INFECTION AND IMMUNITY, Oct. 1991, p. 3708-3714
`0019-9567/91/103708-07$02.00/0
`Copyright © 1991, American Society for Microbiology
`
`Vol. 59, No. 10
`
`Efficacy of Enteric-Coated Protease in Preventing Attachment of
`Enterotoxigenic Escherichia coli and Diarrheal Disease in the
`RITARD Model
`
`TRACEY L. MYNOTT,’* DAVID S. CHANDLER,” anpD RICHARD K.J. LUKE?
`School ofAgriculture, La Trobe University, Bundoora, Victoria 3083,' and Victorian Institute of Animal Science,
`Attwood, Victoria 3049,” Australia
`
`Received 26 April 1991/Accepted 1 July 1991
`
`In this study, we report on a novel approach based on modification of the intestinal surface to prevent
`diarrhea caused by enterotoxigenic Escherichia coliETEC). The removable intestinal tie adult rabbit diarrhea
`(RITARD) model was used to test the efficacy of an enteric-coated protease preparation (Detach; Enzacor
`Technology Pty. Ltd.) in the prevention of bacterial attachment and diarrheal disease caused by colonization
`factor antigen I-positive (CFA/I*) E. coli H10407. Protease was administered orally to rabbits 18 h prior to
`challenge with 10’! bacteria. Four groups of rabbits were inoculated with different ETEC strains which
`produced different combinations of adhesin and enterotoxin or with sterile phosphate-buffered saline.
`Occurrence of diarrhea during the subsequent 24-h incubation period was recorded. Oral administration of
`protease was successful in reducing diarrhea and diarrhea-induced deathin six of seven (86%) rabbits infected
`with CFA/I*, heat-stable and heat-labile toxin-positive E. coli (H10407). Seven of eight (87%) rabbits not
`protected by protease treatment died or developed severe diarrhea. Quantitative analysis of bacterial cultures
`obtained from the small intestine of rabbits showed a significant (P < 0.001) 2,000-fold reduction in CFU per
`centimeter of intestine following treatment with protease. Theefficacy of protease treatment was 99.5%, with
`very wide confidence limits (>0 to 99.9%). The data indicate that the use of protease to prevent ETEC
`diarrheal disease has considerable potential.
`
`Therole of enterotoxigenic Escherichia coli (ETEC)as an
`important etiologic agent in human diarrheal disease is well
`established (31). These organismsare characterized by their
`ability to produce heat-labile toxin (LT) and/or heat-stable
`toxin (ST) (11). Somestrains also produce pilus adhesins
`called colonization factor antigens (CFAs). These adhesins
`permit attachment of ETECstrainsto the intestinal mucosa,
`thereby facilitating colonization and delivery of enterotoxin
`to target epithelial cells.
`CFAsmay be of use as vaccine candidates and for this
`reason have attracted considerable attention. Those identi-
`fied to date include CFA/I and CFA/III and two multivariant
`antigen groups known as CFA/II and CFA/IV. Each of the
`last two possesses a combination of three pilus adhesins,
`designated coli surface antigens (CS). CS1, CS2, and CS3
`are present on CFA/II E. coli, and CS4, CS5, and CS6 are
`present on CFA/IV E.coli (7, 16, 21, 28). Other putative
`colonization factors have also been described previously (5,
`14, 34, 36). As colonization factors are antigenically distinct,
`potential vaccines must be multivalent. A prototype ETEC
`vaccine that contains known CFAsand outer membrane
`antigens associated with ETECis being developed (32).
`Significant protection against diarrhea caused by ETEC
`following vaccination with a combination of formalin and
`heat-inactivated whole Vibrio cholerae cells and the purified
`B subunit of cholera toxin has been reported recently (4). V.
`cholerae and ETEC produceheat-labile enterotoxins which
`are structurally, functionally, and immunologically similar
`and consist of a combination of a biologically active A
`subunit and five B subunits (12, 15, 22).
`Otherefforts to develop vaccines against ETEC infection
`
`* Corresponding author.
`
`havegiven variable results (8, 18, 19), and while the prospect
`of effective immunization against diarrheal diseases of hu-
`mansis appealing, effective vaccines are not expected to be
`available in the near future (16a). Alternative approaches to
`the prevention of ETEC diarrhea are therefore being pur-
`sued.
`Studies with pig ETEC that possess the K88 adhesin have
`indicated that the interaction between ETEC andpig brush
`border membranescan be modified by the use of proteases
`(27). Preliminary challenge experiments and field trials (2a)
`have indicated that an enterically protected protease prepa-
`ration (Detach; Enzacor Technology Pty. Ltd., Melbourne,
`Australia) administered orally is able to modify the mucosal
`surface of the piglet small intestine such that diarrhea is less
`likely to occur.
`A numberof studies have described similarities in mech-
`anisms of pathogenesis of ETEC infection in humans and
`animals (11, 31). This has led us to investigate the use of
`protease in the prevention of human diarrheal diseases.
`Recent experiments (23a) have indicated that binding of
`CFA/I and CFA/II to human intestinal mucosa can be
`prevented in vitro by the use of protease. An effect of
`protease in reducing the binding of LT was also demon-
`strated. It appears that modification of intestinal mucosa to
`prevent humandiarrheal diseases has considerable potential.
`This report describes an experiment in which the removable
`intestinal
`tie adult rabbit diarrhea (RITARD) model de-
`scribed by Spira et al. (29) was used to test the efficacy of
`exogenous protease (Detach) in reducing attachment of
`CFA/I-positive E. coli to rabbit intestinal mucosa in vivo.
`The workrepresents a novel approach to preventing attach-
`ment of ETECto intestinal cells and therefore preventing
`diarrheal disease.
`
`3708
`
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`

`VoL. 59, 1991
`
`PROTEASE AND PREVENTION OF ETEC DIARRHEA
`
`3709
`
`MATERIALS AND METHODS
`
`Animals. Forty-four New Zealand White breed rabbits of
`both sexes from a single breeder were used for the experi-
`ment. Their weights ranged from 1.5 to 2.7 kg. Animals were
`acclimatized to their animal housing facility for at least 1
`weekprior to the start of experimentation.
`Bacteria. ETECstrains used in this study wereoriginally
`isolated in Bangladesh from patients with diarrhea. Strain
`H10407 (serotype 078:K80:H11) and a mutantderivative of
`this strain, H10407-P, were kindly provided by D. G. Evans
`(The University of Texas Medical School at Houston, Hous-
`ton). Strain E1392/75 7A (serotype 06:K15:H16) was kindly
`provided by B. Rowe (Division of Enteric Pathogens, Lon-
`don, United Kingdom). Strain H10407 produces both ST and
`LT and possesses CFA/I. Strain H10407-P produces both ST
`and LT but does not produce CFA/I (7). Strain E1392/75 7A
`is a CFA-negative, nontoxigenic spontaneous laboratory
`derivative of CFA/II* E. coli 1392 (17) and has been shown
`not to colonize or induce diarrhea in the RITARD model
`(35).
`Stock cultures of all strains were suspendedin Trypticase
`soy broth (Oxoid) containing 15% (vol/vol) glycerol and
`stored in multiple aliquots at —80°C. A new aliquot was used
`for each experiment. Bacteria were inoculated onto CFA
`agar (6) and grown at 37°C overnight. Cultures were har-
`vested by means of a flamed Pasteur pipette, washed in
`sterile phosphate-buffered saline (0.01 M, pH 7.2; PBS), and
`diluted to yield the desired optical density measurements.
`The bacterial concentration was confirmed by viable cell
`count on duplicate blood agar plates after serial dilution in
`PBS. Prior to the inoculation of rabbits, all cultures were
`checked for the presence of CFA/I and LT by a specific
`enzyme immunoassay (EIA).
`Antigens. CFA/I waspurified as previously described (6).
`Purified LT (unnicked) was kindly provided by J. D. Clem-
`ents (Walter Reed Army Institute of Research, Washington,
`D.C.).
`Antisera. Specific CFA/I antiserum was produced by
`giving rabbits three subcutaneousinjections with 60 pg of
`purified CFA/I at intervals of 4 weeks. Forthe first immu-
`nization, the antigen was emulsified with Freund’s complete
`adjuvant. Subsequent immunizations were emulsified with
`Freund’s incomplete adjuvant. The animals were bled by the
`ear vein 2 weeksafter the final immunization. LT-specific
`antiserum was similarly produced by injecting 60 pg of
`purified LT. The immunoglobulin G fraction was prepared
`by protein A affinity chromatography with protein A-Seph-
`arose CL-4B (Pharmacia) as specified by the manufacturer.
`Detection of CFA/I and LT by EIA. Disposable polystyrene
`microtiter plates (Nunc, Roskilde, Denmark) were used for
`all assays. CFA/I- or LT-specific immunoglobulin G was
`diluted in sodium bicarbonate buffer (0.1 M, pH 9.6) and
`adsorbed to wells (100 yl per well) by incubation overnight at
`4°C. For CFA/I detection, bacteria were diluted to approx-
`imately 2.5 x 10° cells per ml (Ag); = 1.0) in working dilution
`buffer containing PBS (0.01 M, pH 7.2), bovine serum
`albumin (0.25%; Fraction V; Sigma), Tween 20 (0.05%;
`Sigma), disodium salt EDTA (0.0075%), and sodium azide
`(0.02%). For LT detection, supernatant material was ob-
`tained following centrifugation (at 12,000 < g for 15 min) of
`overnight cultures of bacteria grown in CYE medium (9).
`The bacterial suspension or culture supernatant was inoc-
`ulated into duplicate wells (100 yl per well). Bound material
`was detected with urease-conjugated (CSL, Melbourne,
`Australia) specific immunoglobulin G diluted in working
`
`dilution buffer containing 1% hen egg albumin (Fraction II;
`Sigma). Urea substrate (0.008% bromocresol purple [Sigma],
`0.1% urea [Bio-Rad], 0.0075% disodium salt EDTA; pH 4.8,
`100 pl) was used, and a positive reaction wasindicated by a
`color change from yellow to purple. During each of the
`above steps, plates were incubated at 37°C for 30 min.
`Betweeneach of the steps, supernatant liquid was removed
`from the wells, which were then washed with washing buffer
`(0.01 M PBS, 0.05% Tween 20). Prior to the addition of
`substrate, wells were washed with distilled water to remove
`residual buffer. Developmentof purple color was monitored
`at As4o-
`RITARD model procedure. The RITARD model developed
`by Spira et al. (29) was used with slight modifications. At 18
`h prior to challenge, half of the rabbits from each group (see
`Table 1) were given a single oral dose of 0.42 g of enteric-
`coated protease granules (Detach containing 25% protease;
`Enzacor Technology Pty. Ltd.). The protease granules were
`placed in gelatin capsules and administered by placing the
`capsules at the back of the throat. Food was withheld after
`dosing, but water was made available ad libitum. Before
`surgery, animals were anesthetized with 16 mg of xylazine
`(Rompun; Bayer) and 100 mg of ketamine (Ketapex; Apex
`Laboratories Pty. Ltd) intramuscularly. The incision site
`was anesthetized with 2 ml of lignocaine with adrenalin (20
`mg oflignocaine hydrochloride per ml, 0.01 mg of adrenalin
`[as bitartrate] [Apex Laboratories Pty. Ltd] per ml). The
`cecum was exteriorized through a midline incision and
`ligated permanently as close to the ileo-cecal junction as
`possible with no. 11 umbilical tape (Ethicon). At this time
`the ileum was temporarily obstructed approximately 10 cm
`proximalto the cecal tie with a slipknot tie with umbilical
`tape. Inoculum (10 ml) containing 10" bacteria diluted in
`sterile PBS or sterile PBS alone was injected into the
`duodenum. The intestine was returned to the peritoneal
`cavity, which was sutured with one end of the temporary tie
`left accessible through the incision. Theileal tie was gently
`removed 2 h after bacterial challenge, and the rabbits were
`returned to their cages, where food and waterwereavailable
`ad libitum. All surviving animals were killed 24 h postchal-
`lenge by means of barbiturate overdose; some animals
`infected with the CFA/I* bacteria died within the 24 h.
`Immediately after death, each animal was autopsied and the
`peritoneal cavity was swabbedfor bacterial culture.
`The E. coli enterotoxin and adhesin combinations were
`selected to include a CFA-positive ETEC strain (H10407), a
`CFA-negative ETEC strain (H10407-P), and a CFA-nega-
`tive, nontoxigenic strain (E1392/75 7A). A PBS control was
`included to enable the effect of surgery and protease treat-
`mentin the absence of bacterial challenge to be monitored.
`The challenge rate of 10'' bacteria was based on earlier
`experiments in which such a dose of organisms was required
`to induce diarrhea in 90% of infected animals (1). In the
`present study, surgery was performedon rabbits in groupsof
`eight over a period of 4 weeks (two rabbits from each group
`each week; see Table 1). Two weekslater, 12 rabbits were
`challenged with H10407.
`Monitoring of infection. Rabbits were observed hourly for
`the 24-h postchallenge period for diarrhea, weakness, or
`death. Diarrhea was scored as follows: 0, no diarrhea; 1,
`mild diarrhea with feces softer than normal; 2, moderate
`diarrhea with at least three watery stools; and 3, severe
`diarrhea with multiple watery stools. Fecal swabs were
`collected when feces were passed, and rectal swabs were
`taken from rabbits which did not pass feces. Challenge
`organismswereidentified by means oftypical E. coli colony
`
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`

`3710
`
`MYNOTT ET AL.
`
`INFECT. IMMUN.
`
`Strain
`H10407°
`
`Adhesin
`CFA/I*t
`
`Toxin
`ST* LT*
`
`H10407-P
`
`CFA/I-
`
`ST* LT*
`
`E1392/75 7A CFA/II- ST~ LT™
`
`TABLE1. Diarrheal response in rabbits treated with Detach or
`untreated and challenged with different ETEC strains
`Treat- Diarrheal
`Smallintestine
`ment?
`response’
`fluid vol (ml)
`D
`1/74
`35-50
`Cc
`718¢
`20-105
`D
`1/48
`10-50
`Cc
`1/48
`10-50
`D
`0/4
`15-50
`Cc
`0/4
`15-50
`D
`0/4
`8-12
`Cc
`0/4
`24-40
`
`Nil (PBS)
`
`@ D7,rabbits treated with Detach; C, untreated rabbits.
`>’ Number of animals with diarrhea or death/total number tested. For
`H10407-challenged rabbits, P < 0.001: one of seven Detach-treated animals
`died compared with sevenofeight untreated animals challenged with the same
`doserate.
`© Five rabbits were omitted from the analysis because of death not related
`to diarrhea.
`4 One Detach-treated rabbit died; CFU/cm at $3 was 1.2 x 10’.
`© Onerabbit survived infection; CFU/cm at S3 was 5.8 x 10°.
`The total volume of fluid accumulated in small and large intestines
`combined was 130 to 165 ml.
`® Mild diarrhea (score 1).
`
`morphology and enzyme-linked immunosorbentassaynitro-
`cellulose replica methods as described previously (20).
`Collection of tissue specimens. All animals were sacrificed
`24 h postchallenge, and the intraluminal fluid of the small
`intestine was measured. Large fluid volumes (>60 ml)in the
`small intestine of euthanized or dead rabbits were taken as
`an indication that diarrhea had been a major contribution to
`death (29). Sections (2 by 3 cm) of small intestine were
`collected from five sites: duodenum (S1), proximal jejunum
`(S2), midjejunum (S3), distal jejunum (S4), and ileum (SS).
`Each segment wasopenedlongitudinally and washed exten-
`sively in sterile PBS to determine the numbersof strongly
`adherent bacteria or left unwashed to determine the total
`bacterial numbers present. Quantitative cultures were pre-
`pared by homogenizing tissue for 1 min in a Sorvall homog-
`enizer operated at full speed. Serial dilutions were made in
`PBS, andaliquots (25 1) were plated onto sheep blood agar
`(5%, vol/vol) and CFA agar. After incubation at 37°C for 18
`h, the number of CFU percentimeter of tissue was deter-
`mined. Other specimens were processed promptly for his-
`tology following fixation in 10% neutral-buffered formalin.
`After specimens had been embeddedin paraffin, they were
`stained with hematoxylin-eosin stain and tissue Gram stain.
`Statistical analysis. Bacterial counts were converted by log
`transformation to stabilize variances and analyzed by means
`of Genstat V for analysis of variance. Data are expressed as
`the mean values + the standard error of the mean. The
`efficacy of Detach (protease) protection was determined by
`Fortran-Finney, a program that determines efficacies (per-
`cents) from chemotherapeutic tests (10).
`
`RESULTS
`
`Diarrheal response. None of the rabbits challenged with
`nontoxigenic E1392/75 7A or given 10 ml of sterile PBS
`developed diarrhea (Table 1). At autopsy, the fluid volumes
`in the small intestine (pyloric sphincter to the ileo-cecal
`junction) ranged from 8 to 12 ml in the PBS-protease-treated
`group to 24 to 40 ml in the PBS-treated, non-protease-treated
`group.In rabbits challenged with E1392/75 7A,15 to 50 ml of
`fluid accumulated. Twoof eight rabbits that were challenged
`
`toxigenic H10407-P passed feces
`with the CFA-negative,
`that were softer than normal (score 1). This was only mild
`diarrhea and not considered to be important. One rabbit was
`in the protease-treated group, and one wasin the non-pro-
`tease-treated group. In rabbits challenged with H10407-P,
`the fluid volumes in the small intestine ranged from 10 to 50
`ml. There wasnosignificant difference between the amounts
`of fluid accumulated by the rabbits challenged with the two
`CFA-negative strains.
`In total, 20 rabbits were challenged with the enterotoxi-
`genic CFA/I-positive strain H10407. Five died within 4 h of
`challenge, but none of these had typical acute watery diar-
`rhea. These rabbits were autopsied, and peritoneal swabs
`wereplated on blood agar and MacConkeyagar. Leakage of
`bacteria from the inoculation site was deemed to be the
`cause of death, as colonies of typical E. coli morphology
`wereisolated from the peritoneum of these rabbits. No such
`bacteria were detected in peritoneal swabs from the rabbits
`whichdied in a diarrhea-related manneror in swabsfrom the
`remaining rabbits autopsied at 24 h. These five rabbits were
`omitted from the experiment, leaving seven protease-treated
`rabbits and eight untreated rabbits challenged with H10407.
`Of the eight control (non-protease-treated) rabbits chal-
`lenged with H10407, seven died or developed severe diar-
`rhea. Six rabbits died 5 to 13 h postchallenge, and four of
`these died without passing feces. Of the two rabbits that
`survived the 24-h postchallenge period, one had profuse
`watery diarrhea 18 h postchallenge while the other did not
`pass feces and had noclinical signs of infection. At autopsy,
`the fluid volumein the small intestines of rabbits which did
`not pass feces ranged from 20 to 105 ml. The total volumein
`the small and large intestine combined, however, ranged
`from 130 to 165 ml (in comparison with 10 to 50 mlin rabbits
`inoculated with E1392/75 7A, H10407-P, and PBS). In com-
`parison, the small intestine containedrelativelylittle fluid (35
`to 50 ml)in rabbits that had diarrheaat the time of death. The
`total volumeofintestinal fluid was only 55 to 60 ml. A lack
`of fluid accumulation in rabbits that have passed fecesat the
`time of death has been reported previously (29).
`Of the rabbits treated with protease prior to H10407
`challenge, only one died. This rabbit died 11 h postchallenge
`after passing one loose stool (score 1). Fluid volumesin the
`small and large intestines were 60 and 50 ml, respectively.
`None of the other six rabbits treated with protease had
`diarrhea, and the majority (four of six) had passed formed
`feces by 24h. At autopsy, the contents of the large intestine
`were solid and fluid accumulation in the small intestine
`ranged from 12 to 60 ml.
`Bacterial adhesion. Quantitative cultures (CFU) were pre-
`pared from all animals to determine the adhesion ofchal-
`lenge bacteria in different parts of the small intestine. Chal-
`lenge bacteria were presentatall sites, with CFA/I* H10407
`being the mosteffective colonizer. The mean CFUlevels of
`CFA/I* bacteria at sampling sites in non-protease-treated
`rabbits were lower at S1, S2, S4, and S5 (1.1 x 10°, 6.7 x
`10’, 1.0 x 10°, and 4.0 x 10® CFU/cm,respectively) than at
`S3 (5.5 x 10? CFU/cm). In the analysis which follows, S3
`cultures are used for comparison. The recovery rates of
`different organisms from 1 cm of mucosaare shownin Fig.1.
`The number of CFA/I* bacteria adherent to the mucosa in
`the protease-treated rabbits ranged from 1.3 x 10* (minimum
`count) to 1.2 x 10’ CFU/cm in the rabbit that died (arith-
`metic mean, 2.6 x 10° CFU/cm). Values for control rabbits
`challenged with the same strain were 2,000 times greater (P
`< 0.001). Figure2 illustrates the differences in colony counts
`between protease-treated and untreated animals.
`
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`VoL. 59, 1991
`
`PROTEASE AND PREVENTION OF ETEC DIARRHEA
`
`3711
`
`CFU/cm
`
`H10407
`
`H10407-P
`
`=E1392/75
`7A
`
`PBS
`
`No Diarrhoea
`
`FIG. 1. Range of quantitative cultures at midjejunum (S3) of rabbits challenged with different ETECstrains or given sterile PBS. @, rabbits
`treated with Detach 18 h prior to challenge; O, untreated.
`
`intestinal
`The number of bacteria bound to the small
`mucosaof rabbits infected with the CFA/I” strain H10407-P
`ranged from 1.3 x 10* CFU/cm (minimum count) to 6.6 x
`10’ CFU/em in a rabbit with mild diarrhea (arithmetic mean,
`1.8 x .10’ CFU/cm). Counts for strain E1392/75 7A were
`similar, ranging from 1.3 x 10+ (minimum count) to 2 x 10°
`CFU/cm (arithmetic mean, 3.9 x 10’ CFU/cm). There were
`no significant differences between bacterial numbers in pro-
`tease-treated and non-protease-treated animals challenged
`with either CFA-negative strain.
`In rabbits which received sterile PBS only, there were
`relatively few bacteria in the small
`intestine (1.3 x 10*
`CFU/cm). There were, however, two exceptions. Two con-
`trol rabbits which did not receive protease had colony counts
`of 4.6 x 10° and 5.2 x 10° CFU/cm at S3 (small intestinal
`volumesof 40 and 35 ml, respectively). One of these rabbits
`had 5.1 x 107° and 4.1 x 10?° CFU/cm at S1 and SS,
`respectively. All colonies observed hadtypical E. coli mor-
`phology, and it seems likely that there was cross infection
`from one or more other bacterial challenge groups. These
`colonies were not checked for the presence of CFA/I.
`Williams-Smith and Halls (37) have reported that E. coli are
`not isolated as part of the normal rabbit flora, and it is
`therefore unlikely that the E. coli observed werethe result of
`overgrowth of endogenousbacteria.
`To assess the total numberof bacteria present on the gut
`mucosa, séctions were left unwashed. The mean values of
`washed versus unwashedsections from rabbits challenged
`with CFA/I*are shown in Table 2. These results suggest
`that the organismsare strongly adherent to the mucosa,as
`only 10 bacteria were washed away.
`Fecal excretion of bacteria. Fecal swabs were obtained
`
`from rabbits when feces were passed. In all animals the
`challenge bacteria were excreted. Rectal swabs were taken
`at autopsy. The presence of the challenge strain in the
`rectum was apparentin all rabbits, including those that had
`not passed feces prior to termination of the experiment. In
`all instances, 100% of the colonies cultured were of the
`challenge strain.
`Histology. Histological studies (light microscopy) of small
`intestinal tissue revealed no mucosal abnormality in any of
`the rabbits. Bacteria were only rarely seen on the mucosa,
`suggesting that bacteria boundin particular areas rather than
`being evenly distributed along the mucosa.
`
`DISCUSSION
`
`The decision to investigate the use of protease to modify
`the intestinal surface and thereby prevent diarrhea in hu-
`mans was based on previous prevention of K88* E. coli
`infection in piglets (2a). Several studies have described
`similarities in mechanisms of pathogenesis of ETEC infec-
`tion in humans and animals (11, 31). Most ETECstrains of
`human and animal origin rely on pili for adhesion and
`subsequent colonization of the small intestine. Also, diar-
`rheal disease in both species is dependent ultimately on
`production andefficient delivery of enterotoxin.
`The interaction between K88 adhesin and its intestinal
`receptor is one of the most studied host-pathogen associa-
`tions, while relatively little is known about the interaction
`between CFA adhesins and human brush border mem-
`branes. The epithelial receptor for attachment of K88*
`ETECto the brush border membraneofpiglets is known to
`be a multimeric glycoprotein (30). This and other receptors
`
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`

`3712
`
`MYNOTT ET AL.
`
`INFECT. IMMUN.
`
`Mean (I
`CFU/en"®
`
`)
`
`i
`
`MeerEo
`10
`9
`
`A
`
`8 7 6 5
`
`4 3 2 1 0
`
`Site 1
`
`Site 3
`
`Site 5
`
`,,)
`
`Mean (log
`CFU/cm’°
`
`10-
`
`Site 1
`
`Site 3
`
`Site 5
`
`Mean (lo:
`
`)
`
`_SoorDOOhTHDTTODO
`CFU/cm!®
`
`0AJ?ot
`Site 1
`Site 3
`Site 5
`Site 1
`Site 3
`Site 5
`
`FIG. 2. Mean bacterial counts at duodenum (site 1), midjejunum (site 3), and ileum (site 5) of rabbits challenged with H10407 (A),
`H10407-P (B), E1392/75 7A (C), or sterile PBS (D). Each point represents the mean (log,,) + standard error CFU per centimeteroftissue.
`W@, rabbits treated with Detach 18 h prior to bacterial challenge; O, untreated rabbits.
`
`involved in attachment (and colonization) have been shown
`to be readily inactivated by proteolytic enzymes, including
`those proteases that are normally active in the small intestine
`(23, 27, 30). This was recently investigated in experiments
`observingtheinstability of K88 receptor within pig intestines
`(3a). The variability of receptoractivity in intestinal contents
`was confirmed by EIA as described previously (3), and
`instability of receptor activity could be controlled by addi-
`tion of trypsin inhibitor to sample collection buffers. This
`technique was also used to demonstrate an effect of an
`exogenous enzyme (Detach) on a K88intestinal glycoprotein
`receptor,
`thereby confirming the disruptive influence of
`protease on the binding of K88 adhesin to intestinal tissue.
`The efficacy of Detach under field conditions has been
`confirmedbyfield trials in commercial piggeries where large
`reductions in diarrhea-induced mortality and the incidence
`of diarrhea have been observed.
`Recently we developed an in vitro (EIA) technique for
`monitoring the interaction among brush border preparations
`derived from human small intestine, ETEC, and LT. The
`effects of enzymatic treatment indicate that receptors for
`
`TABLE 2. Quantitative cultures of H10407 bacteria bound
`at five different sites
`
`Small
`. wg
`intestine site
`S1
`
`$2
`
`83
`
`S4
`
`S5
`
`Treatment?
`"
`
`D
`Cc
`D
`Cc
`D
`Cc
`D
`Cc
`D
`Cc
`
`Bacterial count (CFU/cm)°
`Washed
`Unwashed
`5.72 + 0.40
`6.96 + 0.34
`7.25 + 0.35
`8.13 + 0.44
`5.80 + 0.28
`6.90 + 0.46
`7.30 + 0.35
`8.26 + 0.31
`5.56 + 0.40
`7.04 + 0.50
`7.87 + 0.60
`9.29 + 0.38
`6.11 + 0.38
`7.20 + 0.63
`7.66 + 0.24
`9.46 + 0.29
`6.66 + 0.42
`8.80 + 0.58
`7.22 + 0.47
`8.93 + 0.36
`
`2 $1, duodenum; S2, proximal jejunum; $3, midjejunum; S4, distal jejunum;
`SS, ileum.
`+ D, Detach-treated rabbits; C, untreated rabbits.
`© Mean log;) + standard error recovered per centimeter of washed and
`unwashed small intestine at death or 24 h after RITARD challenge. Each mean
`reflects data from six or seven rabbits.
`
`MSNExhibit 1047 - Page 5 of 7
`MSNv. Bausch - IPR2023-00016
`
`

`

`VoL. 59, 1991
`
`PROTEASE AND PREVENTION OF ETEC DIARRHEA
`
`3713
`
`ACKNOWLEDGMENTS
`
`Wethank Alex Hauler and Barry Heywood fortheir invaluable
`assistance and advice. Analysis of the data by Leigh Callinan,
`Victorian Department of Agriculture Biometric Services,
`is also
`greatly appreciated.
`Wealso acknowledge the support of Enzacor Technology Pty.
`Ltd.
`
`REFERENCES
`
`rhea. The efficacy of protection based on the ability of
`protease treatment to reduce colony numbers was deter-
`mined to be 99.5% (>0 to 99.9%)(10). Although based on a
`small numberof animals, the data indicate that the concept
`of surface modification to prevent diarrheal disease has
`considerable potential. Further studies are required to deter-
`mine whetherthis concept can be applied to prevent ETEC-
`induced diarrhea in humans.
`
`CFA/I and CFA/II may besialoglycoproteins on the intesti-
`nal brush border membrane. Receptors for CFA/I on eryth-
`rocytes mayalso besialoglycoproteins (2, 13, 25). Binding of
`CFA/I, CFA/II, and LT to the human mucosain the EIAis
`interfered with by protease.
`The concept of surface modification to control human
`diarrheal disease has now beentested in vivo by means of
`the RITARD model. This model, devised by Spiraet al. (29),
`has previously been used extensively to study the pathogen-
`esis of ETEC andV.cholerae infection and to study protec-
`tive immunity afforded by vaccine candidates(1, 24, 26, 33).
`Thepresent study has demonstrated that oral administration
`of Detach, an enteric-coated protease preparation, was
`successful in reducing diarrhea and diarrhea-induced death
`by 86% (six of seven) in rabbits infected with the CFA/I
`strain H10407. Of the eight control rabbits which did not
`receive protease, seven (87%) died or developed severe
`diarrhea. One of the non-protease-treated rabbits survived
`the infection and did not develop diarrhea despite the
`1. Ahrén, C. M., and A.-M. Svennerholm. 1985. Experimental
`presence of large numbers of CFA/I* bacteria adherent to
`enterotoxin-induced Escherichia coli diarrhea and protection
`the mucosa (5.8 X 10? CFU/cm). Resistance of RITARD
`induced bypreviousinfection with bacteria of the same adhesin
`or enterotoxin type. Infect. Immun. 50:255-261.
`rabbits to large challenge doses of CFA/I* bacteria has been
`2. Bartus, H., P. Actor, E. Snipes, D. Sedlock, and I. Zajac. 1985.
`observed previously (1, 26). This mayreflect physiological,
`Indications that the erythrocyte receptor involved in enterotoxi-
`genetic, or immunevariation. Conversely, one of the seven
`genic Escherichia coli attachmentis a sialoglycoconjugate. J.
`protease-treated rabbits died. It is possible that stress caused
`Clin. Microbiol. 21:951-954.
`by oral dosing and starvation prior to surgery induced gut
`2a.Chandler, D. S., et al. Unpublished data.
`stasis, thereby preventing passage of protease through the
`3. Chandler, D. S., H. M. Chandler, R. K. J. Luke, S. R. Tzipori,
`stomach. Reliable specific protease detection assays are
`and J. A. Craven. 1986. Screening of pig intestines for K88
`currently being developed to enable movement of Detach
`non-adhesive phenotype by enzyme immunoassay. Vet. Micro-
`biol. 11:153-161.
`throughthe gastrointestinal tract to be monitored.
`3a.Chandler, D. S., T. L. Mynott, R. K. J. Luke, and J. H. Craven.
`Wankeand Guerrant(35) have shownthat enterotoxigenic
`Submitted for publication.
`CFA/II* bacteria (strain E1392) induce diarrhea in RITARD
`4. Clemens, J. D., D. A. Sack, J. R. Harris, J. Chakraborty, P. K.
`rabbits and colonize to a level of more than 10°> CFU/cm? of
`Neogy, B. Stanton, N. Huda, M. U. Khan, B. A. Kay, M. R.
`small intestine. None of rabbits given CFA-negative 13927
`Khan, M. Ansaruzzaman, M. Yunus, M. Raghava Rao, A.-M.
`bacteria were colonized with more than 10° CFU/cm?, and
`Svennerholm, and J. Holmgren. 1988. Cross protection by B
`none developed diarrhea. Their report showsthat the thresh-
`subunit-whole cell cholera vaccine against diarrhoea associated
`old for expression of clinical symptomsofdiarrheal infection
`with heat
`labile toxin-producing enterotoxigenic Escherichia
`was 10° CFU/cm? (35). In the present study, rabbits chal-
`coli: results of a large scale field trial. J. Infect. Dis. 158:372-
`377.
`lenged with bacterial strains possessing no knowncoloniza-
`5. Darfeuille-Michaud, A., C. Forestier, B. Joly, and R. Cluzel.
`tion factors did not develop diarrhea and were colonized to
`1986.
`Identification of a nonfimbrial adhesive factor of an
`levels below 5 x 10’ CFU/cm. In these rabbits there was no
`enterotoxigenic Escherichia coli strain. Infect. Immun. 52:468—
`significant difference between colonization of protease-
`475.
`treated and non-protease-treated groups. In contrast, non-
`6. Evans, D. G., D. J. Evans, Jr., S. Clegg, and J. A. Pauley. 1979.
`protease-treated rabbits challenged with the CFA/I* strain
`Purification and characterization of the CFA/I antigen of entero-
`H10407 were colonized to levels well above 10’ CFU/cm
`toxigenic Escherichia coli. Infect. Immun. 25:738-748.
`(arithmetic mean, 6.2 x 10°). Seven ofeight of these rabbits
`7. Evans, D. G., D. J. Evans, Jr., W. S. Tjoa, and H. L. DuPont.
`1978. Detection and characterization of colonization factor of
`either developed severe diarrheaor died in a diarrhea-related
`manner. Rabbits treated with protease and challenged with
`enterotoxigenic Escherichia coli isolated from adults with diar-
`rhea. Infect. Immun. 19:727-736.
`strain H10407 were colonized to levels below 10’ CFU/cm
`8. Evans, D. G., D. Y. Graham, D. J. Evans, Jr., and A. Opebun.
`(arithmetic mean, 2.6 x 10° CFU/cm), i.e., levels similar to
`1984. Administration of purified colonisation factor antigens
`those observed when the challenge strain produced no
`(CFA/I, CFA/II) of enterotoxigenic Escherichia coli to volun-
`known CFA(Fig. 1). This represents a more than 2,000-fold
`teers. Gastroenterology 87:934-940.
`reduction in CFU per centimeter of tissue. It is apparent
`9. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1973.
`therefore that oral treatment with protease was successfulin
`Production of vascu