28
`
`Gut 1996; 38: 28-32
`
`Oral administration of protease inhibits
`enterotoxigenic Escherichia coli receptor activity in
`piglet small intestine
`
`T L Mynott, R KJ Luke, D S Chandler
`
`Abstract
`The virulence of enterotoxigenic Escheri-
`chia coli (ETEC) is attributed to their
`ability to adhere via fimbrial adhesins to
`specific receptors located on the intestinal
`mucosa. A novel approach to preventing
`ETEC induced diarrhoea wouldbe to pre-
`vent attachment of ETECto intestine by
`proteolytically modifying the receptor
`attachment sites. This study aimed to
`examinethe effect of bromelain, a prote-
`olytic extract obtained from pineapple
`stems, on ETEC receptor activity in
`porcine small
`intestine. Bromelain was
`administered orally to piglets and K88*
`ETECattachment to small intestine was
`measured at 50 cm intervals using an
`enzyme immunoassay. K88* ETECattach-
`ment to intestinal sections that were not
`treated with bromelain varied appreciably
`between sampling sites. Variability in
`receptor activity along the intestinal sur-
`face is thoughtto be caused bythe localised
`effects of endogenous proteases. Oral
`administration of exogenous protease
`inhibited K88+ ETEC attachment to pig
`small
`intestine in a dose dependent
`manner
`(p<0-05). Attachment of K88*
`ETEC was negligible after
`treatment,
`resemblingthe levels of attachment of K88
`to piglets of the genetically determined
`non-adhesive phenotype, which areresist-
`ant to K88+ ETEC infection. Serum bio-
`chemical analysis and histopathological
`examination of treated piglets showed no
`adverseeffects ofthe bromelain treatment.
`It
`is concluded that administration of
`bromelain can inhibit ETEC receptor
`activity in vivo and maytherefore be useful
`for prevention of K88* ETEC induced
`diarrhoea.
`(Gut 1996; 38: 28-32)
`
`Keywords: enterotoxigenic Escherichia coli, diarrhoea,
`K88 ETEC,pig intestine, protease.
`
`youngpiglets. K88 occurs in several antigenic
`types or variants,all of which adhereto piglet
`enterocytes.> K88ab, K88ac, K88ad, and
`K88ad(e) variants have been characterised.® 7
`In 1975, Rutter et al® showed that somepigs
`are resistant to colonisation and disease caused
`by K88-positive (K88*) E coli. An adhesive
`phenotype (one susceptible to infection)
`is
`related directly to the ability of K88* bacteria
`to recognise intestinal receptors and attach to
`piglet
`intestinal brush border membranes.
`Intestine obtained from the non-adhesive
`phenotype (disease resistant pigs) do not bind
`K88+t E coli and as a result such animals
`are resistant to K88* E coli infection.? The
`receptors on non-adhesive intestinal brush
`border cells may be absent or non-functional.
`A genetic basis for expression of the adhesive
`or non-adhesive phenotype exists.8!° Differ-
`ent phenotypes may also be distinguished,
`depending on the serological variant of the
`K88antigen.!! Also, in addition to genotype,
`physiological factors, particularly the level of
`intestinal proteolysis within the small intestine,
`mayinfluence the ability of ETEC to attach to
`intestine.!2 13
`The ability of protease to prevent attach-
`ment of ETEC to small intestinal samples in
`vitro is well documented.!3?"!8 Presumably
`attachment ability is prevented because of
`proteolytic cleavage of ETEC receptorsites.
`This
`study aimed to investigate whether
`exogenousprotease, administered orally, could
`inhibit porcine K88+ ETECreceptoractivity
`in vivo and therefore inhibit K88t ETEC
`attachment
`to small
`intestine. The use of
`protease, through its ability to modify intesti-
`nal receptor sites and reduce the binding
`properties of the intestinal mucosa, may be an
`important way of protecting the small intestine
`from microbial colonisation and disease.!? !3
`
`Methods
`
`ANIMALS
`
`Enterotoxigenic Escherichia coli (ETEC) are an
`important cause of disease in young children!
`and young animals.? The virulence of ETEC
`strains is attributed to their ability to adhere,
`via
`fimbrial
`adhesins,
`to highly specific
`receptors located on the intestinal mucosa.?
`These strains also liberate heat labile (LT)
`and/or heat stable (ST) enterotoxins which
`cause fluid secretion and diarrhoea. E coli
`strains which carry the K88 adhesin ontheir
`surface are a significant cause of diarrhoea in
`
`Approval for animal experiments was granted
`by the Victorian Institute for Animal Science
`Animal Experimentation Ethics Committee.
`Five pregnant sows (Large White X Landrace
`X Duroc) were purchased from a commercial
`farm where the incidence of pigs with the non-
`adhesive phenotypeis low (1 in 10).!9 Piglets
`were born within three days of each other in
`the animal housing facility at the Victorian
`Institute of Animal Science (VIAS-Attwood,
`Victoria, Australia). Piglets were weaned at
`approximately 3 weeks of age (weight notless
`
`MSNExhibit 1046 - Page 1 of 5
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`School of Agriculture,
`La Trobe University,
`Bundoora,Victoria
`3083, Australia
`T L Mynott
`RKJ Luke
`Victorian Institute of
`Animal Science,
`Department of
`Agriculture, Attwood,
`Victoria 3049,
`Australia
`TL Mynott
`D S Chandler
`
`Correspondenceto:
`Dr T L Mynott, Digestive
`Diseases Research Centre,
`The Medical College of Saint
`Bartholomew’s Hospital, 4th
`Floor Science Block, Charter
`House Square, London
`ECIM 6BQ. United
`Kingdom.
`Accepted for publication
`31 May 1995
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`K88* enterotoxigenic Escherichia coli (ETEC) attachmentto piglet small intestine
`terminal ileum was 8 to 10 m)ofall pigs was
`removed immediately at death. Brush border
`K88 receptor activity (mean (SD))¢
`Protease
`Non-adhesive
`material was obtained at 50 cm intervals
`StrongS
`Group*
`(mgt
`(Wr
`Non-adhesive
`Moderate
`(n=19 samples per pig) by gently scraping 1
`0
`A
`
`0-409 (0-244) cm sectionsofthe villous surface withasterile
`0-211 (0-178)
`0-078 (0-029)
`29
`0-709 (0-090)
`0-311 (0-108)
`0-069 (0-045)
`cotton-tipped swab. The swabs were then
`0-230 (0-095)
`0-660 (0-096)
`immersed in 3 ml of a working dilution buffer
`0-295 (0-139)
`0-312 (0-187)
`(WDB)consisting of phosphate buffered saline
`(PBS; 0-1 M, pH 7-4) to which bovine serum
`albumin (BSA; 0-25% w/v), EDTA (di-sodium
`salt;
`1 mM), Tween 20 (0:05% v/v) and
`sodium azide (0:02% w/v) had been added.
`The swabs were vortexed to recover the epithe-
`lial cells. Brush border samples were assessed
`for their ability to attach to K88* ETEC
`(strain WG (0149:K91(B):K88ac:H10)2° by
`enzyme immunoassay (EIA)
`as_ previously
`described.!3 2! The EIA gives 95%correlation
`with results obtained by traditional micro-
`scopic adhesiontests for assessing K88 pheno-
`type?! (piglets genetic propensity to produce
`K88receptoronits intestinal surface). Briefly,
`K88+t ETEC are immobilised to wells of a
`microtitre plate and incubated with mucosal
`samples. Mucosal material, bound to the bac-
`teria is detected with antibody (rabbit IgG)
`raised against porcine intestine followed by
`urease-conjugated
`goat
`anti-rabbit
`IgG
`(Sigma) and urea substrate.
`Disposable polystyrene microtitre plates
`(Nunc, Denmark) were used for all assays.
`K88* ETECdiluted to approximately 210°
`bacteria/ml
`in sodium bicarbonate buffer
`(NaHCO,/Na,CO;3; 0-1 M, pH 9-6), were
`adsorbed to wells (100 jl/well) by incubation
`overnight at 4°C. Active binding sites remain-
`ing on wells following coating procedures were
`routinely blocked by incubation (30 min at
`37°C) with BSA (1%w/v) dissolved in PBS
`(200 l/well). Mucosal material bound to
`K88*+ ETEC was detected using rabbit IgG
`raised against a Triton X-100 extract of K88-
`adhesive phenotype brush border vesicles,
`diluted in WDB.?! Anti-rabbit urease-con-
`jugated IgG (Sigma) was diluted in WDB
`containing ovalbumin (from hen egg, gradeII,
`Sigma 1%w/v). All incubation steps, exclud-
`ing the coating procedures, were performedat
`37°C for 30 minutes. Between each of the
`incubation steps,
`supernatant
`liquid was
`removed from the wells which were then
`washed three times with washing buffer (0-1 M
`PBS; 0-05%v/v Tween 20). Before the incuba-
`tion with substrate, wells were washed with
`distilled water to removeanyeffect of residual
`buffer on substrate solutions. Urea substrate
`[bromocresol purple (0-15 mM)urea (16 mM),
`EDTA (disodium salt,
`1 mM); pH 4-8, 100
`pl/well] was used to detect the presence of
`bound, conjugated enzyme. A positive reaction
`was indicated by a colour change from yellow
`to purple which was measured spectro-
`photometrically at Asqonm- All assays were
`standardised by developing the reaction until
`a positive control attained an EIA value of
`0-6.
`intestine
`the
`The K88 phenotype of
`scrapings wasassessed andan adhesion pattern
`for the small intestine of each pig was estab-
`lished. To obtain an overall indication of the
`
`125
`
`250
`
`625
`
`0-359 (0-225),
`
`0-224 (0-195),
`
`57
`
`86
`
`86
`
`86
`
`Oral administration ofprotease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine
`
`29
`
`0-080 (0-099).)
`0-119 (0-071) ¢)
`0-034 (0-014),
`0-055 (0-026)
`0-061 (0-027),
`0-079 (0-057),
`0-039 (0-013).
`0-191 (0-117)
`0-045 (0-017)
`0-044 (0-021)
`0-096 (0-036),
`0-176 (0-118)
`0-033 (0-015)9)
`0-099 (0-115),
`0-034 (0-015)
`0-064 (0-022)
`0-036 (0-015),
`0-059 (0-015).
`0-032 (0-009)(9)
`0-050 (0-040)(9)
`0-033 (0-015)
`0-182 (0-135)
`
`
`1250
`
`0-238 (0-183)
`
`*Numberofpigs per group is 7. tAmountof bromelain administered per dose, three times a day
`for either two (2) days (n=4)or five days (n=3). There was nosignificant difference between
`duration of treatment and reduction in EIA activity (p=0-82). Ability of protease to reduce K88
`receptoractivity was significant (p<0-05; ANOVA). {Values represent the mean (SD)
`absorbanceof the A 549 nm Value of 19 sampling sites per pig. The large SD observed in some
`pigs reflect the variability of receptor activity along the length of the small intestine. |K88*
`ETECattachmentexpressed as strongly adhesive (EIA activities >0-4); moderately adhesive
`GIA activities 0-20 to 0-40); and non-adhesive (EIA activity <0-2). tNumber of K88*t ETEC
`non-adhesive versus numbertested expressed as a percentage. Ofthe total numberofpigs
`treated with protease, 79% (22 of 28) were non-adhesive compared with 29%(2 of 7) pigs not
`treated with protease (p<0-02, Fisher’s exact test).
`
`than 6 kg), and housed in stables with straw
`bedding. Piglets were fed ad libitum a commer-
`cial
`starter
`diet
`(Gropower,
` Barastoc,
`Australia)
`for
`two weeks
`after weaning,
`followed by Grower 8 (Barastoc, Australia) in
`three divided meals (160 g/6 kg of body weight
`daily) until experimentation. Thirty five piglets
`of weight 20 to 25 kg (aged 10 to 16 weeks)
`were used for the study and randomly allo-
`cated between five treatment groups (Table).
`
`PIGLET TREATMENT
`
`cysteine
`a
`is
`Bromelain (E.C. 3.4.22.4)
`stems.
`protease obtained from pineapple
`Bromelain is a glycoprotein and active across a
`wide pH range,thereforeit is ideally suited for
`the gastrointestinal environment. Different
`amounts
`of enteric protected bromelain
`(Detach, Cortecs Ltd, Middlesex UK; 1 g of
`Detach contains 125 mg of bromelain) was
`administered to pigs for two or five days to
`investigate the effect of duration of treatment
`on K88* ETECreceptoractivity and to inves-
`tigate the safety of treatment. The bromelain
`preparation was suspended in water (1g per 5
`ml) and administered by mouth usinga plastic
`syringe, three times a day 15 min before feed-
`ing (Table). Control piglets were untreated
`and administered 25 ml of water. All animals
`were monitored daily to observe any adverse
`clinical effects attributable to treatment. At the
`completion of the experiment animals were
`killed by barbiturate overdose, and autopsied.
`
`ASSESSMENT OF K88 PHENOTYPE BY ENZYME
`IMMUNOASSAY(EIA)
`The small intestine (length from pyloris to the
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`30
`
`K88-adhesivenessof a pig, the mean EJA value
`of all sampling sites (n=19 per pig) was deter-
`mined. The adhesiveness of a particular pig
`could be designated as
`strongly adhesive
`(S; mean EIA>0-4); moderately adhesive
`(M; mean EIA 0-2 to 0-4) or non-adhesive
`(N; mean EIA<0-2). (See Table.)
`
`SERUM BIOCHEMISTRY
`
`Blood samples (10 ml) were collected from
`piglets via the jugular vein one day before
`experimentation and again immediately before
`death. Serum samples were stored at —20°C
`until completion of the experiment, at which
`time they were submitted for biochemical
`analysis. Tests performed include a full bio-
`chemicalprofile, liver function tests, creatinine
`kinase, lipase, and amylase. As normal values
`for biochemical parameters cited in the litera-
`ture vary becauseoffactors such as differences
`in sample handling, assay technique, dietary
`influences or genetic differences between
`animals, pre-treatment values were taken as an
`indication of baseline normal values. These
`values were within the normal ranges cited in
`the literature.2? 23 Differences between serum
`samples before and after treatment were com-
`pared for each pig. In addition,
`the mean
`serum value for each parameterfor the treated
`groups was compared with that for the non-
`treatmentgroup.
`
`HISTOPATHOLOGY
`
`All animals weresacrificed by barbiturate over-
`dose two or five days after beginning treat-
`ment. At autopsy, specimens were immediately
`processed for histological examination after
`fixation with neutral buffered formalin (0°65%
`(wiv) Na,zHPO,, 0-45 (w/v) NaH,PO,, 10%
`(v/v) formalin). Sections of duodenum, mid-
`jejunum,andileum,werestained with haema-
`toxylin and eosin and examined by light
`microscopy
`for morphological
`changes.
`Sectionsof heart, kidney, liver, and mesenteric
`lymph nodeswerealso investigated.
`
`STATISTICAL ANALYSIS
`
`parameters,
`biochemical
`in
`Differences
`between serum samples, before andaftertreat-
`ment, and differences between treatment
`groups were assessed for clinical relevance by
`veterinary pathologists
`at VIAS-Attwood.
`Differences among mean serum biochemical
`values between treatment groups were sub-
`jected
`to
`one-way
`analysis of variance
`(ANOVA)using Microstat. Difference among
`mean EIA values for all treatment groups (0
`mg to 1250 mg) representing mean K88t
`ETEC receptor activity was analysed by
`Genestat V for analysis of variance.
`
`Results
`
`K88+t ETEC ATTACHMENT TO SMALL
`INTESTINE OF UNTREATED (CONTROLPIGS)
`Wefirst examined the ability of K88* ETEC
`
`0-9
`0-8
`0-7
`06
`0-5
`0-4
`0.3
`0.2
`0-1
`0.0
`
`0-9
`0-8
`0.7
`0-6
`0-5
`0-4
`0.3
`0.2
`0-1
`0.0
`
`50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900ileum
`
`S
`
`N
`
`
`
`K88ETECreceptoractivity(Asaonm)
`
`
`
`
`
`Mynott, Luke, Chandler
`
`to attach to the small intestine of pigs that were
`not treated with bromelain (group A). The
`Table summarises the results obtained from all
`untreated pigs. Two pigs were of the strongly
`adhesive phenotype, three pigs were moder-
`ately adhesive and two pigs were of non-
`adhesive phenotype. Figure 1
`shows EIA
`values obtained from intestine of three pigs to
`showthe three phenotypes obtained.
`We next compared the binding of K88*
`ETECto different sections of intestine in indi-
`vidual pigs. K88* ETEC attachment
`to
`intestine varied markedly between samples
`taken at 50 cm intervals (Fig 1). Multiple
`scrapings taken at
`the same site revealed
`similar results indicating that the variability
`between sites was a function of that section of
`intestine, not
`the assay. Earlier, Chandler
`et al'3 observed a similar variation in binding
`between sampling sites and related this
`variation to the state of distention or constric-
`tion of the small intestine at the samplingsite.
`Despite the variation seen between individual
`scrapings, a consistent pattern was observed
`
`0.9
`0.8
`0.7
`0-6
`0-5
`0-4
`0.3
`0.2
`0-1
`0-0 oooooece|co
`WOoONMNOMONMWdMS
`TENNAMYS SLO 550 600 650 700 750 800 850 900
`
`Distance along small intestine (cm)
`Figure 1: Use ofenzyme immunoassay to demonstrate
`variability in K88* enterotoxigenic Escherichia coli
`(ETEC) receptor activity and attachment ofETEC to
`three untreated pigs. Columns represent the mean
`absorbance of the A540 nm values of duplicate wells. S,
`strongly adhesive; M, moderately adhesive; N, non-
`adhesive.
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`Oral administration ofprotease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine
`
`31
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`0-4
`
`p<0-05
`
`and after treatment (data not shown). Pre- and
`post treatment parameters measured remained
`within the normal range.?? 23
`
`
`
`
`
`K88ETECreceptoractivity(Agqopm)
`
`
`
`0
`
`125
`
`250
`
`625
`
`1250
`
`Bromelain dose (mg, 3 times/d)
`Figure 2: K88* enterotoxigenic Escherichia coli ETEC
`receptor activity of small intestine samples ofpigs treated
`with bromelain or untreated. Columns with bars represent
`the mean (SEM) A540 nm values of 19 samples taken from
`each pig (n=7pigs per group). The reduction in enzyme
`immunoassay activity was significant (p<0-05).
`
`intestine
`between segments. The mid-small
`typically had high EIA values,
`indicative of
`high levels of K88 receptor activity and there-
`fore K88+ ETECattachment. In comparison,
`the duodenum andthe ileum had lowerattach-
`mentactivity, as observed previously. !3
`
`K88+t ETEC ATTACHMENT ACTIVITY IN
`PROTEASE TREATED PIGS
`
`To investigate whether exogenous bromelain
`could inhibit K88-attachment to piglet intes-
`tine, we orally administered different amounts
`of the protease and measuredtheability of K88
`to attach to intestine after treatment. Figure 2
`shows mean EIA values for each treatment
`group. A dose-dependent inhibition of K88
`attachment to pig intestine is observed. An
`analysis of variance revealed a significant treat-
`ment effect
`(p<0-05), confirming that
`the
`reduction in K88+ ETEC receptor binding
`activity was dependenton the protease and not
`a result of inherent variation. Bromelain admin-
`istered in amounts of 250 mg or more were
`mosteffective in reducing K88+ ETEC attach-
`ment. In groups C, D and E, which received
`250 mg, 625 mg or 1250 mgof protease per
`dose, six of seven (87%) pigs in each group
`were non-adhesive after treatment (p<0-05) in
`comparison with only two of seven (29%) in
`untreated pigs (Table). Of the 28 pigs receiving
`protease, 22 were non-adhesive (79%) in com-
`parison with 29%in non-protease treated pigs
`(p<0-02, Fischer’s exacttest).
`
`CLINICAL OBSERVATIONS
`
`Because of the direct effect of an exogenous
`protease on theintestine, we conducted some
`preliminary serological and histopathological
`examination of samples obtained from pigs to
`investigate any adverse effects of treatment.
`We observed no abnormal morphological
`changesin tissue specimens, even at the high-
`est protease treatment level. There was no
`clinical or statistical difference (p>0-2) in any
`of the parameters. measured in samples before
`
`Discussion
`Attachment of K88* enterotoxigenic E coli to
`specific receptors located on the intestinal
`brush border is an importantinitial factor in
`the establishment of diarrhoeal disease. The
`receptors
`for K88ac ETEC have been
`described as mucin-type sialoglycoproteins.?4
`In vitro, protease treatmentofintestinal brush
`bordercells containing these receptors inhibits
`receptoractivity and therefore prevents attach-
`ment of K88* ETEC. Similarly, protease
`treatment of human and calf small intestine
`inhibits receptor activity and attachment of
`ETECstrains which carry the CFA/I and
`CFA/II, and K99 adhesins,
`isolated from
`human and calf diarrhoea, respectively.!* 25
`Because of ETEC receptor
`sensitivity to
`protease, one possible way of preventing
`ETEC diarrhoea would be to prevent attach-
`mentofbacteria to intestine by proteolytically
`modifying receptor attachmentsites.!213 In
`the present study, we investigated whether
`oral administration of bromelain, a cysteine
`protease, could inhibit K88+t ETEC receptor
`activity in vivo and therefore inhibit K88
`attachmentto porcine small intestine.
`In pigs that were not treated with bromelain,
`K88receptoractivity varied appreciably along
`the length of the small intestine. This vari-
`ability supports our earlier observations!3 and
`those of others investigating ETEC receptor
`activity in small intestine obtained from calves
`and lambs.76 Variable patterns in receptor
`activity may reflect masking of receptorsites or
`release of receptors from the intestinal epithe-
`lium in vivo.27 Alternatively, the fluctuating
`levels of receptor activity may reflect the action
`of endogenous enzymes in that part of the
`intestine. Several observations support a role
`for protease modification of intestinal recep-
`tors in vivo. ETEC receptor sites have been
`shown to be readily inactivated by trypsin!+
`andbyintestinal contents with high proteolytic
`activity.!3 Also, stabilisation of K88 receptor
`can be achieved by the addition of trypsin
`inhibitor
`to
`sample
`collection
`buffers.!4
`Furthermore, pancreatic proteases are known
`to play a role in the final processing of
`microvillus proteins, and affect the release of
`some membrane-bound proteins
`into the
`lumen of the small intestine.2® 29 Some of the
`proteins that are released may be receptors for
`bacteria.
`To confirm an effect of protease on ETEC
`receptorsites in vivo, we administered various
`amounts
`of protease
`orally
`to
`piglets.
`Exogenous
`protease,
`administered
`orally
`inhibited K88+ ETEC receptor activity and
`therefore ETEC attachmentto small intestine.
`The effect of bromelain on K88 receptor
`activity was dose dependent, in that binding
`activity decreased with increased protease dose
`rate
`(p<0-05). Presumably,
`the protease
`modified K88+ ETEC enterocyte receptor
`sites, such that K88* bacteria could no longer
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`32
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`recognise and attach to the small intestinal
`brush border.
`The pattern of non-adhesiveness observed
`in protease
`treated pigs
`resembled that
`observedin pigs of the genetically determined,
`non-adhesive phenotype. Piglets of the non-
`adhesive phenotype are resistant
`to K88*
`ETECinfection because they lack functional
`receptors for ETEC.® ° Therefore, the inability
`of K88* bacteria to recognise receptorsites on
`the small intestine of bromelain treated piglets
`should renderthe animals resistant to colonisa-
`tion by these bacteria and prevent diarrhoeal
`disease. We have previously shown that the
`oral administration of enteric-coated brome-
`lain to rabbits inhibits colonisation of CFA/I*
`ST+LT* E coli (H10407) and protects against
`diarrhoea and diarrhoea induced death.!?
`Further
`studies
`have
`demonstrated that
`bromelain significantly reduces diarrhoea in
`piglets challenged with K88+ ETEC (Chandler
`and Mynott, manuscript in preparation).
`Thedata in this study support the view that
`increased proteolytic activity in the intestine
`favours
`low receptor activity, and hence
`resistance to ETEC colonisation. In addition
`to acidity in the stomach,
`local
`intestinal
`immunity,
`the flushing action of intestinal
`peristalsis, and competition with commensal
`organisms, intestinal proteolysis may be a pre-
`viously undescribed host-defense mechanism.
`The novel concept of host receptor modifica-
`tion by oral administration of protease is a new
`approachto disease control that could provide
`broad spectrum protection and obviate the
`potential difficulty of antigenic variability of
`microbial virulence determinants.
`
`thank Leigh Callinan, Biometric Services,
`The authors
`Victorian Department of Agriculture,
`(Bendigo, Victoria,
`Australia) for conducting the Genestat analysis. We would also
`like to thank Jim Parsons and Mike Forsyth, VIAS-Attwood
`(Victoria, Australia) for histopathological assessmentof tissues
`and evaluation of serological parameters, respectively. We also
`acknowledge discussions with Ray King, VIAS-Werribee
`(Victoria Australia) on the estimated daily feed intake ofpigs.
`Serum biochemistry analysis were conducted by the Bio-
`chemistry Department at VIAS-Attwood. T M thanks Jim
`Nataro, Christian Engwerda, and Mike Field for comments on
`the manuscript and acknowledges the support of Cortecs Ltd.
`
`1 Cravioto A, Reyes RE, Ortega R, Fernandez G, Hernandez
`R, Lopez D. Prospective study of diarrhoeal disease in a
`cohort of rural Mexican children: incidence and isolated
`pathogens during the first two years of life. Epidem Inf
`1988; 101: 123-34.
`2 Smith HW, Lingood MA. Further observations on
`Escherichia coli enterotoxins with particular regard to those
`produced by atypical piglet strains and by calf and lamb
`strains: the transmissible nature of these enterotoxins and
`of a K antigen Possessed|by calf and lambstrains. 7 Med
`Microbiol 1982; 5: 243-50.
`3 Gaastra W, de Graaf FK. Host specific fimbrial adhesins of
`non-invasive
`enterotoxigenic Escherichia
`coli
`strains.
`Microbiol Rev 1982: 46: 129-61.
`4 Jones GW,Rutter JM. Role of the K88 antigen in the patho-
`genesis of neonatal diarrhoea caused by Escherichia coli in
`piglets. Infect Immun 1972; 6: 918-27.
`5 Cox E, Houvenaghel A.
`In vitro adhesion of K88ab-,
`K88ac- and K88ad-positive Escherichia coli to intestinal
`
`Mynott, Luke, Chandler
`
`villi, to buccal cells and to erythrocytes of weanedpiglets.
`Vet Microbiol 1987; 15: 201-7.
`6 Mooi FR, de Graaf FK. Isolation and characterisation of
`K88 antigens. FEMS Microbiol Lett 1979; 5: 17-20.
`7 KlemmP.Fimbrial adhesins of Escherichia colt. Rev Infect
`Dis 1985; 7: 321-40.
`8 Rutter JM, Burrows MR, Sellwood R, Gibbons RA. A
`genetic basis for resistance to enteric disease caused by
`Escherichia coli. Nature 1975; 257: 135-6.
`9 Sellwood R, Gibbons RA, Jones GW, Rutter JW. Adhesion
`of enteropathogenic Escherichia coli to pig intestinal brush
`borders:
`the existence of two pig phenotypes. 7 Med
`Microbiol 1975; 8: 405-10.
`10 Gibbons RA, Sellwood R Burrows M, Hunter PA.
`Inheritance of resistance to neonatal Escherichia coli
`diarrhoea in the pig. Examination of the genetic system.
`Theor Appl Genet 1977; 51: 65-70.
`11 Bijlsma IGW, de Nijs A, Van der MeerC,Frik JF. Different
`pig phenotypes affect adherence of Escherichia coli to jeju-
`nal brush borders by K88ab, K88ac, or K88ad antigen.
`Infect Immun 1982; 3: 891-94.
`12 Mynott TL, Chandler DS, Luke RKJ. Efficacy of enteric-
`coated protease in preventing attachment of entero-
`toxigenic Escherichia coli (ETEC) and diarrhoeal disease
`in the RITARD model.
`Infect
`Immun 1991; 59:
`708-14
`13 Chandler DS, Mynott TL, Luke RKJ, Craven JA. The dis-
`tribution and stability of the Escherichia coli K88 receptor
`in the gastrointestinal tract of the pig. Vet Microbiol 1994;
`38: 203-15.
`14 Staley TE, Wilson JB. Soluble pig intestinal cell membrane
`components with affinities for Escherichia coli K88* anti-
`gen. Mol Cell Biochem 1983; 52: 177-89.
`15 Laux DC, McSweegan EF, Williams TJ, Wadolkowski EA,
`Cohen PS.Identification and characterisation of mouse
`small intestine mucosal receptors for Escherichia coli K-12
`(K88ab). Infect Immun 1986; 52: 18-25.
`16 Mouricout MA, Julien RA. Pilus mediated binding of
`bovine enterotoxigenic Escherichia colt to calf small intesti-
`nal mucins. Infect Immun 1987; 55: 1216-23.
`17 Metcalfe JW, Krogfelt KA, Krivan HC, Cohen SC, Laux
`DC.Characterisation and identification of a small intes-
`tine mucusreceptor for the K88ab fimbrial adhesin. Infect
`Immun 1991; 59: 91-96.
`18 Mynott TL, Chandler DS. Receptor modification: a novel
`approach to prevent disease caused by enterotoxigenic
`Escherichia coli (Abstract). Proceedings of the 93rd General
`Meeting of the American Society for Microbiology 1993.
`16-20 May; 104.
`19 Chandler DS. Inherited resistance of K88* Escherichia coli in
`pigs. 1986. Ph.D Thesis. School of Agriculture, La Trobe
`University, Bundoora, Victoria, Australia.
`20 Tzipori S, Chandler DS, Smith M, Makin TJ, Hennessy D.
`Factors contributing to postweaning diarrhoea in a large
`intensive piggery. Aust Ver J 1980; 56: 274-8.
`21 Chandler DS, Chandler HM, Luke RKJ, Tzipori SR,
`Craven JA. Screening of pig intestine for K88 non-adhe-
`sive phenotype by enzyme immunoassay. Vet Microbiol
`1986; 11: 153-61.
`22 KanekoJJ (ed.). In: Clinical biochemistry of domestic animals.
`3rd ed. New York: Academic Press, 1980; 790-6.
`23 Mitruka BM, Rawnsley HM (eds.). In: Clinical biochemical
`and haematological reference values in normal experimental
`animals
`and normal humans. New York: Masson
`Publishing, 1982; 216-54.
`24 Erickson AK, Baker DR, Bosworth, BT, Casey TA,
`Benfield DA, Francis DH. Characterisation of porcine
`intestinal receptors for the K88ac fimbrial adhesin of
`Escherichia coli as mucin-type sialoglycoproteins.
`Infect
`Immun 1994; 62: 5404-10.
`25 Mynott TL, Luke RKJ, Chandler DS. Detection of attach-
`mentof enterotoxigenic Escherichia coli ETEC) to human
`small
`intestinal cells by enzyme immunoassay. FEMS
`Immunol Med Microbiol 1995; 10: 207-18.
`26 Sellwood R, Lees D. Adhesion of Escherichia coli patho-
`genic in pigs, calves and lambsto intestinal epithelial cell
`brush borders. In: de Leeuw PW, Guinee PAM eds,
`Laboratory diagnosis in neonatal calf and pig diarrhoea.
`Current topics in veterinary medicine and animal science.
`1980; 13: 163-70.
`27 Dean EA, Whipp S, Moon H.Age-specific colonisation of
`porcineintestinal epithelium by 987P-piliated enterotoxi-
`genic Escherichia coli. Infect Immun 1989; 57: 82-7.
`28 Hauri HP, Quaroni A, Isselbacher KJ. Biogenesis ofintesti-
`nal plasma membrane:post-translational route and cleav-
`age of sucrase-isomaltase. Proc Natl Acad Sci USA 1979;
`76: 5183-6.
`29 Kidder DE, Manners MJ. In: Digestion in the pig. Bristol:
`Wright-Scientechnica, 1978.
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