JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1988, p. 2018-2024 0095-1137/88/102018-07$02.00/0 Copyright C 1988, American Society for Microbiology Vol. 26, No. 10 Vibrio cholerae Non-O1: Production of Cell-Associated Hemagglutinins and In Vitro Adherence to Mucus Coat and Epithelial Surfaces of the Villi and Lymphoid Follicles of Human Small Intestines Treated with Formalin TATSUO YAMAMOTO* AND TAKESHI YOKOTA Department of Bacteriology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan Received 1 April 1988/Accepted 21 June 1988 Clinically isolated Vibrio cholerae non-Ol strains produced more cell-associated hemagglutinins (HAs) on colonization factor antigen agar after ca. 3 h than after ca. 20 h of incubation at 37°C. A high cell-associated HA producer variant of strain TVN-318, grown for 3 h at 37°C, was entrapped in a native mucus coat covering the human ileal mucosa and displayed a striking ability to adhere to the surface of a Formalin-treated mucus coat, in contrast to a poor cell-associated HA producer variant of TVN-318, grown for 20 h at 37°C. Adherence to the Formalin-treated human mucus coat was confirmed with all of the strains tested. V. cholerae non-Ol strains also possessed the ability to adhere to the epithelial surfaces of Formalin-treated human and rabbit ileal or jejunal villi, as well as human lymphoid follicles, in proportion to cell-associated HA levels. The epithelial surface of the lymphoid follicles provided most of the adherence sites for V. cholerae non-Ol strains under the test conditions. We conclude that a mucus coat covering the human small intestinal mucosa is a primary adherence target for V. cholerae non-Ol strains in human intestinal infections and that cell-associated HAs have at least a partial role in the adherence of V. cholerae non-Ol strains to the human small intestine, suggesting a potential role for V. cholerae non-Ol strains in an oral live vaccine. Vibrio cholerae non-Q1 strains, previously called nonag- glutinable vibrios, are widely distributed in the environment, e.g., in estuarine waters and seafood. Some strains are associated with human diarrheal diseases and extraintestinal opportunistic infections (2, 9, 12). Most V. cholerae non-Q1 strains produce a heat-labile hemolysin (7, 11, 18, 20), whereas some strains produce a heat-labile enterotoxin that is very similar to cholera toxin (4, 19, 21, 23), another distinct type of heat-labile enterotoxin (vibrio factor) that is detected by the suckling-mouse assay (15), a heat-stable enterotoxin that is similar to Escherichia coli heat-stable enterotoxin I (1), or a thermostable direct hemolysin (22). Cell-associated hemagglutinins (HAs) and soluble HA-pro- tease are widely distributed in V. cholerae non-Q1 strains, which resemble V. cholerae Q1 strains in this respect (3, 6). Little is known about human colonization by V. cholerae non-Q1 strains. In this study we demonstrated that V. cholerae non-Q1 strains, isolated from patients with diar- rhea, produced cell-associated HAs after as little as ca. 3 h at 37°C and that V. cholerae non-Q1 strains possess the ability to adhere to a mucus coat and to the Formalin-fixed epithe- lial surfaces of the villi and lymphoid follicles of the human small intestine in numbers proportional to the cell-associated HA levels. MATERIALS AND METHODS Bacteria and media. Nine V. cholerae non-Q1 strains were isolated in the Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, Japan, from patients with traveler's diarrhea who had visited Southeast Asian countries in 1986 and 1987. They were kindly provided by S. Matsushita and Y. Kudo. For bacterial growth, colonization factor antigen (CFA) agar (5), consisting of 1% Casamino Acids (Difco * Corresponding author. Laboratories, Detroit, Mich.), 0.15% yeast extract (Difco), 0.005% MgSO4, 0.0005% MnCl2, and 2% agar (pH 7.4), was used. Cell-associated HA assay. V. cholerae non-Qi cells were streaked on the surface of CFA agar plates and grown for 3 to 20 h at 37°C. Bacterial cells grown on the surface were suspended in a modified Krebs-Ringer solution, designated KRT (8), consisting of 7.5 g of NaCl, 0.383 g of KCl, 0.318 g of MgSO4- 7H20, and 0.305 g of CaCl2 in 10 mM Tris hydrochloride (pH 7.4) to a concentration of 300 Klett units (measured in a Klett-Summerson colorimeter with a red filter). Twofold serial dilutions were then made with KRT (pH 7.4), and 100-,ul samples were mixed with 100 ,ul of 3% human (group A) erythrocytes in a 24-well multidish plate (diameter of each well, 15 mm; A/S Nunc, Roskilde, Den- mark). After the samples had stood for 20 min at room temperature (ca. 22°C), HA titers were determined by using a light microscope. The results roughly corresponded to those of hemagglutination on glass slides. The effect of sugar on the HA reaction was examined in the same way, except that 3% human erythrocytes containing 1% (wt/vol) sugar (L-fucose or D-mannose) were used instead of 3% human erythrocytes alone. When the effect of EDTA on the HA reaction was tested, bacterial cells were diluted with KRT (pH 7.4) containing 10 mM EDTA (pH 7.4) and then mixed with 3% human erythrocytes as above. Preparation of small-intestinal specimens. Rabbit jejunal specimens were prepared as described by Nakasone and Iwanaga (13). The jejunum was excised from an adult Japanese White rabbit weighing 2.5 kg, washed with cold (4°C) phosphate-buffered saline (pH 7.4), and fixed with 10% (vol/vol) Formalin in KRT (pH 7.4) (8, 13). Specimens of the human small intestine used in this study were terminal segments of the small intestines excised at Juntendo Hospital from patients (aged 28 to 54 years) with 2018 on March 31, 2020 by guest http://jcm.asm.org/ Downloaded from
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JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1988, p. 2018-20240095-1137/88/102018-07$02.00/0Copyright C 1988, American Society for Microbiology
Vol. 26, No. 10
Vibrio cholerae Non-O1: Production of Cell-AssociatedHemagglutinins and In Vitro Adherence to Mucus Coat and
Epithelial Surfaces of the Villi and Lymphoid Follicles of HumanSmall Intestines Treated with Formalin
TATSUO YAMAMOTO* AND TAKESHI YOKOTADepartment of Bacteriology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan
Received 1 April 1988/Accepted 21 June 1988
Clinically isolated Vibrio cholerae non-Ol strains produced more cell-associated hemagglutinins (HAs) oncolonization factor antigen agar after ca. 3 h than after ca. 20 h of incubation at 37°C. A high cell-associatedHA producer variant of strain TVN-318, grown for 3 h at 37°C, was entrapped in a native mucus coat coveringthe human ileal mucosa and displayed a striking ability to adhere to the surface of a Formalin-treated mucuscoat, in contrast to a poor cell-associated HA producer variant of TVN-318, grown for 20 h at 37°C. Adherenceto the Formalin-treated human mucus coat was confirmed with all of the strains tested. V. cholerae non-Olstrains also possessed the ability to adhere to the epithelial surfaces of Formalin-treated human and rabbit ilealor jejunal villi, as well as human lymphoid follicles, in proportion to cell-associated HA levels. The epithelialsurface of the lymphoid follicles provided most of the adherence sites for V. cholerae non-Ol strains under thetest conditions. We conclude that a mucus coat covering the human small intestinal mucosa is a primaryadherence target for V. cholerae non-Ol strains in human intestinal infections and that cell-associated HAshave at least a partial role in the adherence of V. cholerae non-Ol strains to the human small intestine,suggesting a potential role for V. cholerae non-Ol strains in an oral live vaccine.
Vibrio cholerae non-Q1 strains, previously called nonag-glutinable vibrios, are widely distributed in the environment,e.g., in estuarine waters and seafood. Some strains areassociated with human diarrheal diseases and extraintestinalopportunistic infections (2, 9, 12). Most V. cholerae non-Q1strains produce a heat-labile hemolysin (7, 11, 18, 20),whereas some strains produce a heat-labile enterotoxin thatis very similar to cholera toxin (4, 19, 21, 23), anotherdistinct type of heat-labile enterotoxin (vibrio factor) that isdetected by the suckling-mouse assay (15), a heat-stableenterotoxin that is similar to Escherichia coli heat-stableenterotoxin I (1), or a thermostable direct hemolysin (22).Cell-associated hemagglutinins (HAs) and soluble HA-pro-tease are widely distributed in V. cholerae non-Q1 strains,which resemble V. cholerae Q1 strains in this respect (3, 6).Little is known about human colonization by V. choleraenon-Q1 strains. In this study we demonstrated that V.cholerae non-Q1 strains, isolated from patients with diar-rhea, produced cell-associated HAs after as little as ca. 3 h at37°C and that V. cholerae non-Q1 strains possess the abilityto adhere to a mucus coat and to the Formalin-fixed epithe-lial surfaces of the villi and lymphoid follicles of the humansmall intestine in numbers proportional to the cell-associatedHA levels.
MATERIALS AND METHODS
Bacteria and media. Nine V. cholerae non-Q1 strains wereisolated in the Tokyo Metropolitan Research Laboratory ofPublic Health, Tokyo, Japan, from patients with traveler'sdiarrhea who had visited Southeast Asian countries in 1986and 1987. They were kindly provided by S. Matsushita andY. Kudo. For bacterial growth, colonization factor antigen(CFA) agar (5), consisting of 1% Casamino Acids (Difco
* Corresponding author.
Laboratories, Detroit, Mich.), 0.15% yeast extract (Difco),0.005% MgSO4, 0.0005% MnCl2, and 2% agar (pH 7.4), wasused.
Cell-associated HA assay. V. cholerae non-Qi cells werestreaked on the surface of CFA agar plates and grown for 3to 20 h at 37°C. Bacterial cells grown on the surface weresuspended in a modified Krebs-Ringer solution, designatedKRT (8), consisting of 7.5 g of NaCl, 0.383 g of KCl, 0.318 gof MgSO4- 7H20, and 0.305 g of CaCl2 in 10 mM Trishydrochloride (pH 7.4) to a concentration of 300 Klett units(measured in a Klett-Summerson colorimeter with a redfilter). Twofold serial dilutions were then made with KRT(pH 7.4), and 100-,ul samples were mixed with 100 ,ul of 3%human (group A) erythrocytes in a 24-well multidish plate(diameter of each well, 15 mm; A/S Nunc, Roskilde, Den-mark). After the samples had stood for 20 min at roomtemperature (ca. 22°C), HA titers were determined by usinga light microscope. The results roughly corresponded tothose of hemagglutination on glass slides. The effect of sugaron the HA reaction was examined in the same way, exceptthat 3% human erythrocytes containing 1% (wt/vol) sugar(L-fucose or D-mannose) were used instead of 3% humanerythrocytes alone. When the effect of EDTA on the HAreaction was tested, bacterial cells were diluted with KRT(pH 7.4) containing 10 mM EDTA (pH 7.4) and then mixedwith 3% human erythrocytes as above.
Preparation of small-intestinal specimens. Rabbit jejunalspecimens were prepared as described by Nakasone andIwanaga (13). The jejunum was excised from an adultJapanese White rabbit weighing 2.5 kg, washed with cold(4°C) phosphate-buffered saline (pH 7.4), and fixed with 10%(vol/vol) Formalin in KRT (pH 7.4) (8, 13).Specimens of the human small intestine used in this study
were terminal segments of the small intestines excised atJuntendo Hospital from patients (aged 28 to 54 years) with
V. CHOLERAE HEMAGGLUTININ PRODUCTION AND ADHERENCE 2019
ascending colon cancer. The specimens were donated by T.Kamano, First Department of Surgery, Juntendo University.The intestinal segments were aseptically opened, the muco-sal side of the small intestine (from the muscularis mucosae
to the mucosal epithelium with mucus) was saved, and theremaining tissue was discarded. The mucosa with adherentmucus was washed several times with fresh, cold (4°C)phosphate-buffered saline (pH 7.4). When V. cholerae non-
O1 adherence to the native mucus coat was tested, a slice (1cm by 1 cm) of the washed small-intestinal mucosa (withmucus) was used. For other experiments, the washed small-intestinal mucosa (with mucus) was fixed with 10% Formalinand maintained at 4°C. Prior to the adherence experiments,the Formalin-fixed mucosa (with mucus) was washed inKRT (pH 8.0), and part of the mucus coat covering themucosal surface was carefully removed with soft tissuepaper. The specimens were then cut into squares (0.5 cm by0.5 cm) and washed with cold (4°C) KRT (pH 7.4) with twobuffer changes (500 ml each) for 3 h. The Formalin-treatedmucus coat was somewhat more rigid than the untreatedmucus coat, although it was still viscous.
Adherence tests and scanning electron microscopy. Bacte-rial adherence was evaluated as described previously for V.cholerae O1 adherence to the rabbit mucosa (8, 13). A pieceof the human or rabbit intestinal sample, prepared as above,was immersed into 1.5 to 2 ml of bacterial suspensions at 600Klett units (in KRT [pH 7.4]) and incubated for 10 min at28°C. The intestinal samples were immediately washed fourtimes in KRT (pH 7.4), fixed for 2 h at room temperature ina KRT (pH 7.4) solution containing 2.5% (vol/vol) glutaral-dehyde and 2% (wt/vol) tannic acid, and subsequently post-fixed in 1% (wt/vol) osmium tetroxide for 2 h (or overnight)at 4°C. The fixed samples were dehydrated with acetone andcritical point dried. They were then coated with gold-palladium and analyzed by scanning electron microscopy. Inevery area (epithelial surfaces of the villi or lymphoidfollicles) tested, 30 observation fields (obtained at a magni-fication of 4,000 and covering an area of 23 by 28 ,um) were
randomly chosen and photographed, and the numbers ofbacteria were recorded for each sample. The average num-ber of bacteria per electron-microscopic field (photograph)constituted the adherence index.
Transmission electron microscopy. Bacterial cells grown onCFA agar plates for 3 and 20 h at 37°C were suspended indistilled water. One drop of the bacterial suspension wasapplied to each of several carbon-coated collodion gridscreens (diameter, 3 mm) for 60 s. The adherent bacteria onthe screens were then negatively stained with 2% (wt/vol)uranyl acetate for 60 s. The stained grids were subjected totransmission electron-microscopic analysis.
RESULTS
Cell-associated HA production on CFA agar. When V.cholerae non-O1 strains were grown on CFA agar at 37°C,cell-associated HA production was observed to be greaterafter 3 h of incubation than after 20 h of incubation (Table 1).The cell-associated HAs observed after 3 h of incubationwere divided into those resistant to both L-fucOse andD-mannose and those resistant to L-fucose but moderatelysensitive to D-mannose (Table 1). All HAs were variablysensitive to EDTA (Table 1).Adherence to and entrapment in a mucus coat. An un-
treated (native) human ileal mucosa (with mucus) samplewas immersed for 10 min at 28°C in a bacterial suspension (inKRT [pH 7.4]) of the V. cholerae non-O1 strain TVN-318,
TABLE 1. Bacterial strains and characteristics ofcell-associated HAs
HA activity with following addition:V. cholerae Length (h) ofnon-01 incubation 0.5% 0.5% 5 mMstrain at 37°C None (wt/vol) (wt/vol) EDTA
L-fucose D-mannose
TVN-318b 3 1:32 1:32 1:32 1:1620 <1:1 ND' ND ND
TVN-324b 3 1:64 1:64 1:64 1:1620 1:4 ND ND ND
TVN-332b 3 1:16 1:16 1:32 1:220 1:1 ND ND ND
TVN-334b 3 1:4 1:4 1:8 1:220 <1:1 ND ND ND
TVN-316b 3 1:4 1:4 1:4 1:220 <1:1 ND ND ND
87-530b 3 1:8 1:8 1:8 1:220 <1:1 ND ND ND
87-615d 3 1:32 1:16 1:4 1:220 1:2 ND ND ND
TVN-323d 3 1:32 1:32 1:8 1:120 1:1 ND ND ND
87-715d 3 1:8 1:8 1:4 <1:120 <1:1 ND ND ND
a Data (HA titers) indicate the highest dilution required to yield positiveresults.
b HAs resistant to L-fucose and D-mannose.C ND. Not done.d HAs resistant to L-fucose and moderately sensitive to D-mannose.
which had been grown on CFA agar for 3 h at 37°C. Themucosa was then analyzed by scanning electron microscopy.It displayed marked entrapment of bacteria in the deepmucus coat. The adherence of the same strain to the mucuscoat was demonstrated by using Formalin-fixed human ilealmucosa (with mucus). The high cell-associated HA producervariant of TVN-318, grown for 3 h at 37°C, adhered well tothe surface of à Formalin-treated mucus coat (Fig. 1A andB). In contrast, a poor cell-associated HA producer variantof TVN-318, grown for 20 h at 37°C, adhered at much lowerlevels to the surface of a Formalin-treated mucus coat (Fig.1D and E). Adherence to a Formalin-treated human ilealmucus coat was confirmed with all of the strains (Table 1)tested, although the adherence levels manifested by thestrains (grown for 3 h at 37°C) differed from each other andwere roughly proportional to the cell-associated HA levelsshown in Table 1. Moreover, with each strain, the adherencelevels observed after 20 h of incubation were much lowerthan the adherence levels observed after 3 h of incubation(Fig. 1B and E).Adherence to the Formalin-fixed villus surface. A high
cell-associated HA producer variant of TVN-318, grown for3 h at 37°C, also adhered well to the epithelial surface of theFormalin-treated human ileal villi (Fig. 1A and C) but to alesser extent than to the mucus coat (Fig. 1A and B). A poorcell-associated HA producer variant of TVN-318, grown for20 h at 37°C, adhered to the epithelial surface at a reducedlevel (Fig. 1D and F). A similar relationship between adher-ence and bacterial growth condition (cell-associated HAlevels) was observed with Formalin-treated human jejunalmucosa (Fig. 1G and H).
Formalin-treated human ileal and jejunal mucosa, as wellas Formalin-treated rabbit jejunal mucosa, were exposed tobacterial cells of various V. cholerae non-01 strains, grownfor 3 and 20 h at 37°C. Adherence of bacteria to the epithelialsurface of the villi was examined by scanning electron
V. CHOLERAE HEMAGGLUTININ PRODUCTION AND ADHERENCE 2021
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HA titer(reciprocal)FIG. 2. Relationship between the cell-associated HA levels of V.
cholerae non-O1 strains and the adherence index obtained for theepithelial surfaces of Formalin-treated human ileal and jejunal villiand rabbit jejunal villi. Data were taken from Tables 1 and 2.
(Table 2) and the cell-associated HA levels (Table 1) wasalso confirmed for strains grown for 20 h (Fig. 2). However,when 20- and 3-h-grown strains (different strains) that exhib-ited similar HA levels were compared, the former mani-fested a higher adherence index than did the latter (Fig. 2);e.g., 20-h-grown TVN-324 (HA titer, 1:4; adherence index[human ileum], 16.8 ± 21.4) versus 3-h-grown TVN-316 (HAtiter, 1:4; adherence index [human ileum], 1.4 ± 2.9).
In these experiments, it was also found that human ilealvilli, human jejunal villi, and rabbit jejunal villi were almostequal targets for V. cholerae non-O1 strains in terms ofadherence efficiency (Table 2).Adherence to the surfaces of Formalin-fixed human ileal
lymphoid follicles. Some of the specimens of human ileumcontained lymphoid follicles (or Peyer's patches) (16) sur-rounded by numerous villi (Fig. 3A). A high cell-associatedHA producer variant of TVN-318, after 3 h of incubation at37°C, manifested marked adherence to a mucus coat as wellas to the epithelial surfaces of the Formalin-treated lymphoidfollicles (Fig. 3B to D). Again, adherence to the mucus coatwas much more prominent than to the epithelial surfaces(compare Fig. 3C and D). In contrast, a poor cell-associatedHA producer variant of TVN-318, grown for 20 h at 37°C,adhered less extensively to both the mucus coat and epithe-lial surfaces (Fig. 3E to G).The adherence index determined for the epithelial surfaces
with each of various V. cholerae non-O1 strains grown for 3and 20 h at 37°C is shown in Table 3. All strains displayed agreater adherence index to the epithelial cells than to theepithelial surface (cells) of the ileal villi from the sameintestinal specimen (Table 2). Good correlation existed be-
TABLE 2. Adherence of V. cholerae non-O1 strains to theepithelial cell surface of Formalin-treated human
and rabbit small-intestinal villia
Strain and Adherence index for bacteria grownsmall-intestinal at 37'C for:
87-715Human ileum 11.5 ± 13.0 4.1 ± 9.4Human jejunum 8.1 ± 11.5 NDRabbit jejunum 6.5 ± 6.6 1.1 ± 1.8a The adherence index was 0.0 ± 0.0 when the control human ileum or
jejunum was tested.b ND, Not done.
tween the adherence index (Table 3) and the cell-associatedHA levels (Table 1).
Production of pili. The production of pili of TVN-318,which was grown on CFA agar for 3 and 20 h at 37°C, wasexamined by transmission electron microscopy. When TVN-318 was grown for 3 h, 6 of 119 bacteria observed (5.0%)were positive for piliation, whereas when TVN-318 wasgrown for 20 h, 23 of 71 bacteria observed (32.4%) werepositive for piliation under the conditions used. Thus, theproduction of pili was much more obvious with TVN-318grown for 20 h than with TVN-318 grown for 3 h.
DISCUSSION
The entrapment of V. cholerae O1 strains in the mucus(mucins) of the rabbit small intestine has been demonstratedpreviously, but bacterial adherence was not shown (8, 14).The present study demonstrates that V. cholerae non-O1strains do adhere to and are entrapped in the mucus coatcovering the human ileal villi or lymphoid follicles. On thebasis of our observations that V. cholerae non-O1 strainsadhered to the mucus coat more prominently than to the
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epithelial surfaces of the same intestinal villi or lymphoidfollicles, we conclude that the ileal mucus coat is a primaryadherence target for V. cholerae non-01 strains in humanintestinal infections.
The rigidity of a Formalin-treated mucus coat enables usto study human small-intestinal mucosa mostly free of amucus coat following treatment with Formalin. Such aFormalin-treated human small-intestinal mucosa (villi andlymphoid follicles) was used in the present epithelial adher-ence experiments. Jones et al. (8) have shown that V.cholerae 01 strains adhere to Formalin-treated rabbit brushborders as effectively as to freshly prepared rabbit brushborders. By using the Formalin-treated small-intestinal mu-cosa, we showed that (i) V. cholerae non-01 strains adheredto the rabbit jejunal villus surface to a similar (or evengreater) extent as they adhered to the human jejunal or ilealvillus surface (Table 2); (i) V. cholerae non-01 strainsadhered to the epithelial surface of the human ileal lymphoidfollicles more strikingly than to the epithelial surface of thehuman ileal orjejunal villi (Tables 2 and 3), and therefore theadherence targets of the strains were mucus, lymphoidfollicle epithelial cell surface, and villus epithelial cell sur-face (in order of decreasing adherence efficiency); and (iii)cell-associated HA plays a role, at least in part, in suchadherence (Tables 1 to 3; Fig. 2).
A%.
14.,., A
FIG. 3. Adherence of V. cholerae non-O1 strain TVN-318 to Formalin-treated lymphoid follicles in the human ileum. (A) Control humanileal mucosa with lymphoid follicles surrounded by the villi; (B to D) epithelial surfaces of the Iymphoid follicles which were adhered to byTVN-318 grown for 3 h at 37°C; (E to G) epithelial surfaces of the lymphoid follicles which were adhered to by TVN-318 grown for 20 h at37°C. Panels C and F show mucus; they are higher magnifications of panels B and E, respectively. Panels D and G show the epithelialsurfaces; they are higher magnifications of panels B and E, respectively. The mucus-producing goblet cells, which lack the brush borders, areobserved as holes surrounded by numerous brush borders (panels B, D, E and G). Numbers indicate length of scale bars in micrometers.
a The adherence index was 0.0 ± 0.0 when the control human ilealiymphoid follicle epithelium was tested.
b ND, Not done.
The production of pili (of TVN-318) was more markedafter 20 h of incubation at 37°C, when no detectable cell-associated HAs were produced (Table 1) and low levels ofadherence were observed (Tables 2 and 3), than after 3 h ofincubation at 37°C, when levels of cell-associated HAs aswell as adherence were markedly high (Tables 1 to 3). Thus,piliation of V. cholerae non-01 strain TVN-318 did not seemto strictly correlate with adherence or cell-associated HAactivities detected, although the piliation may account forthe considerably higher adherence index observed withbacteria grown for 20 h at 37°C (Fig. 2).
V. cholerae non-01 strains (e.g., TVN-318, TVN-324, and87-615) adhered strikingly to the epithelial surface of theFormalin-treated human lymphoid follicles (Fig. 3B to D).There existed some morphologically distinct epithelial cellsthat covered the tissue, e.g., cells having long microvilli(brush borders) similar to the absorptive cells on the ileal orjejunal villi, cells having short and irregular microvilli ormicrofolds (M cells [16]), and, occasionally, goblet cellsproducing mucus. V. cholerae non-01 strains adhered al-
FIG. 4. Adherence of V. cholerae non-Qi strain TVN-324,
grown for 3 h at 370C, to epithelial cells with long and short
microvilli in Formalin-trcated human lymphoid folicles. The num-
ber indicates the length of the scale bar in micrometers.
most uniformly to the surfaces of these morphologicallydifferent epithelial cells (Fig. 4). Since M cells conveybacterial pathogens from the intestinal lumen into intestinallymphoid tissue and thus play a role in the initial step of thelocal immune responses (17), the ability to adhere to M cellsmay be an important factor for oral live vaccines, such aslive cholera vaccine (10). In view of this situation, V.cholerae non-01 strains which possess the ability to strik-ingly adhere to the epithelial surface of the human ileallymphoid follicles (Table 3) may be a potential vector forconstruction of genetically manipulated live vaccines againstintestinal infections.
Finally, the possibility that V. cholerae non-Q1 strains canalso adhere to the native epithelial surfaces of the humanlymphoid follicles (or Peyer's patches) much more promi-nently than to the native epithelial surfaces of the humanileal villi is under investigation.
ACKNOWLEDGMENTS
We thank Y. Kudo and S. Matsushita for the bacterial strains, T.Kamano for human small-intestinal specimens, K. Sato and M.Uchimura for assistance in transmission electron microscopic anal-ysis, and M. Iwanaga for advice.
This work was supported by a grant-in-aid for scientific researchfrom the Ministry of Education, Science, and Culture of Japan.
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