GASTROENTEROLOGY 68:270-278, 1975 Vol. 68, No.

2
Copyright © 1975 by The Williams & Wilkins Co. Printed in U.S.A.

PATHOPHYSIOLOGY OF SHIGELLA DIARRHEA IN THE RHESUS

MONKEY: INTESTINAL TRANSPORT, MORPHOLOGICAL,
AND BACTERIOLOGICAL STUDIES

W. R. ROUT, M.D., S. B. FORMAL, PH.D., R. A. GIANNELLA, M.D., AND G. J. DAMMIN,

M.D.
Department of Gastroenterology, University of Kentucky College of Medicine,
Lexington, Kentucky; the Departments of Gastroenterology and Applied Immunology,
Walter Reed Army Institute of Research, Washington, D. C.; and the Department of
Pathology, Harvard Medical School and Peter Bent Brigham Hospital, Boston,
Massachusetts

In contrast to the "toxigenic diarrheas" caused by Vibrio cholerae

and Escherichia coli, the site and mechanism of fluid loss in shigellosis
are unknown. The occurrence of watery diarrhea in shigellosis suggests
involvement of the small bowel. Therefore, jejunal, ileal, and colonic
water and electrolyte transport was studied in Shigella flexneri
2a-infected monkeys. Infected animals fell into three groups: dysentery
alone, diarrhea alone, or diarrhea and dysentery. In controls, net water,
sodium, and chloride absorption was seen in the jejunum, ileum, and
colon. All infected animals demonstrated diminished colonic absorption
or net colonic secretion. In monkeys with dysentery alone, this was the
only transport defect observed. In contrast, animals with diarrhea,
either alone or in combination with dysentery, exhibited net jejunal
secretion. Ileal transport was normal in all animals. A severe colitis
with intramucosal shigellae was seen in all symptomatic animals. In
the jejunum or ileum, however, morphological changes were minimal
and bacterial invasion was not seen. Therefore, unlike the "toxigenic
diarrheas," shigellosis is both a small and large intestinal disease.
Mucosal invasion of the colon is essential to the development of a
morphological and transport defect. Dysentery results from a colonic
transport defect, while diarrhea is secondary to jejunal secretion
superimposed on the defect in colonic absorption.

The watery diarrhea due to intestinal
infection with Vibrio cholerae and some
Received May 22, 1974. Accepted July 29, 1974.
Presented in part at a meeting of the American
Gasttoenterological Association, San Francisco, May
25, 1974, and published in abstract form.'
Address requests for reprints to: Dr. R. A. Gian-
nella, Department of Gastroenterology, University of
Kentucky School of Medicine, Lexington,
Kentucky 40506.
The authors wish to thank Mr. Peter Manty, Sp 4
John Schubert, and Mr. Smiley Austin for their
expert technical assistance, Dr. Douglas Tang for help
with statistical evaluation, and Miss Francine
Wisotzkey for secretarial help.
In conducting the research described in this report,
270
Escherichia coli strains is the result of
colonization of the small intestine by non-
invasive, toxigenic bacteria. 2-3 These
enteropathogens elaborate an enterotoxin
that stimulates mucosal adenyl cyclase
causing an increase in intracellular levels
of cyclic AMP and secretion of water and
electrolytes. 2 - 4 Small intestinal morphol-
ogy remains normal. 5 Colonic transport is
unaffected and diarrhea results because
the investigators adhered to the "Guide for Labora-
tory Animal Facilities and Care," as promulgated by
the Committee on the Guide for Laboratory Animal
Resources, National Academy of Sciences-National
Research Council.
February 1975 PATHOPHYSIOLOGY OF SHIGELLA DIARRHEA
the volume of small intestinal secretion
overwhelms the absorptive capacity of the
colon. 5
In contrast to our level of understanding
of these "toxigenic" diarrheas, little is
known of the pathophysiology of shigella
dysentery and diarrhea. 6-8 It is well estab-
lished that bacterial invasion of and multi-
plication within the colonic mucosa is es-
sential to the pathogenesis of shigellosis. 6. 7
Colitis and dysentery result from this inva-
sive process. 7 Less well appreciated is the
observation that watery diarrhea fre-
quently preceeds the dysenterY,9-I2 and, in
fact, diarrhea may be the only intestinal
manifestation of shigellosis. 11-14 These ob-
servations suggest that the small intestine
may be involved in this disease. I5 To evalu-
ate this possibility and to examine further
the pathophysiology, we studied shigellosis
in the rhesus monkey, a natural host for
shigella. I6 Shigellosis in the monkey closely
resembles that occurring in man. 7 • 11. 12, 16
Specifically, we examined the changes in
fluid and electrolyte transport occurring in
both the small intestine and colon, and the
relationship of these changes to alterations
in intraluminal shigella concentrations and
in intestinal morphology.
Methods
Animals. A detailed description of the experi-
mental design, surgical, and perfusion proce-
dures has been reported. 17 In brief, male rhesus
monkeys, Macaca mulatta, weighing 3 to 4 kg
and whose stools were free of enteric pathogens,
were orally infected with Shigella flexneri 2a
(strain M42-43). This strain has been used
previously in both animal and human stud-
ies. 7 , 11, 18, 19 Infection was induced after an
18-hr fast by orogastric tube feeding of 5 x 10 10
agar grown organisms suspended in 20 ml of
nutrient broth. Controls were fed an equal
volume of sterile broth, Symptomatic monkeys
were studied 48 to 72 hr later when clinical
findings were most severe. Monkeys with
shigellosis fell into one of three groups on the
basis of stool characteristics: dysentery alone,
diarrhea alone, and a combination of both diar-
rhea and dysentery. In animals with both diar-
rhea and dysentery, the diarrhea usually pre-
ceeded the appearance of dysentery by 12 to
24 hr. Diarrhea was defined as two or more
copious, watery bowel movements on each of 2
successive days, and dysentery as two or more
271
mucoid, bloody bowel movements for at least 1
day.
Perfusion technique. In anesthetized mon-
keys, 15-cm segments of proximal jejunum,
terminal ileum, and transverse colon were iso-
lated, biopsied, and luminal contents were cul-
tured. The three segments were then simultane-
ously perfused in situ at a rate of 0,5 ml per min.
The perfusion solution, pH 7.5 and kept at
37°C, contained (in millimoles per liter): Na
150; K 5; CI 125; HCO. 30; and d-mannitol 16;
and was equilibrated with 5% CO 2 -95% O 2 for 30
min before and during perfusion. Polyethylene
glycol 4000 (PEG), at a concentration of 600 mg
per 100 ml, was included as a nonabsorbable
water marker. Osmolality of the perfusion solu-
tion was 305 milliosmoles per kg. Blood samples
were taken at the beginning and end of the
experiment for electrolyte and osmolality deter-
minations. Serum electrolytes and osmolality
were not affected by perfusion, and the os-
molalities and serum electrolyte concentrations
observed in the three disease groups were not
significantly different from the control values or
from each other.
After a 2-hr equilibration period, the perfu-
sion solution was separately collected for three
1-hr intervals and measured to the nearest 0.1
ml. At the termination of the experiment, the
abdomen was reopened and the bowel exam-
ined. If a test segment appeared to be dilated or
cyanotic, it was excluded from the study. The
perfused intestinal segments were removed, the
length recorded, and a 2-cm piece was removed
from the distal end of each segment for histolog-
ical examination. The animal was then killed by
intravenous overdose of sodium pentobarbital.
All chemical analyses were done in duplicate.
PEG concentrations were determined by a mod-
ification of Hyden's turbidimetric method 20 ;
sodium and potassium concentrations by flame
photometry; chloride by coulometric titration;
osmolality by freezing point depression; and
total protein estimated by the method of War-
burg and Christian. 21 Net fluxes of water and
electrolytes were calculated from changes in the
concentration of PEG by standard formulae. 22
PEG recoveries in all segments studied were
greater than 95%. Values for transport were
expressed as microliters or microequivalents per
hour per 15 cm of bowel. Each 60-min perfusion
period was analyzed separately, and the mean
value for the three I-hr perfusion periods was
calculated. Negative values signify absorption,
net luman to blood transport, while positive
val ues signify secretion, net transport from
blood to lumen.
Bacteriology. Stool samples were cultured
272 ROUT ET AL. Vol . 68, No . 2

twice daily during the 48 to 72 hr prior to transport was markedly impaired in most
perfusion. At the time of perfusion, shigella animals . Striking differences in jejunal
concentrations in the jejunum , ileum , and colon
transport among animals became apparent
were quantitated . Luminal contents were seri- when animals were grouped according to
ally diluted in broth, and the various dilutions
disease characteristics , i.e., diarrhea
were plated in duplicate on MacConkey agar.
Lactose-negative colonies were enumerated and and/or dysentery. In animals with dysen-
tery alone, jejunal transport remained nor-
confirmed to be S . flexneri 2a by slide aggluti-
mal. In animals with diarrhea, alone or in
nation using specific antisera . Numbers of shi-
gellae are expresse d as the geometric mean combination with dysentery, net jejunal
(lOglO) per gram of intestinal content. secretion was regularly observed.
Histology. Specimens for histological study Net sodium and chloride transport par-
were fixed in 10% buffered formalin, processed alleled water transport in jejunum, ileum ,
for light microscopy, and stained with hematox-and colon in both control and shigella-
ylin-eosin and Giemsa stains. Coded slides wereinfected animals (fig. 1).
read (G. J. D.) without knowledge of the history
Intestinal perfusion protein concentra-
of the monkey from which the specimen was
obtained .
tions (table 2). Protein concentrations in
the intestinal perfusion solutions were
Statistical evaluation. All data are expressed
as mean ± 1 SE. Transport data and intestinal measured to assess the possibility that
secretion might be due to exudation of
protein concentrations were subjected to statis-
fluid from damaged bowel. No increase in
tical analysis by utilizing the Kruskal- Wallis
analysis of variance 23 and bacterial flora by protein concentration was noted in jejunal
anal ysis of variance or paired t-test, 24 as appro-
or ileal perfusion solutions of infected ani-
priate. Correlation between intestinal protein mals when compared to controls. On the
concentration and water transport was calcu- other hand, differences in response among
lated by linear regression by least squares."
the groups were observed in the colon. A
Results significant (P < 0.02) increase in protein
concentration was seen in those animals
Shigellosis in rhesus m onkeys. Of the 76 with dysentery only. There was no correla-
monkeys orally inoculated in this study, 31 tion between colonic protein concentration
(40.8%) developed clinical evidence of shi- and alterations in colonic net water trans-
gellosis, i.e., dysentery, diarrhea, or a com- port, r = 0.2495 .
bination of diarrhea and dysentery. Of the Bacteriology (table 3). Control monkeys
31 with obvious disease, 29% manifested
dysen tery alone, 32% diarrhea alone, and
39% a combination of the two. It should be TABLE 1. Net water transport in control and
pointed out that the incidence of clinical shigella·infected monkeys"
disease varies substantially among groups Jejunum Ileum Colon
of monkeys , a reflection, perhaps of prior
exposure of the animals to this natural Controls (8) -799 -1483 -1389
± 135 ± 202 ± 290
pathogen. Animals were studied 48 to 72
'9
Shigella monkeys (16) +319" - 1185 + 220"
hr after infection . If observed for a longer ± 266 ± 223 ± 130
period, many monkeys solely with diarrhea Dysentery (6) -585 -1370 + 426"
at 48 to 72 hr would eventually develop ± 2121 ± 251 ± 269
dysentery. Diarrhea (5) + 656" - 984 - 103"
Net transport of water and eLectroly tes . ± 277 I ± 328 ± 162
As expected, control monkeys (table 1), Diarrhea and dys· + 1066" [ - 1163 + 294"
demonstrated net water absorption in the entery (5) ± 541 ± 615 ± 168
jejunum, ileum , and colon. In contrast, all "Mean ± 1 SE Jlliters per hr per 15 cm . Negative
symptomatic shigella-infected monkeys, values indicate absorption (net lumen to blood move-
regardless of clinical presentation, demon- ment) and positive values indicate secretion (net
strated markedly diminished colonic ab- blood to lumen movement) . Numbers in parentheses
sorption or net colonic secretion. Ileal are numbers of animals studied .
transport was little affected and jejunal " P < .{).01 compared to control monkeys.
February 1975 PATHOPHYSIOLOGY OF SHIGELLA DIARRHEA 273
SODIUM
ileum, and colon, with the greatest concen-
JEJU UM ILEU COLON
tration being found in the colon. When
animals were grouped according to the
JOO
nature of disease, animals with diarrhea
alone differed from the other two groups.
In these animals, few shigellae (1.3 ± 0.8
organisms per g logIo) were found in the
jejunum. In general, the concentration of
shigellae in both small intestinal sites of
animals with diarrhea only were signifi-
o CONTROl. DOIAAIIHEA cantly lower than that seen in animals of
200 the two other clinical groups. Colonic con-
. D'I'lENTERY D=~RYANO centrations of shigellae did not signifi-
cantly differ among the three animal
CHLORIDE groups.
JEJ U ILE COLON
Intestinal morphology. Intestinal biop-
JOO
sies were taken before and after perfusion
to assess the effect of this procedure on the
intestinal mucosa. In control monkeys, the
only alterations attributable to the proce-
dure were (1) a dilation of small intestinal
lacteals, and (2) an increase in nuclear
pyknosis and karyorrhexis of cells in the
lamina propria, especially in the villus
tips. The mucosal pattern in infected ani-
mals appeared to be less altered by the
FIG. 1. Net intestinal sodium and chloride trans-
perfusion procedure.
port in control and shigella-infected monkeys. The All symptomatic monkeys exhibited an
size of each group is denoted by the number at the acute colitis (fig. 2) with a large number of
base of each bar. P values determined by comparison shigellae in colonic epithelial cells (fig. 3)
with control monkeys. and an acute pyogenic reaction in the
lamina propria. Monkeys with dysentery
alone tended to have the most severe and
TABLE 2. Intestinal perfusion protein concentration extensive colitis. Monkeys with diarrhea
in control and shigella-infected monkeysa
alone had a less severe colitis than that
Jejunum Colon seen either in animals with dysentery alone
mg/ml
Control (7) 0.17 ± 0.05 0.08 ± 0.03 I 0.10 ± 0.02 TABLE 3. Intestinal shigella concentrations in
Dysentery 0.09 ± 0.02 0.03 ± 0.01 0.37 ± 0.10" shigella-infected monkeysa
(5)
Jejunum Ileum Colon
Diarrhea (5) I 0.08 ± 0.03 0.04 ± 0.01 0.10 ± 0.05
Diarrhea 0.09 ± 0.02 0.03 ± 0.01 0.12 ± 0.03 All shigella 4.0 ± 0.5 6.3 ± 0.3" 6.9 ± 0.5"
and dys, monkeys (15)
entery (5) Dysentery (6) 5.4 ± 0.3< 16.8 ± 0.5 d 7.0±1.0
Diarrhea (4) 1.3 ± 0.8 5.3 ± 0.6 6.8 ± 0.6
a Mean ± SE. Numbers in parentheses indicate
Diarrhea and 4.7 ± 0.3< 6.5 ± 0.5" 6.8 ± 0.6
number of animals.
dysentery (5)
" P < 0.02 compared to control monkeys.
a Mean ± 1 SE. Number of shigellae per gram of

intestinal content (lOglO). Numbers in parentheses are

were free of enteric pathogens. When all numbers of animals studied.
symptomatic shigella-infected monkeys " P < 0.001 compared to jejunum.
were considered as a group, large numbers < P < 0.001 compared to animals with diarrhea.

of shigellae were observed in the jejunum, " P < 0.05 compared to animals with diarrhea.
274 ROUT ET AL. Vol. 68, No.2

or with dysentery and diarrhea (compare the jejunum were minimal (fig. 4A). They
fig, 2, A and B). consisted of slight to moderate reductions
In contrast to the striking colonic pathol- of the villus-crypt ratio, expansion of the
ogy, morphological alterations observed in villi, hyperplasia of the crypt epithelium

.. .. ~' . . .
B "
,J'.'- I' ",
FIG . 2. A, colon from shigella-infected animal with diarrhea only . Surface epithelium is intact but abnormal
in that cells are cuboidal and flat. There is focal dilation of crypt glands which lack mucus. The lamina propria
is edematous and increased in cellularity. B, colon from shigella-infected animal with dysentery only. Surface
epithelium is markedly attenuated and hyperchromatic with small area of ulceration in center. Note marked
focal dilation of crypts and mucus depletion. The lamina propria is markedly edematous and increased in
cellularity (Giemsa, x 150).
February 1975 PATHOPHYSIOLOGY OF SHIGELLA DIARRHEA 275

FIG. 3. High power view of superficial colonic epithelium of shigella-infected monkey. Note abundant bacilli
within epithelial cells and increased exfoliation (Giemsa, x 1300).

with a reduction in mucus content, and an of jejunal transport abnormalities de-

increase in interepithelial cells. There were
distinctly fewer abnormalities in the ileum,
and in many animals, this segment was
completely normal (fig. 4B). No pyogenic
foci or bacterial invasion was seen in the
jejunum or ileum.
Discussion
In shigella-infected monkeys, the most
consistent alterations in fluid and elec-
trolyte transport were observed in the
colon. Regardless of the nature of the
disease, i.e., diarrhea and/or dysentery, all
symptomatic monkeys demonstrated
markedly diminished colonic absorption or
net colonic secretion. Indeed, in monkeys
with dysentery alone, this was the only
transport defect observed. The occurrence
pended upon the nature of the disease. In
monkeys with diarrhea, alone or in combi-
nation with dysentery, jejunal transport
was consistently abnormal, every animal
demonstrating net jejunal secretion. Ileal
transport was little affected. Thus, these
results suggest that dysentery results pri-
marily from colonic dysfunction, and that
diarrhea results from abnormalities in jeju-
nal transport superimposed on a defect in
colonic absorption.
All symptomatic monkeys exhibited a
severe acute colitis with a large number of
shigellae in colonic epithelial cells. Al-
though those animals with dysentery alone
tended to have the most severe colitis, no
differences in colonic transport or colonic
intraluminal shigella concentrations were
276 ROUT ET AL. Vol. 68, No . 2

FiG. 4. A ; jejunum from shigella-infected monkey . }<;ssentially normal except for slight shortening of villi ,
mild hypertrophy of crypts, and depletion of goblet cell mucus. B, ileum from shigella-infected monkey .
Essentially normal (Giemsa, x 125) .

apparent among the three clinical groups.
Our observations that a severe colitis and a
colonic transport defect regularly occurs in
shigella-infected monkeys is consistent
with the presently accepted view that the
establishment of disease is dependent upon
the ability of shigellae to invade and multi-
ply within the colonic mucosa. 6 - 8 • 1 2 The
events occurring subsequent to mucosal
invasion which result in alteration in co-
lonic transport are not known. They may
involve impaired epithelial absorption,
passive transport through a damaged mu-
cosa, and/or an active epithelial secretory
February 1975 PATHOPHYSIOLOGY OF SHIGELLA DIARRHEA
process. Reduced absorption could occur as
a result of a damaged colonic epithelium,
presumably the reason for the reduced
absorption seen in ulcerative colitis. 25, 26 In
view of the invasive nature of shigellae and
the morphological alterations evoked by
this pathogen, passive transport (transuda-
tion-exudation) through a damaged mu-
cosa could contribute to the fluid lost in the
colon. 15 , 17. 27 The tendency for animals
with dysentery alone to have the most
severe colonic inflammatory reaction and
the highest perfusion protein concentration
is compatible with this view. However,
even the colonic perfusion protein concen-
tration observed in monkeys with dysen-
tery is only one-quarter to one-half the
concentration expected if a transudative-
exudative mechanism was operative. 17 ,27
Of course, net colonic secretion may result
from a combination of impaired absorption
and/or an active secretory process superim-
posed on a transudative-exudative mecha-
nism. These experiments were not de-
signed to discriminate among these possi-
bilities.
In contrast to the colon, invasion of
jejunal or ileal mucosa was not seen and
minimal morphological abnormalities were
observed. Yet, substantial numbers of shi-
gellae were found in the lumen of both the
jejunum and ileum of most animals. Al-
though ileal transport was little affected,
net jejunal secretion occurred in 10 of 16
animals studied. The strain used in this
study, S. flexneri 2a, unlike S. dysenteriae
1, does not elaborate a detectable
enterotoxin 8 (S. B. Formal, P. Gemski, R.
A. Giannella, unpublished observations),
and thus, jejunal secretion cannot be read-
ily attributed to an enterotoxin-mediated
process. The pathogenesis of the jejunal
transport abnormalities is not known, but
it apparently differs from that in the colon
where mucosal invasion by shigellae and
morphological alterations are regularly
seen. A similar defect in jejunal fluid and
electrolyte transport, in the absence of
jejunal morphological abnormalities, has
been observed in two other colonic disor-
ders, i.e., ulcerative colitis 28 and salmo-
nella-infected monkeys. 17 It is possible
that an as yet undetected bacterial "en-
277
terotoxin" or the release of a "colonic fac-
tor" by a damaged and inflamed colon
may mediate the observed jejunal trans-
port abnormalities. Since inflammatory
reactions release prostaglandins 29 and
these compounds can induce jejunal se-
cretion,30 their participation in these dis-
orders must be considered.
The alterations in intestinal transport
observed in shigella-infected monkeys bear
some similarities and exhibit some differ-
ences from other enteric infectious diar-
rheal disorders. Like salmonellosis, a
prominent colonic transport defect and
colitis accompanies shigellosis. 17 Unlike
salmonellosis, however, ileal transport in
shigellosis remains normal. 17 In both of
these invasive diarrheas, alterations in je-
junal transport occur in some animals.
Unlike the enterotoxin-mediated diarrheas
of V. cholerae and some E. coli strains, the
invasive diarrheas of shigellosis and sal-
monellosis are characterized by striking
colonic morphological and transport abnor-
malities and an acute colonic inflamma-
tory reaction. 2, 3, 5
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