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,1* DAVID S. CHANDLER,2 AND RICHARD K. J. LUKE'
`School of Agriculture, La Trobe University, Bundoora, Victoria 3083,' and Victorian Institute ofAnimal Science,
`Attwood, Victoria 3049,2 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 coli (ETEC). 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 death in 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. The efficacy 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.
`
`The role of enterotoxigenic Escherichia coli (ETEC) as an
`important etiologic agent in human diarrheal disease is well
`established (31). These organisms are characterized by their
`ability to produce heat-labile toxin (LT) and/or heat-stable
`toxin (ST) (11). Some strains also produce pilus adhesins
`called colonization factor antigens (CFAs). These adhesins
`permit attachment of ETEC strains to the intestinal mucosa,
`thereby facilitating colonization and delivery of enterotoxin
`to target epithelial cells.
`CFAs may 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 CFAs and outer membrane
`antigens associated with ETEC is 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 produce heat-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).
`Other efforts to develop vaccines against ETEC infection
`
`* Corresponding author.
`
`have given variable results (8, 18, 19), and while the prospect
`of effective immunization against diarrheal diseases of hu-
`mans is 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 and pig brush
`border membranes can 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 number of 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 human diarrheal 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 work represents a novel approach to preventing attach-
`ment of ETEC to intestinal cells and therefore preventing
`diarrheal disease.
`
`3708
`
`MYLAN EXHIBIT - 1047
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`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
`week prior to the start of experimentation.
`Bacteria. ETEC strains used in this study were originally
`isolated in Bangladesh from patients with diarrhea. Strain
`H10407 (serotype 078:K80:H11) and a mutant derivative 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
`E. coli 1392 (17) and has been shown
`derivative of CFA/II1
`not to colonize or induce diarrhea in the RITARD model
`(35).
`Stock cultures of all strains were suspended in 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 was purified 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 subcutaneous injections with 60 p.g of
`purified CFA/I at intervals of 4 weeks. For the 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 weeks after the final immunization. LT-specific
`antiserum was similarly produced by injecting 60 ,ug 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 CFAII 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 pl per well) by incubation overnight at
`4°C. For CFA/I detection, bacteria were diluted to approx-
`imately 2.5 x 109 cells per ml (A625 = 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 x 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 ,ul 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 ,ul) was used, and a positive reaction was indicated by a
`color change from yellow to purple. During each of the
`above steps, plates were incubated at 37°C for 30 min.
`Between each 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. Development of purple color was monitored
`at A540.
`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 of lignocaine 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
`proximal to the cecal tie with a slipknot tie with umbilical
`tape. Inoculum (10 ml) containing 1011 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. The ileal tie was gently
`removed 2 h after bacterial challenge, and the rabbits were
`returned to their cages, where food and water were available
`ad libitum. All surviving animals were killed 24 h postchal-
`lenge by means of barbiturate overdose; some animals
`infected with the CFAII+ bacteria died within the 24 h.
`Immediately after death, each animal was autopsied and the
`peritoneal cavity was swabbed for 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-
`ment in the absence of bacterial challenge to be monitored.
`The challenge rate of 1011 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 performed on rabbits in groups of
`eight over a period of 4 weeks (two rabbits from each group
`each week; see Table 1). Two weeks later, 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
`organisms were identified by means of typical E. coli colony
`
`

`

`3710
`
`MYNOTT ET AL.
`
`INFECT. IMMUN.
`
`TABLE 1. Diarrheal response in rabbits treated with Detach or
`untreated and challenged with different ETEC strains
`
`Strain
`
`Adhesin
`
`Toxin
`
`H10407C
`
`CFA/I+
`
`ST+ LT+
`
`H10407-P
`
`CFA/1-
`
`ST+ LT+
`
`E1392/75 7A CFA/II- ST- LT-
`
`Nil (PBS)
`
`Diarrheal
`Treat-
`responseb
`menta
`1/7d
`D
`7/8
`C
`1/49
`D
`C
`1/49
`0/4
`D
`0/4
`C
`0/4
`D
`0/4
`C
`a D, rabbits treated with Detach; C, untreated rabbits.
`b 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 seven of eight untreated animals challenged with the same
`dose rate.
`c Five rabbits were omitted from the analysis because of death not related
`to diarrhea.
`d One Detach-treated rabbit died; CFU/cm at S3 was 1.2 x 107.
`e One rabbit survived infection; CFU/cm at S3 was 5.8 x 109.
`f The total volume of fluid accumulated in small and large intestines
`combined was 130 to 165 ml.
`g Mild diarrhea (score 1).
`
`Small intestine
`fluid vol (ml)
`35-50
`20-105f
`10-50
`10-50
`15-50
`15-50
`8-12
`24-40
`
`with the CFA-negative, toxigenic H10407-P passed feces
`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 was in 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 was no significant 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
`were plated on blood agar and MacConkey agar. Leakage of
`bacteria from the inoculation site was deemed to be the
`cause of death, as colonies of typical E. coli morphology
`were isolated from the peritoneum of these rabbits. No such
`bacteria were detected in peritoneal swabs from the rabbits
`which died in a diarrhea-related manner or in swabs from 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 no clinical signs of infection. At autopsy,
`the fluid volume in the small intestines of rabbits which did
`not pass feces ranged from 20 to 105 ml. The total volume in
`the small and large intestine combined, however, ranged
`from 130 to 165 ml (in comparison with 10 to 50 ml in rabbits
`inoculated with E1392/75 7A, H10407-P, and PBS). In com-
`parison, the small intestine contained relatively little fluid (35
`to 50 ml) in rabbits that had diarrhea at the time of death. The
`total volume of intestinal fluid was only 55 to 60 ml. A lack
`of fluid accumulation in rabbits that have passed feces at 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 volumes in 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 24 h. 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 of chal-
`lenge bacteria in different parts of the small intestine. Chal-
`lenge bacteria were present at all sites, with CFA/I+ H10407
`being the most effective colonizer. The mean CFU levels of
`CFA/I+ bacteria at sampling sites in non-protease-treated
`rabbits were lower at S1, S2, S4, and S5 (1.1 x 108, 6.7 x
`107, 1.0 x 108, and 4.0 x 108 CFU/cm, respectively) than at
`S3 (5.5 x 109 CFU/cm). In the analysis which follows, S3
`cultures are used for comparison. The recovery rates of
`different organisms from 1 cm of mucosa are shown in Fig. 1.
`The number of CFA/I+ bacteria adherent to the mucosa in
`the protease-treated rabbits ranged from 1.3 x 104 (minimum
`count) to 1.2 x 107 CFU/cm in the rabbit that died (arith-
`metic mean, 2.6 x 106 CFU/cm). Values for control rabbits
`challenged with the same strain were 2,000 times greater (P
`< 0.001). Figure 2 illustrates the differences in colony counts
`between protease-treated and untreated animals.
`
`morphology and enzyme-linked immunosorbent assay nitro-
`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 (Si), proximal jejunum
`(S2), midjejunum (S3), distal jejunum (S4), and ileum (S5).
`Each segment was opened longitudinally and washed exten-
`sively in sterile PBS to determine the numbers of 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, and aliquots (25 ,ul) were plated onto sheep blood agar
`(5%, vol/vol) and CFA agar. After incubation at 37°C for 18
`h, the number of CFU per centimeter of tissue was deter-
`mined. Other specimens were processed promptly for his-
`tology following fixation in 10% neutral-buffered formalin.
`After specimens had been embedded in 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 E1392/5 7A, 15 to 50 ml of
`fluid accumulated. Two of eight rabbits that were challenged
`
`

`

`VOL. 59, 1991
`
`PROTEASE AND PREVENTION OF ETEC DIARRHEA
`
`3711
`
`Diarrhoea
`
`No Diarrhoea
`
`0U
`
`0
`
`.
`
`cica
`
`0
`
`U a
`
`0
`
`0U
`
`0
`
`U 8
`
`0
`
`UU
`
`a0
`
`0 9
`
`000 8 U
`
`U U U U
`
`No
`
`CFU/cm
`
`1010
`
`109
`
`108
`
`107
`
`106
`
`105
`
`104
`
`H10407
`
`H10407-P
`
`E1392/75
`7A
`
`PBS
`
`FIG. 1. Range of quantitative cultures at midjejunum (S3) of rabbits challenged with different ETEC strains or given sterile PBS. *, rabbits
`treated with Detach 18 h prior to challenge; 0, untreated.
`
`The number of bacteria bound to the small intestinal
`mucosa of rabbits infected with the CFA/I- strain H10407-P
`ranged from 1.3 x 104 CFU/cm (minimum count) to 6.6 x
`107 CFU/cm in a rabbit with mild diarrhea (arithmetic mean,
`CFU/cm). Counts for strain E1392/75 7A were
`1.8 x .
`similar, ranging from 1.3 x 104 (minimum count) to 2 x 108
`CFIJ/cm (arithmetic mean, 3.9 x 107 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 104
`CFU/cm). There were, however, two exceptions. Two con-
`trol rabbits which did not receive protease had colony counts
`of 4.6 x 106 and 5.2 x 10' CFU/cm at S3 (small intestinal
`volumes of 40 and 35 ml, respectively). One of these rabbits
`had 5.1 x 1010 and 4.1 x 1010 CFU/cm at Si and S5,
`respectively. All colonies observed had typical 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 were the result of
`overgrowth of endogenous bacteria.
`To assess the total number of bacteria present on the gut
`mucosa, sections were left unwashed. The mean values of
`washed versus unwashed sections from rabbits challenged
`with CFA/I+ are shown in Table 2. These results suggest
`that the organisms are strongly adherent to the mucosa, as
`only 102 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 apparent in 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 bound in 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 ETEC strains 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 and efficient 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+
`ETEC to the brush border membrane of piglets is known to
`be a multimeric glycoprotein (30). This and other receptors
`
`

`

`3712
`
`MYNOTT ET AL.
`
`INFECT. IMMUN.
`
`BX, I
`
`Mean (log I
`CFU/cm
`10-
`
`-
`
`9 8
`
`7-
`6-
`
`5-
`4-
`3-
`2-
`
`I
`
`1 A
`
`Mean (log l)
`CFU/cm
`
`10:1-g_9
`
`'k
`
`8 -
`7 -
`6 -
`5-
`4-
`3-
`2-
`I1-
`
`Site 1
`
`Site 3
`
`Site 5
`
`Site 1
`
`Site 3
`
`Site 5
`
`Mean (log 1O)
`CFU/cm
`10 -
`
`D
`
`. -
`
`I~~~~-
`
`8-
`7 -
`
`6 5
`
`-
`4-
`
`3-
`2-
`1-
`0
`
`T
`
`/
`
`Mean (log 10)
`CFU/cm
`10-
`
`C
`
`-
`
`9 8
`
`7-
`
`6 5
`
`-
`4-
`3-
`2-
`1
`
`Site 1
`Site 3
`Site 3
`Site 1
`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 (log1o)
`standard error CFU per centimeter of tissue.
`*, rabbits treated with Detach 18 h prior to bacterial challenge; 0, untreated rabbits.
`
`Site 5
`
`Site 5
`
`TABLE 2. Quantitative cultures of H10407 bacteria bound
`at five different sites
`Bacterial count (CFU/cm)c
`tb
`TreatmenWashed
`Unwashed
`
`Small
`intestine sitea
`
`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
`observing the instability of K88 receptor within pig intestines
`(3a). The variability of receptor activity 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 K88 intestinal 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
`confirmed by field 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
`
`S4
`
`S5
`
`Si
`
`S2
`
`S3
`
`6.96 + 0.34
`5.72 + 0.40
`D
`8.13 ± 0.44
`7.25 t 0.35
`C
`6.90 ± 0.46
`5.80 ± 0.28
`D
`8.26 ± 0.31
`7.30 ± 0.35
`C
`7.04 ± 0.50
`5.56 ± 0.40
`D
`7.87 ± 0.60
`9.29 ± 0.38
`C
`6.11 ± 0.38
`7.20 + 0.63
`D
`9.46 ± 0.29
`7.66 ± 0.24
`C
`8.80 ± 0.58
`6.66 ± 0.42
`D
`8.93 ± 0.36
`7.22 + 0.47
`C
`a Si, duodenum; S2, proximal jejunum; S3, midjejunum; S4, distal jejunum;
`S5, ileum.
`b D, Detach-treated rabbits; C, untreated rabbits.
`c Mean loglo + 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.
`
`

`

`VOL. 59, 1991
`
`PROTEASE AND PREVENTION OF ETEC DIARRHEA
`
`3713
`
`CFA/I and CFA/II may be sialoglycoproteins on the intesti-
`nal brush border membrane. Receptors for CFA/I on eryth-
`rocytes may also be sialoglycoproteins (2, 13, 25). Binding of
`CFA/I, CFA/II, and LT to the human mucosa in the EIA is
`interfered with by protease.
`The concept of surface modification to control human
`diarrheal disease has now been tested in vivo by means of
`the RITARD model. This model, devised by Spira et al. (29),
`has previously been used extensively to study the pathogen-
`esis of ETEC and V. cholerae infection and to study protec-
`tive immunity afforded by vaccine candidates (1, 24, 26, 33).
`The present 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
`presence of large numbers of CFA/I1
`bacteria adherent to
`the mucosa (5.8 x 109 CFU/cm). Resistance of RITARD
`rabbits to large challenge doses of CFA/I1
`bacteria has been
`observed previously (1, 26). This may reflect physiological,
`genetic, or immune variation. Conversely, one -of the seven
`protease-treated rabbits died. It is possible that stress caused
`by oral dosing and starvation prior to surgery induced gut
`stasis, thereby preventing passage of protease through the
`stomach. Reliable specific protease detection assays are
`currently being developed to enable movement of Detach
`through the gastrointestinal tract to be monitored.
`Wanke and Guerrant (35) have shown that enterotoxigenic
`CFA/II1
`bacteria (strain E1392) induce diarrhea in RITARD
`rabbits and colonize to a level of more than 108.5 CFU/cm2 of
`small intestine. None of rabbits given CFA-negative 1392-
`bacteria were colonized with more than 108 CFU/cm2, and
`none developed diarrhea. Their report shows that the thresh-
`old for expression of clinical symptoms of diarrheal infection
`was 108 CFU/cm2 (35). In the present study, rabbits chal-
`lenged with bacterial strains possessing no known coloniza-
`tion factors did not develop diarrhea and were colonized to
`levels below 5 x 107 CFU/cm. In these rabbits there was no
`significant difference between colonization of protease-
`treated and non-protease-treated groups. In contrast, non-
`protease-treated rabbits challenged with the CFA/I+ strain
`H10407 were colonized to levels well above 107 CFU/cm
`(arithmetic mean, 6.2 x 109). Seven of eight of these rabbits
`either developed severe diarrhea or died in a diarrhea-related
`manner. Rabbits treated with protease and challenged with
`strain H10407 were colonized to levels below 107 CFU/cm
`(arithmetic mean, 2.6 x 106 CFU/cm), i.e., levels similar to
`those observed when the challenge strain produced no
`known CFA (Fig. 1). This represents a more than 2,000-fold
`reduction in CFU per centimeter of tissue. It is apparent
`therefore that oral treatment with protease was successful in
`modifying the surface of the rabbit mucosa such that colo-
`nization of CFA/I1
`bacteria was significantly (P < 0.001)
`reduced.
`In summary, oral administration of an enteric-coated
`protease preparation was successful in reducing diarrhea and
`diarrhea-induced death by 86% (P < 0.001) in rabbits chal-
`lenged with CFA/I-positive H10407. Quantitative analysis of
`the cultures obtained from the small intestine of these rabbits
`showed a significant (P < 0.001) treatment effect, with a
`greater than 2,000-fold reduction in colonies per centimeter
`of intestine. The reduction brings the number of bacteria
`below the threshold required for the development of diar-
`
`rhea. The efficacy of protection based on the ability of
`protease treatment to reduce colony numbers was deter-
`mined to be 99.5% (>O to 99.9%) (10). Although based on a
`small number of animals, the data indicate that the concept
`of surface modification to prevent diarrheal disease has
`considerable potential. Further studies are required to deter-
`mine whether this concept can be applied to prevent ETEC-
`induced diarrhea in humans.
`
`ACKNOWLEDGMENTS
`We thank Alex Hauler and Barry Heywood for their invaluable
`assistance and advice. Analysis of the data by Leigh Callinan,
`Victorian Department of Agriculture Biometric Services, is also
`greatly appreciated.
`We also acknowledge the support of Enzacor Technology Pty.
`Ltd.
`
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