In Vitro Adhesion Specificity of Indigenous Lactobacilli within the Avian Intestinal Tract

In vitro adherence of Lactobacillus strains to cell and tissuetypes along the chicken alimentary tract and to ileal mucuswere determined. Fresh isolates from chickens adhered to theepithelium of crop and, in a strain-dependent manner, to follicle-associatedepithelium and the apical surfaces of mature enterocytes ofintestinal villi. No adherence to the apical surfaces of undifferentiatedenterocytes, the mucus-producing goblet cells, or the ilealmucus was detected.

INTRODUCTION

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Introduction

Species of Lactobacillus belong to the normal bacterial floraof gastrointestinal and genital tracts of humans and animals.Early studies on chicken microflora (2, 5-7) showed that theLactobacillus flora lining the crop of the chicken gastrointestinaltract becomes established within a day after hatching and thatthe specific adherence of avian-associated lactobacilli ontothe crop epithelium plays a role in the colonization. Overall,the tissue, cell, and molecular specificities of Lactobacillusadherence have remained poorly characterized, and the role ofadherence in intestinal colonization by lactobacilli has remaineda controversial topic (19). We initiated this study to analyzethe cell and tissue tropism in the in vitro adhesion of commensallactobacilli at different surfaces of the chicken intestinaltract, which offers a well-characterized tissue with structurallyand functionally varied epithelial surfaces (9).

Isolation of lactobacilli from chicken intestine.

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Isolation of lactobacilli from...

Lactobacillus crispatus strains ST1, A33, and 134mi, Lactobacillusreuteri CT7, and Lactobacillus gasseri CT5 were isolated fromthe crop of 2- to 12-day-old chickens by enrichment under anaerobicconditions on selection agar plates (LBS; Becton Dickinson MicrobiologySystems, Cockeysville, Md.), and their identification was basedon the 16S ribosomal DNA sequence determination (1). The strainsL. crispatus ATCC 33820, L. reuteri ATCC 53609, L. gasseri ATCC33323, and Lactobacillus casei ATCC 393 were obtained from theAmerican Type Culture Collection (ATCC). L. reuteri 1063 wasobtained from Stefan Roos, Uppsala, Sweden (16). For testing,the bacteria were cultivated in static MRS broth overnight at37°C (20).

Bacterial adhesion to intestinal tissue.

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Bacterial adhesion to intestinal...

Double staining of frozen tissue sections with bacteria andtissue markers has been successfully applied to determine tissuetropism in the adhesion of a variety of pathogenic bacteriaand their adhesion proteins (reviewed in reference 11). Tissuesamples of crop, duodenum, jejunum, ileum, cecum, and colonfrom four 12-day-old chickens, two males and two females, wereexcised and frozen as detailed in references 17 and 18. Thefluorescein isothiocyanate (FITC)-labeled bacteria were testedat concentrations of 10⁸ and 10⁹/ml. Tetramethyl rhodamine isothiocyanate(TRITC)-conjugated wheat germ agglutinin lectin (20 µg/mlin phosphate-buffered saline [PBS]; Vector Laboratories, Inc.,Burlingame, Calif.) was used as a general stain and a markerfor chicken epithelium. Anti-chicken type III collagen immunoglobulinG (IgG) (0.02 µg/ml in PBS; Developmental Studies HybridomaBank, University of Iowa, Iowa City, Iowa) was used as a markerfor extracellular tissue, anti-chicken T-cell receptor ${alpha}$ /ßVß₁ (TCR2) IgG (10 µg/ml in PBS; Southern BiotechnologyAssociates, Inc., Birmingham, Ala.) was used for lymphoid organs,and anti-chicken ß1-integrin IgG (50 µg/ml inPBS; Sigma, St. Louis, Mo.) was used for basolateral epithelia.

L. crispatus ST1 showed a strong adherence to the epithelialareas of the stratified squamous epithelium of the crop, tothe simple columnar epithelium of intestine, and to the connectivetissue areas along the lower intestinal tract (Fig. 1; Table1). The pericellular tissue reactive with the anti-type IIIcollagen monoclonal antibody (MAb) contained only a few adherentbacteria (Fig. 1B). The bacterium was observed to not adhereonto the endothelium. The epithelium-connective tissue biasof ST1 adhesion was different in the lower parts of the alimentarytract. In the small intestine (Fig. 1C through E), the adherenceof L. crispatus ST1 was strong to the lamina propria of thevilli and the basolateral poles of the villus enterocytes. Bacteriaadhered strongly to the apical pole of the epithelium only atthe areas of mature enterocytes at the villus tips (Fig. 1C and D).At the crypts containing undifferentiated columnar enterocytes,the bacteria adhered specifically to basolateral poles of theenterocytes but did not show any adherence to the apical poles(Fig. 1E). The adhesion of ST1 to the cecal epithelium and underlyingtissues was less efficient and less distinctively localizedthan in the other parts of the digestive tract (Fig. 1F). Thebacteria did not adhere to the indigenous debris remaining onthe glass surface during preparation of the cecal sections (Fig.1F). In the colon, the bacteria adhered to the lamina propriaand less efficiently to the luminal poles of the villi. Theadhesion patterns of the strains A33 and ST1 were highly similar(Table 1). The strain ATCC 33820 showed only a weak adhesionin the colon (Fig. 1G) as well as in the other parts of theintestine (data not shown). In contrast, L. crispatus strain134mi adhered only to the crop and the duodenal epithelium (Table1). The isolate CT7 of L. reuteri showed adherence only to thecrop epithelium (Table 1). The adherence pattern shown by theisolate CT5 of L. gasseri was highly similar to those of L.crispatus strains ST1 and A33 (Table 1). The strains ATCC 33323of L. gasseri, ATCC 53609 of L. reuteri, and ATCC 393 of L.casei showed only poor adhesiveness to the chicken tissue (datanot shown).

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FIG. 1. Adherence of L. crispatus strains ST1 (AI through FI) and ATCC 33820 (GI) to regions of the chicken alimentary tract. The adherence was visualized by double staining of frozen intestinal sections with FITC-conjugated bacteria (I panels) and with indirect immunofluorescence using a primary MAb against chicken type III collagen (BII, EII, and GII) or chicken ß1-integrin (CII and DII). The tissue sections were from crop (A and B), ileum (C to E), cecum (F), and colon (G). Panels BIII, DIII, EIII, FII, and GIII show light microscopy images of the tissue areas. Arrowheads indicate outermost layer of the epithelium, and the arrow indicates sloughing enterocytes at the villus tip. e, epithelial layers; C, connective tissue; LP, lamina propria; Cr, crypts; asterisks, cecal mucus and unwashed digesta. The thickness of the crop epithelium is indicated in the B panels. Bars, 50 µm.

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TABLE 1. Adherence of freshly isolated lactobacilli to frozen tissue sections of chicken alimentary tract

In the ileum, the follicle-associated epithelium (FAE) and thePeyer's patches of the mucous-associated lymphoid tissues areimportant sites for pathogen entry as well as for immune stimulation(8, 12). One application of orally administered lactobacillihas been to prevent or diminish intestinal infections. We thereforeassessed the bacterial adherence to lymphatic follicles andFAE of the chicken ileum. The follicles and FAE were identifiedby their morphology, i.e., the presence of germinal centersand lack of tall columnar enterocytes, as well as the high numberof cells recognized by the anti-T-cell receptor MAb. L. crispatusST1 adhered to the follicles and the germinal center regionsof the follicles as well as to the FAE located immediately abovethe follicles (Fig. 2A). Adherent bacteria could be locatedat the luminal FAE surfaces (Fig. 2B) but were absent at theluminal surfaces of enterocytes at the same ileal region (Fig.2C and D). The adhesion of L. crispatus A33 was also efficienton the ileal Peyer's patches and the overlying FAE; L. gasseristrain CT5 showed moderate adhesion to the lymphoid follicles,whereas the adhesiveness to the FAE was poor (Table 1). Thestrain L. reuteri ATCC 53609 exhibited selective adherence tothe tissue domains of ileal Peyer's patches, whereas poor adherenceto the intestinal mucous-associated lymphoid areas was observedwith other Lactobacillus strains (data not shown).

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FIG. 2. Adherence of L. crispatus ST1 to the ileal lymphoid follicles, its overlying FAE, and to columnar epithelial cells in the ileum. The tissue sections were double stained with FITC-labeled bacteria as well as MAbs against chicken T-cell receptor 2 (TCR ${alpha}$ /ß Vß₁) and TRITC-labeled secondary antibodies. (A) Adherence to the lymphoid follicle, with the characteristic germinal center (GC) rich in B cells and surrounded by the T-cell zone strongly positive for the tissue marker. (B and C) Bacterial adherence to the FAE (B) and to the corresponding ileal epithelium (e) not associated with lymphoid follicles (C). (D) Adherence to FAE and enterocytes in the same microscopic tissue section. Arrows indicate FAE surface rich in intraepithelial T cells reactive with the tissue marker; arrowheads indicate the apical surfaces of tall columnar enterocytes with only a few intracellular T cells. The asterisk indicates a mucus-producing goblet cell. Bars, 50 µm.

Mucus adherence.

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Mucus adherence.

The procedure to prepare the tissue sections used in this studyremoves the protective mucus layer covering epithelial surfaces(4). Isolates of Lactobacillus have been reported to bind tointestinal mucus or fecal glycoproteins (16, 21); we thereforecompared the strains for adhesiveness onto immobilized mucusscraped from the chicken ileum (18). For comparison, we alsotested adhesion to an unrelated protein, type IV collagen, andthe study also included strain 1063 of L. reuteri, which waspreviously found to adhere to mucus (16). Strains A33 of L.crispatus and ATCC 53609 of L. reuteri showed adhesiveness tomucus which, however, was low compared to that seen with theadhesive strain 1063. The other test strains showed nondetectablelevels of adhesion (Fig. 3). The poor adhesion by chicken-associatedlactobacilli is in line with the finding that the crop epitheliumdoes not contain mucus-producing cells (9).

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FIG. 3. Adherence of chicken-associated lactobacilli isolates to immobilized ileal mucus. Adherence to mucus preparations is shown by filled symbols, and the adherence to type IV collagen is shown by open symbols. (A) Adherence of L. crispatus strains ST1 (•), A33 ( ${blacktriangledown}$ ), 134mi ( ${blacksquare}$ ), and ATCC 33820 ( ${blacklozenge}$ ). (B) Adherence of L. reuteri strains CT7 (•) and ATCC 53609 ( ${blacktriangledown}$ ). (C) Adherence of L. gasseri strains CT5 (•) and ATCC 33323 ( ${blacktriangledown}$ ). (D) For comparison, the adhesion by strains 1063 of L. reuteri (•) and ATCC 393 of L. casei ( ${blacktriangledown}$ ) are shown.

Our results on the in vitro tissue and cell specificity of lactobacillaradherence are in agreement with the findings of Fuller and Turvey(7) and others (13). We studied the tissue distribution of thein vivo colonization by lactobacilli in the chicken intestine.Our data also give support for the observation (5) that adherenceto crop epithelial cells is a major determinant of lactobacillarcolonization in the chicken intestine. The importance of thecrop epithelium as an adhesion target for lactobacilli was stressedby the behavior of L. reuteri CT7, which was isolated from thechicken intestine in this study and adhered efficiently to thecrop epithelium but very poorly to the other regions of thechicken intestinal tract. We observed that the isolates ST1and A33 of L. crispatus as well as CT5 of L. gasseri adheredto the apical aspects of mature enterocytes at the tips of villi.Fuller and Turvey (7) observed massive bacterial colonizationin the chicken cecum, where a thick layer of bacteria coveredthe epithelium. We found that the lactobacilli adhered to thececal epithelium only poorly. On the other hand, it has beensuggested that the bacterial adherence plays a less importantrole in cecal colonization due to the slow peristalsis of cecalloops, which gives a longer time for bacteria to multiply (6).

The M cells located in the FAE are important for pathogen invasioninto the circulation and for presentation of antigens to theimmune system (14). The adherence of lactobacilli to FAE observedin this study may denote presentation of lactobacilli to theimmune cells and is in line with the recent report of associationof certain lactobacilli with Peyer's patches of the mouse (15).The light microscopy techniques employed here do not allow identificationof individual M cells in the chicken ileum (3, 10), and thepossible interaction of Lactobacillus with M cells requiresa more detailed study. Our ongoing research (18) has shown thatavian pathogenic Escherichia coli adhere to the crop epitheliumand the FAE by using their mannose-binding type 1 fimbriae andthat the strain ST1 efficiently inhibits such binding in vitro.

In summary, our results show that Lactobacillus isolates exhibitconsiderable cell and tissue tropism in their adherence to thetissue domains of the chicken intestine. Isolates from the chickenshared adhesiveness to the crop epithelium but showed differencesin their adherence to enterocytes at the villi and to the FAEin the ileum. With the lack of knowledge on the adhesin moleculesincluded in lactobacillar adhesion, we have no explanation atthe present for the low adhesiveness of the lactobacilli obtainedfrom the culture collection. Our results support the hypothesis(7) that bacterial adherence to the crop is important for colonizationin the chicken and suggest, on the other hand, that some lactobacillarisolates colonize the crop and simply pass through the otherparts of the chicken intestine.

ACKNOWLEDGMENTS

This study was supported by the Academy of Finland (grant numbers44168, 44600, and 50725).

We thank Brita Mäki and Raili Lameranta for skilled technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Division of General Microbiology, Department of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Finland. Phone: 358-9-19159260. Fax: 358-9-19159262. E-mail: timo.korhonen{at}helsinki.fi.

REFERENCES

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