:35,? on etioioznr, 9:31ng 121010351 and antibiotic
therapy 0. infantile diarr’2193, with particular reference
to cert3in serotypes of “MM-"1‘3“ 9.9.3.1- 9.2.3. J. i’uo.
39911313, 311, 1990-1395.

,Bartrnfil 4, L. F., 2-31: 3. fig, 2:31.11'10052 :1. R0, 9.11.51

-9‘.>9:3..:..3 C. Li. 1932 Sta-3193 0:111:31;ng nation and
3.9:311M -3 by . 2‘39 .99 .- 31-3111 antisera. J. Liz-pt}... £193.,

22, 1-15.

-9 , M. 1952 Demonstration of 911391;" 391:; 512111 05’) and '
0111 antigens by means of the lamnbztimuion teat. Fran.
303. ESXQ'Cl. 7.3101. am} :‘:Gdo’ 22’ 2) :“257.

2.3:": Do, £1318U3si1’ H. Jo. m Bertram II. F. 1952
InhibiEion of bacterial (A99321’1313 3:19.11) moglificationgl of
grytIn‘ocytes. PPOO. .09. fixgtlc 3101. m3. iifid.’ 122,60

Q

 

   

331W3:51,1u J. 6221513313803, L". U. 19,)3

- -' 1-mm-3331utin9tion response of 9391!: 901119--
{3:39:93 to inmated 1511;; 9“ a £311 055139. Pro-c. Soc.
E}I?)tlo 3101. and {stQ’ 32, 219-31“ 0

 

 

011153221, In; AvinoryifS. 3.; and 392193931951, J. 191+]. Lenka-

99:11:: 90 ion of d faron microorgani— as. J. Lxmnol" 111,
3519373.

Orslzov, F. and Boklmari, S. 3-1. .1. 1951 0.3391191“ of am
$21.19. strains isolated from 09398 09191129 scours.

.999. 133.11. a» 13.1"?0b101g SGWO’ a, 3?3"373.

P9519: 9, 1.. :4. 1-1., and Cook, 6. T. 1950 A specific sax-0105:1991
typa of 311.519,]; found in infants' home in absence of
epidemic diarrima. Brit. Med. J... 2, 1929195.

RBiBtPiQA. Ho, and. Topley‘, W. U. C. 1931* lWZing fractions
{gale-£336 from am W.Br1tu J. Exptl. Path... 1.5,
3’ o

Pxeiuann, H. H.,Pr199, A. hi. and {$03.30!}, J. H. 191019 negative
results in studies of 931392119 diarrhea, nausea and 99311;.-
1:33 of unknmm cause. Faro . Soc. Exptl. Bio]... and £991.,

55’ 233“23"+0

Rogars, K. B. 1951 Spread of infantile gastro-ontaricis in
a cubic 19d ward. J. hm... 512, 519-522.

110

R0 are, K. 3., and $033133, 3. J. 19‘71 Intor-hosdital cross
11—7faction of epidogic inf333 L113 gastro-anteritis a933ciatad
with type strains of 133;..QQLL. J. Ly3.,.fifl, 132-161.

3_3 Roe u181', o. J. and Gerrard, J.1943 Chlorann
phr 111001 in trentzzont ofnf..nt113 3.13 tree-91* teritis. Pro-
lifiimry report. drift. “0'3. Jo, a, 7/u‘7‘3‘3.

Sakula, J. 1993 An nub breaL of gastro-entaritis in the nav-
born. Lama‘s“? a, 7);; .7JO o

aavag L. 3., Lachnan, D.“ 3., and Smolens, J. 1933 ‘Eho 130-
la on 01 tha consonants of afitono cal nucleoprotoin
in sorologie ally active for:. J. u o.nau.,‘;2& kgSah36.

Sovitt, S. 19%3 Th3 et10133y of infantile enteritis. Pros.
305’. 303. {$93.10, J, 1-3}-1_.)0

Sb““pe, fl. 3. 19W Invas tigations of figgggfigqggggg f3qggTia
ard its as iluiOfl‘Lita g.stro~ent3ritis. J. dy3., 3333.,

:2, ‘33)" 1:?

th, D. E. 1997 Stu£ica 03 thn patzo 3n133tf 3? 331
fram bovine sources. J. Lu3u1. L3G.,}1_, 199-131.

3 Mi *;1, J. 1999 The association of certain types (.\ and D) of
33:.jggflgku1tn infantila gastro—entort is. J. H'3.,.&2,

“'- r- 3..)

t9?“

_, u3110¥f~".5y, to x... and 3991r3, 9L. 11.1350 In. {inu110
WuurO'GQua“1CL3 mith5p33131 reforenca to th specific
seroloaieal tvne 0-95 Bg.flo (Data tyne) of 2333. 39:1.

J. “ya-..33. “73.433.

3

‘p

 

.D

T. 1&3219 relation of the caps ular 3333*1330 of
.3321; to antibody production. J. prtl. Led.,.33,
21‘39U-

_- .3. 1927 Studies. on time pati-o; en131ty of P333. gall
f1 ‘01:! bovine SOUI'“GS¢ 1.39121. 319110, En, lul’lflo

8:1

'-

9
3”,

04;

3a 139 on the pat.079313 ty
3. J. 3.5;:3t1. 2:03., L1,

 

3 , and 9ry3nt, u.1927 St
of ‘13‘ 333;; from bovine source

, anfi Litt e, K. 3.1927 Studias on tha patho- 3
:nwl' t? 0? ’l“*- “f ro:3 Dev 33 souraas. J. Lthl. Ladoo
L.,, 13>-131.“

Eta even? on, J. 8. 1990 ”99*3‘92l1 D #33 onses of dizzrrhoa in
adults: J; “it. :94. I)...” 13’ 115-133.

 

111

St3v3320n, J. S. 1992 Furth3r obsarV3ticns on tha o~~nrr3n~e
o- $13.111LLDJH33 in adult £9333. Brit. fi-33. J.,‘£, 12¢-
0

u.

T?

Stafl‘t t,” Q. A. «Mi is. 21.. £1113 3:11.013 A3. Lia. 193 3
.OLationuulps of Qiiform or33nisus. J. P3: t.,.31, 391-410.

T31, 3., and OlitsLl L. 1993 P~33tians fran anti,3n of
«H “av-2m Iuvnsn-fin H. J. 113111133... F?” -g, 33’ML11.

Taylor, J. 1931 nis3ussion on infantile gastro-entoritis.
Proc. 30?. 333-3. i 3a., 1:1: 2'13“}190
LA___- Pow311,3. 1., and ¥right J. 19' 9 In antila
diarrnaa and voaitin5;. A clinica and b3~t3r1010113a1 in»
V03 tifiatlon. liritt 230-2. JC’ 3’ 117-1230

Vahlno, G. 1995 Serological typi.v of the colon ba3taria.
Leta path. at micrablal. 53333., Supp. KLII.

wallisk, 3., and Stuart, 3. A. 19h3 Antigen 313 relationships
Of at. 223.1. J. Bflfit:’ :13, 121-1? '90

laraurton, M. F.. Eioogh, B. V., 1n3 11113.33, 3. i. 1999
A homagglutination.test far the diagnor is of influenzal
"'1 “jStls. £101. Jo EEUStralo’ 1’ le‘l33.

fl 3. W_ , and Fisfer, S. rho haeuazglutinin of ’39-
lggngilu§.n1z§uga;g. 1951 Ans tral. J. L.xptl. 3101. and
heal. ”01., £2, 3:33;)‘272.

 

'N

W19 13? , 2:. a. ., Luhby L. 3., and 33h311,1.. L. 1950 The
a3t10n&ofe “ y;3as in bandqluuinatmu aye. 2935. J. Inuunol.,

394+
31531, F. 1395 Emil. Soc. fiéd. 869. do Paris, 13, 539.

€130.2an, D. K. 1931 T220 inJucti on of lymphocytosis and
lymphatic hyperpla 81a b" moans of a arenterally aaminisw
torod protain. J. prtl. hed.,,§3, 99-510.

Wong, 3. C. 1933 Polyaacchariles of 3n33,r nsulatel bacillus.
Ejroc. Soc. ‘1? ~E3t1. “’1010 {no :Aeil’ 31’ 107-11- 3.

U: rugbyz G. 1993 Investigations into the antigenic structure
of133*. 111; isclatQJ from calves. Upgsala: Appelbergs
trygkevia.t* obolag.

202 a a, H. 1932 1:2.rane reactions botwean polysacahar dos,
and some bacterial antisera. . Exptl. L03 ., 555 3) 3-330.

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