Gut 1995; 36: 209-214

Rat intestinal mucosal responses to a microbialflora and different diets

R Sharma, U Schumacher, V Ronaasen, M Coates

AbstractThe effects of diet on the histochemicalcomposition of intestinal mucosubstancesand the morphology of the villi and cryptswere investigated by comparing thedata of germ free and conventionallymaintained rats fed either a purified dietor a commercial diet. The influence ofintestinal microflora was evaluated bycomparing the germ free rats and thoseharbouring either a conventional rat floraor a human microbial flora. In both germfree rats and those maintained conven-tionally, feeding a purified diet resulted inshallower crypts in the small intestinebut deeper crypts in the large intestinecompared with their counterparts fed onthe commercial diet. The preliminary dataobtained with association of human florashowed a reduction ofthe villus height andcrypt depth in the small intestine and, tosome extent, the amount ofneutral mucinsin the goblet cells of both small and largeintestine and an increase in the amountof sulphated mucins in the large intestine.In rats given the commercial diet theperiodic acid Schiff staining for neutralmucins was more intense in the uppercrypts of the small intestine than in thelower crypts, and to a lesser extent inthe upper crypts of the large intestine.These results provide evidence that thedietary composition, microbial flora, aswell as the interactions between thedietary constituents and microbial florachange the mucosal architecture and themucus composition and therefore alter thefunctional characteristics of the intestinaltract.(Gut 1995; 36: 209-214)

Keywords: diet, microbial flora, intestinal mucosa.

Human Morphology,University ofSouthamptonR SharmaU Schumacher

School of BiologicalSciences, University ofSurrey, Guildford,SurreyV RonaasenM Coates

Correspondence to:Mr R Sharma, HumanMorphology, University ofSouthampton, BassettCrescent East, Southampton.Accepted for publication29 April 1994

A distinguishing feature of the gastrointestinalmucosa is the ability of its epithelial cellsto synthesise and secrete the mucus glyco-proteins which protect and lubricate thesurface epithelium and act as a stabiliser forthe association of some enteric bacteria.' Inrecent years, the interest in intestinal mucinshas grown because changes in their biosyn-thesis or degradation are important factors inthe aetiology of diseases such as colonicadenocarcinoma and ulcerative colitis.2 Inaddition, studies of intestinal mucin are ofspecial interest because changes in dietaryhabits may modify their chemical composi-tion.3 Since dietary factors that either affectthe production of mucin or enhance its

degradation would make the intestinalmucosa vulnerable to toxic substances, it ispossible that appropriate modifications of thediet may protect the intestinal mucosa fromvarious diseases.

Studies in various animal models, as well asin humans, have shown that many bacterialspecies colonise the gastrointestinal tract byattachment to the mucus layer,4 5 and degrademucin glycoproteins in vivo6 and in vitro.7Little is known, however, about the influencethe gut microbial flora may exert on thesecretory pattern of the intestinal mucins.The histochemical characterisation of

intestinal mucins in laboratory animals and inhumans has received growing attention (forreview, see: rat8 9; human'0) and differentchemical compositions of these mucinshave been observed in different parts of thegastrointestinal tract.' -13 Furthermore, theglycoproteins secreted by the goblet cells inthe lower part of the crypt differ from those ofthe upper part.'4 The question whether gutmicrobial flora or diet, or both in combinationhave an effect on the synthesis and secretion ofintestinal mucins, or in the proportion ofsulphated and sialomucins in the goblet cells ofdifferent regions of the gastrointestinal tracthas not received sufficient attention.

It has been reported that diets containingfibre can modify the shape of villi'5 in the smallintestine ofnewly weaned rats and alter the celltumover rates in the intestinal crypts.3 It is alsowell known that the turnover rate of epithelialcells is faster in conventional animals than intheir germ free counterparts. In all speciesexamined the villus:crypt ratio is higherin germ free animals, indicating that lessproliferating tissue is required to keep the germfree mucosa intact.'6Our objective in this investigation was to

evaluate whether the microbial flora, or diet, orboth, affect the nature and distribution of thecarbohydrates in the mucus secreting cells of arat model and also to determine whether theintestinal morphology was altered by dietarymodification. Two different types of diet wereused. One was a commercial rodent dietcomposed of natural materials and the otherwas a nutritionally adequate mixture ofpurified ingredients. Because the compositionof the indigenous gut microflora is characteris-tic of each species, the suitability of the con-ventional rat as a model for man is open toquestion. In an attempt to produce a modelsystem more closely related to man severalworkers have used rodents born germ free andthen associated with a flora of human origin(see review'7). To obtain some preliminaryevidence on possible species differences, our

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TABLE I Composition of diets fed to rats

Component Concentration (g/kg)

Purified diet:Casein 250Maize starch 380Potato starch 100Sucrose 50Cellulose powder 80Soya bean oil 60Mineral mix 60Vitamin mix 20

Commercial diet*:Cereal products (barley, maize,wheat and wheatfeed) 775

Extracted soya bean meal 107Fish meal 98

Mineral and vitamin supplement 20

* Calculated crude fibre content 4-1!%.

study included one group of rats thatharboured a flora of human origin. Theyreceived the purified diet; isolator space was

inadequate to accommodate a similar group

given the commercial diet. Their histochemicaland morphological characteristics were

compared with those of a corresponding groupof germ free rats.

Methods

ANIMALS AND ASSOCIATION OF INTESTINALMICROFLORATwenty five, 9 week old male rats were usedfor the study when they were killed. The germfree animals (n= 10) were born and maintainedin plastic isolators while the conventionalcontrols (n= 10) were kept in the openlaboratory. Another five germ free rats were

inoculated one week after weaning (that is, at4 weeks of age) with a suspension of humanfaecal organisms by oral intubation (HFA) as

described by Mallet et al.'8 They were alsomaintained in an isolator.

DIETSFive germ free and five conventional animalswere fed on a commercial rat diet (diet GR3;Special Diets Service, Witham, Essex) (TableI). Five germ free rats and five conventionalrats were maintained on a purified diet (TableI). The five HFA rats also received this diet.

PREPARATION OF TISSUES AND HISTOCHEMICAL

METHODSAnimals were killed at 9 weeks of age and

samples from the mid-region of the smallintestine (jejunum) and proximal largeintestine (2 cm from caecum) were fixed in10% buffered formalin and embedded inparaffin wax.

Serial 5 gm sections were subjected to thefollowing procedures for the identification ofmucosubstances:

(1) The periodic acid Schiff (PAS) reactionfor studying unsubstituted a-glycol richneutral mucins.

(2) 1% Alcian blue, pH 2-5 followed byPAS (AB 2.5/PAS) top allow neutral (pink)and acidic (blue) mucins to be differentiated.19With this procedure a purple colour isobtained when neutral mucins are also present,and deep purple when neutral mucins are

mainly present.20(3) 1% Alcian blue, pH 1 0 (AB 1.0) for the

selective characterisation of sulphomucins.21The Alcian blue dyes and the techniquesused in this study were pretested in a dotblot assay for their suitability to label carboxy-lated and sulphated mucins, respectively.22Semiquantitative staining intensities were

based on a scale ranging from 0, unreactive to+ + +, intensely stained.

QUANTITATIVE MORPHOLOGYTen longitudinally oriented villi and cryptsfrom the small intestine and 10 crypts fromthe large intestine were selected randomlyfrom each animal for measurement. Villuslength was measured from the tip to the baseof villus and the crypt length was measuredfrom the bottom of the crypt to the opening ofthe crypt. The results were expressed as mean

(SEM), and statistical differences betweengroups were analysed for significance byStudent's t test.

Results

HISTOCHEMICAL STUDIESThe histochemical staining pattern of carbo-hydrate-containing surface mucus, surfacegoblet cells, and upper and lower crypt gobletcells is summarised in Tables II and III.

Effects of dietThe effects of diet were assessed by comparingthe staining characteristics of germ free and

TABLE II Effects of diet and human flora on histochemical characteristics of intestinal mucins in germ free rats

Germ free fed conventional diet Germ free fed purified diet HFA inoculatedfed punfied diet

Intestine and cell type PAS AB 2-5/PAS AB 1.0 PAS AB 2-5/PAS AB 1.0 PAS AB 2-5/PAS AB 1.0

Small intestine:Surface mucus + +purple (+) + + +pink+ +DP (+) + + +pink+DP 0Surface goblet cells + + +++DP + +++ +DP + + + + ++DP + +

Upper crypt goblet cells +++ +++DP + + + + + +purple + + +++ +purple +++Lower crypt goblet cells + + +DP 0 + + + +DP + (+) +++DP (+)

Large intestine:Surface mucus (+) +purple (+) + + +purple (+) (+) +purple + +Surface goblet cells + + +pink 0 + + +pink (+) + + + +pink +

+ +purple +purpleUpper crypt goblet cells + +pink +++ +++ ++DP + + + + +DP +++Lower crypt goblet cells + + + +blue (+) + + + +blue (+) (+) + + +blue (+)

Intensity of reaction: 0 no reactivity; (+) weak reactivity: + moderate reactivity; + + strong reactivity; + + + intense reactivity. DP=deep purple. PAS=periodicacid Schiff; AB=Alcian blue.

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TABLE IIII Effects of intestinal microflora on histochemical characteristics of intestinal mucins in conventional rats

Conventional rats fed commercial diet Conventional rats fed purified diet

Intestine and cell type PAS AB 2-5/PAS AB 1J0 PAS AB 2.5/PAS AB 1J0

Small intestine:Surface mucus ++ +purple 0 (+) +pink 0

+ +purpleSurface goblet cells +++ + +DP (+) + + + +DP+purple + +Upper crypt goblet cells +++ +purple + + + + +pink +

+ +purpleLower crypt goblet cells + + + +DP (+) + + +purple (+)

++DPLarge intestine:

Surface mucus + +purple (+) 0 +purple 0Surface goblet cells +++ +pink (+) + + +purple + +

+purple +pinkUpper crypt goblet cells +++ + +DP + + + + + +purple + +

+ +pinkLower crypt goblet cells + + + +blue + (+) + +blue (+)

+purple

Intensity of reaction: 0 no reactivity; (+) weak reactivity; + moderate reactivity; + + strong reactivity; + + + intense reactivity.DP=deep purple. PAS=periodic acid Schiff; AB=Alcian blue.

conventional rats receiving either the com-mercial or the purified diet.

Surface mucus. The PAS reaction of thesurface mucus of the epithelium lining both thesmall and large intestines was more intense inthe germ free rats fed on the purified diet. WithAB 2.5/PAS procedure in the germ free ratsfed on the purified diet there was no purplestaining in the surface mucus of the smallintestine. In contrast, in conventional animalsthe pink and purple staining of surface mucusin the small intestine of rats fed on a purifieddiet indicates the presence of both neutral andacidic mucins.

Goblet cells. In germ free rats fed the purifieddiet, overall staining intensity was higher in thesurface goblet cells and in the upper cryptgoblet cells of the large intestine than in theircounterparts fed on the commercial diet. Incontrast to germ free rats, the goblet cells in thesmall and large intestine of conventional ratsfed the commercial diet were found to bestrongly PAS positive indicating abundantpresence of neutral mucins.The combined AB 2.5/PAS procedure

showed appreciable differences between therelative proportions of acidic and neutralmucins in the goblet cells of rats fed ondifferent diets. The surface and crypt gobletcells in the small intestine of the germ free ratsfed the commercial diet stained deep purple

Figure 1: Mucosal epithelium of (A) small intestine and (B) large intestinal crypts fromgerm free rats fed on a commercial diet stained by Alcian blue-periodic acid Schiff tech-nique. Surface goblet cells and crypt goblet cells in the small intestine are stained deep purpleindicating that they contain both neutral and acidic mucins. Those in the large intestine are

predominantly stained pink. (Bar= 50 ,um)

(Fig 1 (A)), an indication that the neutralmucins predominant in these animals. Thestaining was less intense in the upper cryptgoblet cells of the germ free rats fed purifieddiet. The surface goblet cells and upper cryptgoblet cells in the large intestine of the germfree rats fed commercial diet contained noacidic mucins (Fig 1 (B)), but they werepresent in the lower crypt goblet cells. In con-trast, the staining reactions in the upper cryptgoblet cells of the rats fed on the purified dietindicated the presence of acidic mucins, andthe more intensely blue reaction in the deepcrypt goblet cells of the large intestine of thegerm free rats fed the purified diet indicatesabundant presence of both sulphated andcarboxylated acidic mucins. With thecombined AB-PAS procedure, the surfacegoblet cells and upper crypt goblet cells ofboththe small and large intestine of conventionalrats fed on a purified diet stained purple, anindication that the acidic mucins predominatein these animals. In the crypt goblet cells ofconventional rats fed a commercial diet, theneutral mucins were found only in conjunctionwith the acidic ones but the pink reaction insome upper crypt goblet cells of the small andlarge intestine of conventional, purified dietfed rats indicates the presence of neutralmucins (Fig 2(A)).AB staining at pH 1.0 for assessment of

sulphated mucins showed a higher sulphatecontent in the goblet cells of the small intestineof germ free rats fed a purified diet comparedwith those of conventional diet fed counter-parts. In the large intestine the staining inten-sity was greater in the upper crypt goblet cellsof germ free, commercial diet rats. The surfacegoblet cells of conventional rats fed onthe purified diet stained more intenselywith AB 1.0 than those of their commercialdiet fed counterparts (Fig 2(B)). Goblet cellsat the bases of the crypts contained little or nosulphated mucins.

Effects of the microfloraTo assess the effects of conventional microfloraand of the interactions of the luminal nutrientswith the conventional microflora on intestinalmucins, histochemical staining intensities were

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Figure 2: Large intestinal crypts of conventional ratsfed on a purified diet sAlcian blue-periodic acid Schiffprocedure and with Alcian blue atpH 1 0.in the upper crypt containing neutral mucins and (B) surface goblet cells co;sulphated mucins are arrowed. (Bar=50 gm)

compared between germ free andrats receiving the commercial dietgerm free and conventional ratspurified diet. The effects of humsassessed by comparing germ free a

receiving the purified diet.Surface mucus. The PAS reactivi

face mucus of the epithelium linirand large intestines was more

conventional, commercial dietthan in their germ free counterpaxthe staining reaction in conventioon the purified diet was less intencorresponding germ free countintensity of PAS reaction of the siwas more intense in germ free,animals than in HFA, purified c

significant differences in the stairof acidic mucins were detected bfree and conventional commerciaand between gernn free and HFAgroups with the combined AB-PAHowever, in rats fed on a purisurface mucus of conventional ri

less neutral mucins than thcorresponding germ free rats.The surface mucus of HFA an

more intensely with PAS than in c

purified diet fed rats. In HFA ratmucus of the large intestine cor

sulphate than that of conventionaGoblet cells. The surface and

goblet cells of conventional ratscommercial diet stained more irPAS than those of the correspond

rats. In contrast, overall PAS staining intensity

animals fed on the purified diet than in theirgerm free counterparts. There was no

regionally consistent difference between germ

free and HFA rats fed purified diet; forinstance, in the upper crypt cells the intensitywas greater in the small intestine but less in the

large intestine. PAS staining intensity was

higher in the crypt goblet cells of the largeintestine in germ free rats.With the AB-PAS procedure no significant

differences were seen between the con-: ~~ventional and germ free rats fed commercial

diet and between germ free and HFA rats fedstained with purified diet. However, in rats fed on a purified(A) Gobletcells diet the purple staining reaction in gobletntaining cells of both small and large intestine of con-

ventional rats indicated the presence of acidicmucins, and the more intensely blue reaction

conventional in the lower crypt goblet cells of HFA ratsand between indicates the presence of both sulphated andreceiving the carboxylic acidic mucins.an flora were Examination after AB staining at pH 1.0md HFA rats showed that goblet cells on the surface and in

the upper and lower crypts of the small andity of the sur- large intestines of HFA, purified diet ratsig both small contained more sulphate than those of the

intense in corresponding germ free rats.fed animals In rats associated with a human flora, overallrts. However, PAS reactivity was higher in the goblet cellsonal rats fed than in animals harbouring a conventional ratise than their flora. With the AB-PAS procedure the uppererparts. The crypt and surface goblet cells in the small andarface mucus large intestine of conventional animals showedpurified diet abundant presence of acidic mucins. In HFAdiet rats. No rats, however, the acidic mucins were predom-iing intensity inant in the deep crypt goblet cells of the largeetween germ intestine. AB staining at pH 1.0 detected morel diet groups sulphate in the upper goblet cells of both thepurified diet small and large intestines of HFA rats than.S procedure. those of the conventional rats.fied diet theats containedlose of the

imals stainedconventional,ts the surfaceitained morerats.upper crypt

s fed on theitensely withLing germ free

QUANTITATIVE MORPHOLOGYThe quantitative data on the length of villi andcrypts and villus:crypt ratio of the smallintestines and length of crypts of the largeintestines are summarised in Table IV. Thegerm free rats fed on a purified diet comparedwith those fed on a commercial diet hadsignificantly reduced (p<0002) small intesti-nal crypt lengths but increased crypt lengths(p<005) of the large intestine. No differencesin villus lengths were observed between the

TABLE IV Effects of diet and intestinal microflora on rat intestinal morphology (values, mean (SEM))

Commercial diet Purified diet

Germ free Conventional Germ free Conventional Human florarats flora rats rats flora rats rats

Small intestine:Villus length (gm) 562-56 (29 2) 604-64 (23-1)* 504-60 (65 7) 468-48 (8)4 396-96 (50-1)Crypt length (,um) 182-00 (9.1) 180-56 (11.7)* 148-10 (5.8)t 150-72 (3.5)4 96-92 (12-4)Villus:crypt ratio 3-12 (0.2) 3 40 (0.2) 3-37 (0.3) 3-14 (0 1)§ 4-12 (0.2)

Large intestine:Crypt length (gm) 195-52 (6.6) 187-73 (11-8)* 238-5 (12-8)t 244-4 (8.4)4 152-08 (21-3)

For feeding regimes and experimental groups see table I and methods.*Significantly different at p<0O001 from corresponding value for conventional rats fed a purified diet.tSignificantly different at p<0 01 from corresponding value for rats inoculated with human faecal flora.iSignificantly different at p<0O001 from corresponding value for rats inoculated with human faecal flora.§Significantly different at p<001 from corresponding value for rats inoculated with human faecal flora.

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two dietary groups. In rats harbouring aconventional microflora, the mean values ofvillus and crypt lengths of small intestine weresignificantly higher in the group fed on thecommercial diet but there was a reduction inthe crypt length in the large intestine.The mean values of villus and crypt lengths of

small intestine and crypt lengths of large intes-tine were similar in germ free and conventionalrats fed commercial diet and in germ free andconventional rats fed the purified diet. Therewas no difference in the ratio of the villus lengthto crypt length in the small intestine. In rats fedon the purified diet association with the humanflora resulted in a reduction in the villus andcrypt length in the small intestine and of cryptlength in the large intestine.The rats associated with human flora

when compared with those harbouring a con-ventional flora had significantly reduced lengthsof small intestinal villi and reduced lengths ofboth small and large intestinal crypts.

DiscussionThe present studies have confirmed theprevious histochemical observations8 14 23 thatthe composition of secretory glycoproteins inthe intestinal mucosa of rats differs with theregion and with cell type. Our results in TablesII and III indicate that these differences inmucin composition are also influenced by dietand the presence or absence of a microbialflora in the intestinal lumen.

Neutral mucosubstances are the predomi-nant type seen in the small intestine, whereasacid mucosubstances were predominant inthe large intestine of conventionally rearedanimals. Furthermore, the goblet cells in theupper part of the large intestinal crypts differedfrom those in the lower part in that theycontained AB pH 1-0 reactive sulphatedmucins. Although similar findings havebeen described in previous histochemicalinvestigations, 8 9 13 24 25 the significance ofthe differences in mucin distribution patternsthroughout the intestinal tract and functions ofvarious classes of intestinal mucins is still notwell understood.The influence of diet on the composition of

intestinal mucins was explored in germ free andconventional rats given either a diet of finelypowdered purified ingredients, including cellu-lose as a source of fibre, or a more coarselyground diet of natural ingredients containingcrude fibre of mainly cereal origin. Our findingsindicated generally less neutral mucins in thesmall intestine and the presence of sulphatedmucins in both small and large intestines ofanimals fed on the purified diet, which might beaccounted for by the different characteristics ofthe two diets, or of their fibrous components.Although histochemical studies on responses todietary fibres have so far been limited to jejunalgoblet cells of pigs,26 there are other morpho-logical studies which report that specific dietaryfibres may increase the secretory activity ofgoblet cells in rodents.3 27The comparisons between germ free

rats and their counterparts harbouring their

indigenous flora showed that, independently ofdiet the intestinal tract responded to intralumi-nal contamination by depletion of neutralmucins from the globlet cells of the lowercrypts of the small and large intestines. Fromthe reactions observed to AB staining, it isobvious that the presence of a microflora influ-ences the relative proportions of sulphated andsialylated species of acidic mucins.When the intensity of PAS reaction of germ

free rats was compared with that in conven-tional rats, the surface mucus and goblet cellsin rats harbouring a conventional flora weremore intensely stained. It is well known thatthe amount of mucus in the intestinal lumen ofgerm free rats is greater than that in theirconventional counterparts, because of thedegradative effect of bacterial mucinase.However, as the parameter of staining intensitywas analysed in this study, a direct analogybetween staining activity and the amount ofmucus cannot be drawn. The degree of glycosy-lation of mucus glycoproteins and hydration ofsecreted mucus may alter the staining intensityand it is possible that changes in the hydrationof mucus may have been partly responsible fordifferent staining intensities in our animals.

Changes in the carbohydrate composition ofintestinal mucins have been known to occurduring development28 29 and during the migra-tion of crypt cells toward the epithelial sur-face.9 It is well known that the migration ofepithelial cells along the villi is faster in con-ventional than germ free animals. In this study,in the presence of a conventional flora, gobletcell mucins in the large intestine became moresulphated along the crypt villus axis. This isconsistent with studies in the neonatal mouse,where similar effects of the conventional floraon the production of mucins have beendescribed.30 According to Heneghan16 thevillus:crypt ratio is higher in germ free animalsthan conventional animals. This is in contrastwith our findings which indicate comparablelengths ofvillus column and crypts in germ freeanimals and conventional animals fed on eitherthe purified or the commercial diet. Similarresults to ours have been observed in studies byIshikawa et al 31 in the upper region of the smallintestine. When the morphological effects ofthe two diets were compared in our germ freeanimals, the most noticeable changes wereseen in the crypt lengths of small and largeintestines. Crypt size was decreased in thesmall intestine of rats fed a purified diet, andalthough the villus height did not changesignificantly, it was clear that the increasedcrypt length in the large intestine observed inthis group of animals reflected an increasedamount of neutral mucins. Our finding ofno changes in villus lengths contrasts withthe results of a morphological study onconventionally fed rats by Sigleo et al,32 andthis discrepancy suggests that there may havebeen an interaction between the diet andmicrobial activity33 in their studies.

Although the mechanisms responsible forstructural and chemical changes in the intes-tinal mucosa are unknown, our results showthat the presence of a microbial flora and the

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214 Sharma, Schumacher, Ronaasen, Coates

nature of the diet can determine the shape andmucus secretion of the intestinal epitheliumand its supporting stroma. Since the thicknessof the surface mucus and the chemical com-position of surface mucins is linked to adynamic equilibrium between the continuoussecretion of mucins from the goblet cells andtheir degradation within the intestinal lumen,it is appropriate, therefore, to assess the com-position of the surface mucus in response tointraluminal stimuli of different diets anddifferent species of gut flora. The evaluation ofsurface mucus is prone to artifacts of fixationand should be considered with caution. Itseems reasonable, however, to assume that thecomparison between staining intensities ofsurface mucus in our animal groups, if allsamples are processed at the same time andanalysed by standardised histological methods,are valid. The effects of the human flora on thegut structure and mucus composition were inmany ways similar to those of the indigenousrat flora. There were, however, somedifferences, for instance in the composition ofthe surface and goblet cell mucus and thelength of the large intestinal crypt cells, whichmerit more detailed investigation. Althoughthey may reflect real differences in response tothe two types of flora, it is also possible thatwhen a human flora is inoculated in the germfree animal, its establishment and subsequentinteraction with the intestinal tract mayinduce changes not seen with an indigenous

flora where colonisation is not subject toexperimental manipulation.The findings of altered mucosal morphology

and mucin biosynthesis with particular dietarypatterns in the presence of an indigenous or ahuman microbial flora described in this workstrongly emphasises the use of the rat asa model system for pathogenesis of intestinaldisease. Our results on human flora rats arepreliminary and more detailed studies areneeded to ascertain whether the HFA rat is amore appropriate model for man than theconventional rat. Further studies of changes inmucin composition by various intraluminalstimuli will help in understanding themechanisms of intestinal disorders and indeveloping probes for detection of boweldisease. Morphometric analysis of goblet cellglycoproteins using an image processor isnow being carried out in our animal model tofurther elucidate the interaction betweenthe mucin secretion and intestinal luminalcomponents.The authors are grateful to the EC-FLAIR Concerted ActionProgramme No 9 which sponsored part of this work and to DrYoshio Saito, Calpis International Flora Laboratory,Kanagawa, Japan, for providing the purified diet.

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