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PART V. PANEL DISCUSSION: NEW KNOWLEDGE OF THE ENTEROTOXINS PRODUCED BY ENTEROPATHOGENIC STRAINS OF ESCHERICHIA COLI
DISCUSSION : SOLUBLE ANTIGENS OF ENTEROPATHOGENIC
ESCHERICHIA COLI
K. A. Bettelheim * and Joan Taylor
Salmonella Reference Laboratory Colidale, London, England
That certain strains of E. coli will cause enteritis has been shown by feeding living cultures experimentally. As long as the number of ingested organisms was large enough, the effect was produced in both infants and A parallel method of investigating enterotoxic effects has been the use of the ligated rabbit gut loop first described by De and colleagues and applied to the study of E. coli by Taylor and coworkers.4 By this method it was shown that while certain serotypes derived from infants with diarrhea produced dilatation of the rabbit gut loop, the factors responsible were other than the classical 0, K, and H antigens. Whereas chloroform-killed suspensions of strains of E. coli derived from infants with diarrhea produced a positive reaction in the ligated loop of rabbit gut, similar preparations of antigenically identical orga- nisms from healthy infants did Using strains of E. coli of non-human origin and testing their enteropathogenicity on both homologous and heterolo- gous animal species, Smith and Halls concluded that several enterotoxic factors were responsible.6-*
Further work suggested that some of the factors involved in enteropatho- genicity might be antigenic9 This study is concerned with the relation of antigens derived from the ligated loop of gut and extracts of E. coli cultures to sera derived from various strains of E. coli.
METHODS AND MATERIALS
Preparation of Antisera
Two types of antisera were prepared in rabbits: The first was made by the method described by Ewing for the preparation of K antisera.lO Intravenous inoculations of cultures killed with formalin were followed by inoculations of living cultures. This antiserum was known to contain both 0 and K anti- bodies and was known as L or “live.” A similar series of inoculations was used in making the second type of antiserum, but the organisms were killed with absolute alcohol. Cultures were grown on Hartley digest broth and harvested into about 1 ml sterile saline to which 100 ml absolute alcohol was then added. This was shaken, allowed to stand overnight, and then centrifuged. A sample was tested for sterility and the remainder suspended in 0.05% formalin in physiological saline buffered at pH 7.6 to give about 5 x lo8 organisms/ml.
* Present address: Dept. of Biochemistry, Imperial College, London, S.W.7, England.
301
302
The suspension was again tested for sterility. It was known that this method preserved 0 and K antigens. This antiserum was known as D or “dead.”
Annals New York Academy of Sciences
The list of antisera prepared is shown in TABLE 1.
Preparation of Antigens
Two different kinds of material were used as antigens: one was derived from the contents of the ligated loop of gut and the other from cultures of E. coli.
If dilatation of the gut loop occurred, the resulting exudate in the lumen
TABLE 1
ANTISERA PREPARED FOR THE STUDY OF Escherichia coli SLUBLE ANITGENS
Intestinal Antiserum Inoculum Loop Reaction Code Name Strain Inoculum Serotype of Strain
~
E65L E65 L * 026K60H- + $ E65D E65 D t 026K60H- + Aberdeen p Aberdeen p L 055K59H6 + Aberdeen p Aberdeen p D 055K59H6 + D2101L D2101 L 01 11K58H 12 - § D2101D D2101 D 01 11K58H12 - E122L El22 L 01 11K58H2 + D5301L D5301 L 0128K67H2 + D5301D D5301 D 0128K67H2 + E21L E2 1 L 0 128K67H2 -
L
D
* L. = living vaccine. t D = dead vaccine. $ f = strains producing dilatation in rabbit gut loop. 5 - = strains failing to produce dilatation in rabbit gut loop.
was used. When the loop contained no exudate, the mucous membrane was gently scraped and washed with 2-3 drops of sterile normal saline.
The bacterial antigen was prepared as follows: Previous work had suggested that cultures grown in a milk medium produced the maximum effect in the ligated loop test.ll As an opaque medium cannot be used in gel diffusion, a medium was used that corresponds roughly to the chemical composition of milk. This consisted of 20 g casein hydrolysate and 30 g dried whey made up in distilled water to 100 ml. This solution was steamed for one hour, filtered, adjusted to pH 7.0, and finally sterilized by Seitz filtration. For use 1 volume of this stock medium was diluted aseptically with 9 volumes sterile distilled water and bottled in 100-ml and 5-ml amounts in bottles with tightly fitting screw caps with rubber washers.
Bettelheim & Taylor : Discussion 303
The stock cultures used in this part of the work were those used previously in an earlier study of the ligated They had been stored on Dorset's egg medium. Subcultures were made into 5 ml amounts of the whey medium and incubated at 37" C. for seven to eight hours. The contents of the bottle were then transferred to 100 ml of the same medium, which was incubated at 37" C for 16 hours. The metal caps on the bottles were always screwed down as tightly as possible to reduce gaseous exchange.
After incubation, the cultures were centrifuged at 13,000 r.p.m. to pack the bacteria into a tight plug. The supernatant fluid was discarded. The plug of bacteria was transferred to 0.5 ml azide buffer (0.02 M sodium phosphate buffer at pH 6.0 containing 0.01 % w/v sodium azide) and shaken mechanically for one hour at 37" C. The suspension was then centrifuged at ca. 1,000 r.p.m. for one hour. The clear supernatant fluid, i.e., the antigen, was kept in a screw capped bottle at 4" C until wanted. All antigens were tested five days after preparation and some after 18 months storage: the results obtained were iden- tical.
Immunoelectrophoresis
An ex-Admiralty Rectifier Unit, des.95, Ser. No. 158., Patt., 58754 was used as a power pack with a Shandon electrophoresis tank. A volt meter and milliammeter were included in the circuit. The experiments were performed on glass microscope slides, prepared as follows: Ion agar no. 2 (Oxoid) was added to azide buffer to give a concentration of 1.2%. This mixture was heated to boiling point with continuous stirring. After bottling in 100-ml amounts, it was cooled to about 60" C. A few drops of the agar were spread on the slide and allowed to dry and then 5 ml was run on to the dried surface. Treated in this way, the film did not slip off the slide. A trough 1 mm wide and a hole 1 mm in diameter were cut with a L.K.B. Gel Punch set, the holes being 2.1 mm from the trough. The antigen preparations were placed in the antigen holes and the slides transferred at once to the electrophoresis tank. Whatman No. 1 filter paper soaked in azide buffer was used to establish electrical contact between the slide and the buffer compartment. The power pack used did not permit of control of voltage or amperage, but when eight slides were put in the tank in parallel and the current allowed to flow for the standard period (two hours), the voltage varied consistently 3 10-320 V and the milliamperage, 15-23.
At the end of two hours the slides were removed and the antiserum was added to the trough. The slides were incubated at room temperature for a week in a humid chamber and were examined daily. After washing for a further week in three changes of azide buffer and drying by a warm air current, they were stained with amido black B. This stain was dissolved in a solvent consisting of distilled water 5 parts, ethanol 4 parts and glacial acetic acid 1 part (all v:v:v) and the same solvent was used to differentiate the stained slides. After drying overnight at room temperature, the slides could be handled without difficulty and kept well when stored in boxes.
Preliminary tests were done on the exudates from gut loops to determine the pH at which separation of the precipitin lines was optimal. Between pH 4.0 and pH 9.0, it was found that pH 6.0 gave maximum differentiation.
304 Annals New York Academy of Sciences
RESULTS
Material from Ligated Gut Loops
When the whey casein medium and scrapings from gut loops that had not been inoculated were used as antigens, no precipitin lines were observed with any sera used.
All material from the gut loops was cultured before testing. Material from uninoculated loops was sterile. The inoculated loops consisted of exudate, rabbit cells, culture medium, and the test organism in pure culture.
When an exudate was tested against an L or D antiserum specific for the organism that had been inoculated, a strong precipitin line always appeared on the negative side of the antigen. If the same exudate was tested against anti- sera prepared from organisms whose antigens were unrelated according to the international antigenic scheme,12 precipitin lines were absent. This distinction occurred irrespective of whether the antiserum had been prepared with living or dead inocula or whether or not the strain had caused dilatation of the gut loop. This precipitin line was therefore assumed to be the somatic (0) line through it probably represents surface (K) antigens as well. There was never any suggestion of a line relating to the H antigen in these preparations.
On the positive side of the antigen well several lines appeared, some of them at a distance of ca. 1 cm (FIGURE 1 ) . There was no obvious relation between these and the known antigenic structure of the organism in the exudate and the known serum antibodies. As an example, an exudate containing strain E65 with the structure 026K60H- was tested against an L serum prepared from strain El22 with the structure OlllK58H2. Two lines were formed on the positive side in spite of the fact that the antigenic structure of the E. coli strain and the antiserum were apparently unrelated.
One of the many lines on the positive side was of particular interest. This was formed when material from a dilated gut loop was tested against the L antiserum prepared from any strain of E . coli that caused dilatation of the loop. This line will be referred to as line 1 . Line 1 was never formed by any exudate when tested against D sera, nor was it formed by washings from a gut loop that had been inoculated with a strain of E . coli that did not cause dilatation of the loop. The results are shown in TABLE 2.
Extracts of Cultures
In the first experiments the cultures tested were those that had been used in previous work.5
As in the experiments on exudates, an 0 precipitin line was formed on the negative side when E . coli extract was tested against both L- and D-specific sera containing homologous 0 antibodies. On the positive side of the antigen up to four lines appeared: Line 1 has been described and, in addition, there were lines 2, 3, and 4. Line 4 was very thin, with a wide sweep towards the trough of antiserum and was formed by most antigens when tested against the majority of both L and D sera. Line 2 was also thin; it appeared rarely and only when line 1 was seen. Line 3 was short and appeared closer to the posi- tive pole. In only one test were all four lines formed from a single antigen. Their approximate positions are shown in FIGURE 2.
Bettelheim & Taylor : Discussion 305
LINE 1 ........................
........ OT t-I E R /*. LINES
Extracts were made from eight cultures. Four (labeled “positive”) had been isolated from babies with enteritis and all had produced dilatation of the gut loop. Four (“negative”) came from healthy babies and had failed to produce a reaction in the gut loop. These eight extracts were tested against ten antisera (TABLE 3). It will be seen that line 4 was formed when both L and D sera were used. I t bore no relation to the known antigens in the extract nor to the enteropathogenicity of the strain from which the extract was derived.
.> ............
..... .....
.ANTIGEN HOLE .... ....
““.SERUM TROl GH
+ FIGURE 1. Immunoelectrophoresis and rabbit gut exudates, showing precipitin
lines.
Line 3 was formed only when L sera were tested, but again this line bore no relation to the known antigens nor to enteropathogenicity. The results suggest that line 1 alone was related to ability to cause enteritis since it was formed by all positive strains when tested against L sera prepared from the positive strains E65 and E122. Line 1 was formed only by strains that produced dilatation of the gut loop when tested against L antisera prepared from certain strains having the same property. Whether tested against L or D sera, a strain that failed to cause dilatation of the gut loop never produced Line 1.
> T
AB
LE
2
RE
SUL
TS WITH
EX
UD
AT
ES OR
W
AS
HIN
GS
FR
OM
R
AB
BIT
G
UT
LOOPS
Lin
es w
ith S
era
E E 5 01
0
14
4 4
-
1 -
14
-
01
0 3
-
1 -
1%
0 0
-
-
08
I$
Whe
y ca
sein
SL K
Gut
E65
02
6K60
H-
+ A
bP
055K
59H
6 4-
D53
01
0128
K67
H2
+ D
2101
0
11 1K
58H
12
-
El2
2 01
1 lK
58H
2 +
E2 1
01
28K
67H
2 -
Stra
in
Sero
type
R
eact
ion
E65
L
E65
D
Abp
L
Abp
D
D21
01L
D21
01D
D
5301
L
D53
01D
E
122L
8
13 1
Of
12
1 -
1 -
0 0
(1)*
-
01
-
-
012
-
1 -
-
-
-
-
-
s -
-
0 0
-
-
-
-
r
Uni
nocu
late
d -
-
-
-
-
-
-
-
-
-
loop
s, 2
st
erile
med
ium
st
erile
-
-
-
-
-
- -
-
-
-
* ( 1
) =lin
e po
orly
dev
elop
ed.
t O
=som
atic
line
. $ 0
Bettelheim & Taylor: Discussion 307
SURVEY OF STRAINS OF E. coli FROM VARIOUS SOURCES
Method
The antigenic structure of all the strains was identified and extracts pre- pared as previously described. The investigation of the diffusible antigens was carried out in three stages.
All antigens were tested for line 1 production by immunoelectrophoresis with the two antisera that had given the best results with the antigens derived
0
LI
LI LI
LI
+ FIGURE 2. Immunoelectrophoresis and culture extracts, showing precipitin lines.
from gut loops. These were E65L prepared from E. coli 026K60H- and E122L from OlllK58H2. Those antigens that produced line 1 with one or both sera were then tested by simple gel-diffusion against a further six antisera. Three sera were prepared using alcohol-killed inocula of E65, Aberdeen p and D5301 and another three using living inocula (D2101, Aberdeen /3 and D5301). The antigenic structure of these strains is shown in TABLE 1. The antisera Aberdeen B L and D5301L contained line 1 antibodies (TABLE 3) but the anti- genic structure of these strains was different from that of E65 and E122. The four D sera contained the same 0, K antibodies as one of the line 1 and line 4 sera but lacked any line 1 antibodies. I t was hoped that this test would reveal
W
TA
BL
E 3
PREC
IPIT
IN
LIN
ES
DE
TE
CT
ED
WITH
CU
LT
UR
E
EXTR
AC
TS
TE
STE
D
IN
IMM
UN
OE
LE
CT
RO
PHO
RE
SIS
SIW
DIE
S AGAINST
VA
RIO
US
AN
TIS
ER
A
Prec
ipiti
n L
ines
Dev
elop
ed a
gain
st Serum
E65
L
E65
D
Abp
L
Abp
D
E12
2L
D21
01L
D
2101
D
D53
01L
D
5301
D
E21
L
“Pos
itive
stra
ins”
* E
E65
3 (0
55:K
59:H
6)
12
4
4 09
1 0,
4 1,
3 4
...
...
...
44
E
l22
% D
5301
8
E68
6 0,
..
.
(026
: K60
:H-)
0,
1,2,
3,4
0,4
t 4
1,3
4 4
..
4 <
Abe
rdee
n p
..
.
(01 1
1 :K
58:H
2)
1A4
4 .
.
0,1
04
0
4 ..
. ..
.
(01 2
8 : K
67 : H
2)
1,3,
4 4
1,3,
4 4
..
.
0~
4
0 0,
4
(026
: K60
: H32
) 0
0 ..
.
(055 : K
59 :H
-)
3 74
4
0 0,
4 3 9
4 4
..
.
4 .
.
(01 l
l:K
58:H
12)
3,4
4 .
..
4
0,4
0,4
0 4
..
(0 12
8 :K
67:H
2)
3,4
4 ..
. 4
..
.
4 .
..
0,
4 0.
4 0.
4
Fo li
“Neg
ativ
e str
ains
” E2
8 w
.
..
.
..
..
. ...
...
...
...
D21
01
E2 1
* Pos
itive
str
ains
are
tho
se c
ausi
ng d
ilatio
n in
rab
bit
gut
loop
. N
egat
ive
stra
ins
do n
ot.
i .
..
=no
lin
es d
etec
ted.
Bettelheim & Taylor: Discussion 309
serological relationships not indicated by the antigenic structure and, at the same time, act as a control on the production of line 1.
The gel-diffusion was performed on agar-coated slides prepared as for immunoelectrophoresis. A template was used for siting the punched holes (FIGURE 3 ) . The antigen was placed in the central hole and the antisera in the peripheral ones. The slides were incubated at room temperature in a humid atmosphere for seven days, washed in buffer, dried, and stained with amido black.
Any antigen that produced one or more precipitin lines was then tested by immunoelectrophoresis against those antisera that gave positive reactions, in the hope that further differentiation would help to assess the significance of the lines.
Precipitin lines were obtained not only when the known antigens of the strain under examination corresponded to the known serum antibodies but occasionally when there was no recognized relationship between the two. Such lines where the reagents had no known relationship included line 1, but true differentiation could be obtained only by immunoelectrophoresis.
SURVEY OF 375 STRAINS OF E . coli FROM HUMAN SOURCES
Three hundred and seventy-five strains of known antigenic structure were tested. Three hundred and sixteen were from the feces of babies with diarrhea, 39 from the feces of healthy babies, 17 from infected urine, two from vaginal swabs and one from cerebrospinal fluid (CSF). None of these from healthy babies, urine, vaginal swabs, or CSF produced line 1. Ninety-nine out of 316 strains from babies with diarrhea produced line 1 (TABLE 4). The antigenic structure of some strains that gave line 1 is shown in TABLE 5, together with the number of those from both sick and healthy babies, which were found to be antigenically identical but which failed to give line l.13
SURVEY OF 25 STRAINS OF E. coli OF ANIMAL ORIGIN
These strains came from mice, pigs, calves, monkeys, a puma, a rabbit, chickens, turkeys, and pigeons. The details of their origin were seldom known except that some of the animals had died of septicemic infection. E. coli from one calf that had been scouring (i.e., had diarrhea) produced line 1: strains from three other calves whose history was unknown did not. On the other hand a strain from a scouring sow failed to produce line 1, as did strains from three other pigs about which there was no information. None of the other animal strains produced line 1.
DISCUSSION
E . coli is found normally in human feces both from healthy subjects and from those suffering from diarrhea. The majority of strains can be typed according to the international antigenic scheme. Certain serotypes appear to be especially associated with diarrhea, but these same strains are also isolated from healthy subjects. That some of these are the actual causative agent of diarrhea
310 Annals New York Academy of Sciences
ANTISERUM BACTERIAL A N T I G E N
1 I \
0 0 0
0 0 0 0 0 0
0 0
__
DIAMETER 1
FIGURE 3. Gel diffusion test plate.
is suggested by the epidemiological evidence, by the results of human feeding experiments, and by the fact that some strains that appear to be associated with the production of diarrhea cause dilatation of the ligated rabbit gut loop. Because the gut loop test is clumsy, time-consuming, and not always reliable, other tests for enterotoxicity were explored. The possibility of preexisting en- terotoxic agents in the gut of the test animal cannot be excluded and some test to be performed in vitro was to be preferred.
Certain of our laboratory strains of E . coli had been used repeatedly in experimental work and their effect on the gut loop was known. Although this effect tends to get weaker after storage, the strains were known to retain their activity. When exudate formed in the gut loop is inoculated into a second gut loop, the amount of exudate produced tends to increase, and this effect may be observed over several passages. It is a reasonable supposition that with each passage a larger quantity of the enterotoxin is produced. This effect might be due to the enhancement of the toxin-producing powers of the organism or to some toxin resulting from the interaction between organisms and tissue. It was with these thoughts in mind that it was decided to use gut extracts as well as cultures of E . coli in the first set of experiments.
A similar line of thought determined the methods used for the preparation of antisera. If the enterotoxin was formed (or the amount of it increased) by the reaction between the organism and the tissues of the host, it was clearly desirable to use a living vaccine. Moreover, previous work had shown that enterotoxin is so labile that a killed vaccine might fail to stimulate the forma- tion of antibodies to it. For these reasons four antisera were prepared with
TABLE 4
RESULTS OF TESTS FOR LINE 1 ANTIGEN IN 355 ROUTINE FECAL STRAINS OF Escherichia coli ISOLATED FROM BABIES
Number of Strains Tested
Condition of Baby
Number of Strains Showing Presence ( + )
or Absence ( - ) of Line 1 Antigen
39 316
Healthy Diarrheal Disease
o+, 3 9 - 99 +, 217 -
Bettelheim & Taylor: Discussion 311
living vaccines. Vaccines killed with alcohol are known to be capable of pro- ducing antibodies to the 0 and K somatic antigens and, because these antigens might be related to the production of enterotoxin, four antisera were made using alcoholized vaccines of the identical strains. In addition, two other strains that had the same 0, K structure as two of the other four were used to prepare antisera in the belief that these would help in the elucidation of the precipitin lines due to somatic or other antigens. The reaction of all six strains in the gut loop was known and the assumption made that a positive reaction indicated the production of enterotoxin. On the passage of an electric current, the somatic antigens moved towards the negative pole and all others in the reverse direc- tion. The somatic precipitin line appeared only when the antigen was tested against sera prepared with a vaccine made from an organism, alive or dead, of the same 0, K serotype. On the positive side several lines were produced but, of these, only one (line 1 ) was produced by exudates from the dilated gut and
TABLE 5
RELATION OF SEROTYPE LINE 1 ANTIGEN AND ASSOCIATION WITH HUMAN DIARRHEAL DISEASE (BABIES)
Number of Strains with Line 1 Present ( + ) or Absent ( - )
Serotype diarrhea healthy
026K60H 1 1
O55K59H7 01 llK58H12
026K60H-
0128K67H-
14+, 11- 2- 5+, 7- . . .
9 + , 20 - 1- 3 + , 7- 2- 4+, 1- 3 -
*
Total: 35+, 46 - 8-
Total, all types: 99+, 118- o+ 19-
* . . . =no strains.
by strains that produced this effect, but by no other antigen. This line appeared to be unrelated to the antigenic structure of the strain tested and uniquely asso- ciated with the production of enterotoxin. Material from the gut of the un- inoculated rabbit and from media produced no lines at all.
Line 1 was produced only by strains that produced dilatation of the rabbit gut when tested against L sera made from gut-dilatating strains. The other lines (3 and 4) bore no relation to the known antigenic structure of the strains nor to their enteropathogenic properties.
These findings are illustrated by the following. Extracts of strains E65, Aberdeen /3, El22 and D5301, all known to produce gut dilatation, produced line 1 when tested against antisera E65L and E122L but not against E65D and E122D. L antisera prepared from Aberdeen /3 and D5301 produced line 1 only when tested against homologous extracts. It is possible that these two strains produce less line 1 antibody than do E65 and E122. Strain D2101 did
312
not produce line 1 in either L or D homologous sera. This strain (and E28, E686, and E21) came from healthy babies and failed to produce dilatation of the gut loop.
The evidence appeared to justify the belief that the production of line 1 was related to the enteropathogenic effect of a strain of E. coli and therefore the experiments were extended to strains isolated from babies with diarrhea. These came from many different laboratories; no more than one strain from any one baby was included. It was impossible to know whether the strain sent to us was that responsible for the diarrhea or whether some organism other than E. coli might have been responsible. The antigenic structure of each strain was determined and the tests performed as described. TABLE 4 shows that only 99 out of 316 strains from babies with diarrhea gave line 1. All 39 strains from healthy babies failed to produce line 1.
TABLE 5 shows that the strains producing line 1 belonged to 24 different 0 groups, many of which had never been known to be associated with diarrhea. On the other hand many serotypes that had been isolated from babies with diarrhea failed to give line 1, nor did the Same serotypes isolated from healthy babies.
The investigation of epidemic infantile diarrhea and feeding experiments have both shown that certain serotypes, e.g. 0 1 1 1 and 055, cause diarrhea. These same types, however, have been isolated from healthy babies, which suggests that any one serotype may sometimes be enteropathogenic and some- times may not. We believe that by the use of immunoelectrophoresis with anti- sera produced from living enterotoxic strains, as has been described, it may be possible to recognize enterotoxic strains of E . coli more easily and reliably than by other methods. The method requires further investigation. Line 1 may be an indication of only one enterotoxic factor out of several. It should be possible to produce a more potent antigen and anti-enterotoxic serum. Attempts to do so using an azide extract for immunization were a failure.
It is of particular interest that one strain, E. coli 026K60H11, isolated from a scouring calf, produced line 1. This particular strain had not been tested in the rabbit gut loop but other strains from calves have given a positive reaction. It may be that the enterotoxin produced by some calf strains is related to that formed by human strains.
Annals New York Academy of Sciences
SUMMARY
Because the existing methods of detecting enteropathogenicity in strains of E. coli are not entirely satisfactory, the possibility of using immunoelectro- phoresis for this purpose was explored. This method was applied to bacterial cultures and to the exudates from dilated rabbit gut loops. All the strains of E. coli used were identified by the international antigenic scheme. With this method, a precipitin line, referred to as line 1, was produced by the gut exudates and by extracts of cultures of strains that produced gut exudates as long as the antisera were the product of living vaccines. Antisera prepared by the use of dead vaccines of strains known to be enterotoxic never gave line 1, nor was the latter produced by the media used, by washings from the normal rabbit gut, or by extracts of E . coli that lacked the property of dilating the rabbit
The study was extended to a survey of the results of this test with strains of gut loop.
Bettelheim & Taylor: Discussion 313
E. coli f rom healthy babies and babies with diarrhea and to E. coli f rom other human sources and from various species of animals.
REFERENCES
1. NETER, E. & C. E. SHUMWAY. 1950. E. coli serotype D433: occurrence in intestinal and respiratory tracts. Cultural characteristics, pathogenicity, sensi- tivity to antibiotics. Proc. SOC. Exp. Biol. Med. 75: 504.
2. FERGUSON, W. W. & R. C. JUNE. 1952. Experiments on feeding adult volun- teers with Escherichia coli 11 1 B4, a coliform organism associated with infant diarrhoea. h e r . J. Hyg. 55: 155.
3. DE, S. N., K. BHATTACHARYA & J. K. SARKAR. 1956. A study of the pathogenicity of strains of Bacterium coli from acute and chronic enteritis. J. Path. Bact. 71: 201.
4. TAYLOR, J., M. P. MALTBY & J. M. PAYNE. 1958. Factors influencing the response of ligated rabbit-gut segments to injected Escherichia coli. J. Path. Bact. 76: 491.
5. TAYLOR, J. & K. A. BETTELHEIM. 1966. The action of chloroform-killed sus- pensions of enteropathogenic Escherichia coli on ligated rabbit-gut segments. J. Gen. Microbiol. 42: 309.
6. SMITH, H. W. & S. HALLS. 1967a. Observations on the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J. Path. Bact. 93: 499.
7. SMITH, H. W. & S. HALLS. 1967b. Studies on Escherichia coli enterotoxin. J. Path. Bact. 93: 531.
8. SMITH, H. W. & S. HALLS. 1968. The transmissible nature of the genetic factor in Escherichia coii that controls enterotoxin production. J. Gen. Microbiol. 52: 319.
9. BETTELHEIM, K. A. 1969. Thesis. University of London. 10. EWING, W. H. 1956. Production of Escherichia coli antiserums. Publ. Hlth.
1 1 . TAYLOR, J., M. P. WILICINS & J. M. PAYNE. 1961. Relation of rabbit-gut reaction
12. KAUFFMANN, F. 1969. The bacteriology of the Enterobacteriaceae. 2nd ed.:
13. BEITELHEIM, K. A. & J. TAYLOR. 1970. Soluble antigens of enteropathogenic
Lab. 14: 138.
to enteropathogenic Escherichia coli. Brit. J. Exp. Path. 42: 43.
35-40. Copenhagen.
Escherichia coli. J. Med. Microbiol. In press.
