Vol. 21, No. 2

INFECTION AND IMMUNITY, Aug. 1978, P. 392-397 0019-9567/78/0021-0392$02.00$0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

In Vitro Adhesion of Escherichia coli to Porcine Small Intestinal Epithelial Cells: Pili as Adhesive Factors R. E. ISAACSON,`* P. C. FUSCO,2 C. C. BRINTON,2 AND H. W. MOON' National Animal Disease Center, Agricultural Research Service, U.S. Department ofAgriculture, Ames, Iowa 50010,' and Department of Life Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 152602

Received for publication 27 March 1978

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99', or type 1 pilus' strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99' strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.

Enterotoxigenic Escherichia coli (ETEC) colonize the porcine small intestine by attachment or adhesion to small intestinal epithelial cells (1, 2, 7, 14). Various lines of evidence are consistent with the hypothesis that the attachment of bacterial cells to epithelial cells is mediated by pili (10, 11, 15). Three serologically different E. coli pili have so far been recognized as pili associated with bacterial colonization. They are K88 (16), which is found on porcine ETEC, and K99 and 987P, which are found on both porcine and bovine ETEC (13; R. E. Isaacson, H. W. Moon, and R. A. Schneider, Am. J. Vet. Res., in press). A fourth pilus, called type 1 pilus (3), is a hemagglutinin and promotes bacterial cell attachment to solid surfaces and, therefore, may also be important as a colonization and adhesive factor. Type I pili have been found on some ETEC which induce diarrhea in humans (4a). The study of the adhesion of K88+ ETEC to porcine intestinal epithelial cells was simplified by the development of several in vitro adhesion assays (12, 19, 20). However, at the time that these assays were developed, it was shown that K88- ETEC did not adhere. Subsequently, we reported on the in vitro attachment of a 987P+ ETEC strain to porcine intestinal epithelial cells (15), and Burrows, Sellwood, and Gibbons (4) reported on the attachment of K99+ bovine ETEC strains to bovine intestinal epithelial 392

brush borders. Using the isolated intestinal epithelial cell in vitro adhesion assay, we wanted to confirm the observation that a 987P+ ETEC strain adheres and to determine whether K99+ and type 1 pilus+ E. coli strains would also adhere. In addition, we wanted to quantitatively describe the adhesive process in vitro and to determine whether these specific colonization pili acted as adhesive factors in the in vitro adhesion assay and whether these pili acted with biological specificity. MATERUILS AND METHODS E. coli strains and growth conditions. The E. coli strains used are listed in Table 1. The pili on these strains have been described previously (3, 8, 10). Stock cultures of strains used for the in vitro adhesion assays were maintained at 4°C on blood agar plates for the 987P+ strain (987), on tryptone broth agar plates (9) for the K99+ strains, and on minimal glucose agar plates (18) for the type 1 pilus strain (BAM). Cells from individual colonies were tested for the presence of the described pilus by slide agglutination before inoculation into tubes of Trypticase soy broth (Baltimore Biological Laboratory). Piliated or nonpiliated clones were incubated for 18 to 24 h at 37°C with vigorous shaking. Before a culture was used for an in vitro adhesion assay, it was centrifuged at 10,000 x g and resuspended in 0.15 M NaCl (saline). The cells were then tested for specific pilus production by slide agglutination. In vitro adhesion assay. Isolated intestinal epi-

VOL. 21, 1978

ADHESION OF E. COLI

TABLE 1. E. coli strauis used Strain

Pilus type

Origin

B41

K99

Bovine

B44 431 987

K99 K99 987P

Bovine Porcine Porcine

140

987P

1474

K99

1475

None

Comments

K99 reference strain

Prototype 987P strain Laboratory Acapsular mutant of strain 987 Laboratory Constructed by introduction of K99 plssmid from B41 into 1475 Laboratory K-12 strain (711)

393

with Fab fragments was performed in 0.4-ml volumes. Bacteria were concentrated twofold, and 0.1 ml of bacteria was mixed with 0.1 ml of Fab. These mixes were preincubated 15 min as above followed by the addition of 0.2 ml of epithelial cells and an additional 15-min incubation. Purified pili. Type 1 pili from E. coli BAM and 987P from E. coli 987 were purified as previously described (3). K99 was purified from E. coli 1474 as previously described (8). Fab fragments. Fab fragments were prepared by the method of Porter (17) by using gamma globulins obtained from rabbits previously immunized with K99, 987P, or type 1 pili or strain 1475 cells.

RESULTS In vitro adhesion to porcine intestinal epithelial cells. Strains 987 (987P+), 1474 thelial cells were prepared from 1-day-old hysterec- (K99+), and BAm (type 1 pilus+) were each grown tomy-derived, colostrum-deprived pigs by a modifica- overnight (inoculating from a colony of piliated tion of the procedure of Wilson and Hohmann (20). bacteria) and used undiluted (109 to 2 x 109/ml) Pigs were starved overnight and sacrificed, and the for the in vitro adhesion assay using ileal epitheentire small intestines were removed. For initial ex- lial cells. Each assay was run in triplicate (on periments, the small intestines were divided into separate days). The results were that 35.1 bacthirds, the middle third was discarded, and the other terial cells adhered per epithelial cell when two segments were treated separately. The remaining strain 987 was used, 22.5 bacterial cells adhered pieces of small intestine were placed in phosphate- per epithelial cell when strain 1474 (K99+) was buffered saline (PBS) containing 8 mM Na2HPO4, 15 used, and 25.0 bacterial cells adhered per epithemM KH2PO4, 2.7 mM KC1, and 0.137 M NaCl (pH 7.5) for 30 to 60 min at 40C. Portions of small intestine lial cell when strain Bm (type 1 pilus') was used. Nonpiliated cultures of strains 987 and were everted onto spiral coils of copper tubing (20), and the tubing was attached to a Pollenex 3D orbital BAm or strain 1475 failed to adhere to leal epithehial cells when used at the same approximate massager. After the tissue was submerged into fresh, cold PBS, the epithelial cells were removed by shaking concentrations as the piliated cells (less than one (vibrating) for 5 min. Epithelial cells were collected by bacterium attached per epithelial cell). Thus, centrifugation at 200 x g for 10 min at 40C and washed 987P+, K99+, and type 1 pilus+ organisms adonce by centrifugation after resuspension in PBS. The hered in vitro to porcine intestinal epithelial epithelial cells were then adjusted to 2 x 106/ml by cells. addition of cold PBS and stored at 40C. Concentration leal versus jejunal in vitro attachment of epithelial cells was determined by microscopic evaluation. At 1- to 2-week intervals, the epithelial cells by E. coli Previously, we (14) and Hohmann and Wilson (7) showed that ETEC strains lackwere washed with PBS. Upon storage for 2 months, the epithelial cells had not lost either the ability to ing K88, which colonize and adhere to porcine allow bacterial cells to adhere to them or the specific- small intestine, preferentially attach in vivo to ity of adhesion. ileal epithelium. However, when ligated small In vitro adhesion assays were performed as previ- intestinal loops were used, these ETEC adhered ously described (20) except that 0.2-ml aliquots of to both ileal and jejunal epithelium. Because epithelial and bacterial cells were used. Bacterial con- one of the aims of the work described here was centration was determined by plate count of diluted to quantitatively describe bacterial attachment samples. The number of bacteria adhering to individual epithelial cells was determined by microscopic in vitro to epithelial cells, it was necessary to observation; a Leitz orthoplan microscope with No- determine whether quantitative differences in marski optics was used. The first 20 well-defined epi- epithelial cell receptor activities for bacterial thelial cells observed were used. Aggregates or sheets cells were detectable between leal and jejunal of epithelial cells were disregarded. Competition ex- epithelial cells. Two bacterial strains, 987 periments were performed in a final volume of 1.0 ml. (987P+) and 1474 (K99+), each of which has pili The epithelial and bacterial cells were concentrated associated with ileal colonization, were used to fivefold. A 0.6-ml quantity of competitor pili (in saline) test this possibility. Various amounts of cells of was mixed with 0.2 ml of epithelial cells. These mixes either strain were mixed with a constant amount were preincubated for 15 min at 370C with gentle of ileal or jejunal epithelial cells for the detershaking, followed by the addition of 0.2 ml of bacterial mination of bacterial attachment. For either cells, and then incubated for an additional 15 min. no differences were detected in bacterial strain, as above. evaluated were Mixes microscopically Inhibition of bacterial attachment to epithelial cells the number of bacterial cells attached to either

BA. Type 1 Laboratory Prototype type 1 strain

394

INFECT. IMMUN.

ISAACSON, BRINTON, AND MOON

ileal or jejunal epithelial cells (Table 2). This supports the concept that no differences in receptor activities exist between epithelial cells of the ileum and jejunum and that the preferential attachment of bacteria to the ileum in vivo results from other factors. In subsequent experiments, epithelial cells were pooled from the entire small intestines. In vitro saturation adherence ofE. coli to intestinal epithelium. Figure 1 shows typical saturation adherence curves obtained by incubating various amounts of piliated and nonpiliated 987 and BAM or 1474 (K99+) and 1475 (K99-) with a constant amount of pooled epithelial cells. In each experiment, epithelial cells were saturated by the piliated strains when approximately 30 to 40 bacterial cells had attached. In no instance did comparable numbers of nonpiliated bacteria attach to epithelial cells. These experiments show that the adherence of bacterial cells to epithelium is not only a saturable process, but is also dependent upon the presence of a pilus on the bacterial surface. The reason that saturation is achieved in these experiments cannot be determined from the data. Saturation could arise as a result of the saturation of specific epithelial cell receptors or as a result of physical space depletion. In vitro adherence of other K99+ E. coli to intestinal epithelium. Three other K99' strains of bovine and porcine origin were tested for the degree ofattachment to porcine intestinal epithelial cells (Table 3) and were compared with the "prototype strain" 1474. Each strain adhered; however, the number of bacterial cells of strain B44 that adhered was lower than that of the other K99' strains. The role of K99 in the attachment process was further explored by determining the degree of attachment which occurs when these bacterial strains were grown statically at 180C instead of shaking at 370C. At 180C, it can be demonstrated serologically that these strains do not produce detectable quantiTABLE 2. Comparison of in vitro adhesion to ileal and jejunal epithelial cells by E. coli strains 987 and 1474 Bacterial strain (no. of bacteria/ml)

No. of bacterial cells attached/epithelial cell

Jejunum

987 (5 x 107) 987 (1 x 108) 987 (5 x 108)

1474 (5 x 108) 1474 (1 x 109) 1474 (5 x 10W)

14.3 28.7 35.6

Ileum 13.1 28.9 34.6

8.1 9.0 18.5 18.1 33.3 31.8 a Each number is the arithmetic mean of two deter-

minations.

w

a

40 I

*

2

4

3

w J30 I

20

W

10

z

0

40

--om9m

'r

I

2

3

1

4

5

NUMBER BACTERIA ADDED (X10-8)

I-

b

-Jo

ow204 W%. Co

2

w

10(

xh

0o-

I

2

6

4

I

8

lUMBER BACTERIA ADDED (X 10-9) C 0

40

- 30 c

CL Z.20

aao 2

I

I

4

6

8 10 INUMBER BACTERIA ADDED ( X 10-9 )

FIG. 1. Saturation binding curves of piliated (0) and nonpiliated (0) strain 987 (a), BAAg (b), or 1474 and 1475 (c) to porcine intestinal epithelial cells. Piliated and nonpiliated colony types were identified as previously described (10) and used to prepare test cultures. In vitro adhesion assays were performed as described in the text; varying bacterial cell concentrations were used. Each point represents the mean of three assays conducted on different days.

ties of K99. There was a dramatic reduction in the number of attached bacteria grown at 180C when compared to 370C-grown bacteria (Table 3). The reduction in attached bacteria was 88 to 96% depending upon the strain. Competitive inhibition of bacterial attachment. If the in vitro attachment of E. coli to intestinal epithelial cells is mediated by specific pili that interact with specific receptors on the epithelial cell surface, then it should be possible to inhibit bacterial attachment with

VOL. 21, 1978

ADHESION OF E. COLI

purified homologous pilL. Figures 2 and 3 show the results of competition experiments with stains 987 and BAm. Purified 987P successfully competed for 987 attachment sites on epithelial cell surfaces as shown by the decrease in the number of 987 bacteria attached to epithelial cells with increasing pilus concentration (Fig. 2). On the other hand, the two heterologous pili (K99 and type 1 pili) were not capable of competing against 987. Conversely, purified type 1 pili competed for BA.m attachment sites on epithelial cells, but the heterologous pili (987P and K99) did not (Fig. 3). These experiments, therefore, add to the evidence indicating that 987P and type 1 pill are adhesive factors and also demonstrate a biological specificity in the binding reaction. Similar competition experiments were performed with strain 1474 and yielded unexpected results. The heterologous pili (987P and type 1 pili) had no effect on 1474 binding to epithelial TABLE 3. In vitro adhesion of K99a E. coli strains to porcine intestinal epithelial ceUs and the effect of bacterial growth at 180C on attachment No. of bacterial cells attached/epithelial ceub

Strain

370C-grown bacteria 180C-grown 370Cgrowbaceria teria bacB41 45.7 5.5 B44 7.5 0.8 42.6 431 3.6 34 1.1 1474 'Bacteria were concentrated to 7 x 10i to 8 x 109/ml before in vitro adhesion was assayed. bEach number is the arithmetic mean of three determinations. 1101I00

z zW

-p60

4W

40

OW tICFL4 Z 20200-

500 1000 1500 2000 COMPETITOR PILI (PAg) FIG. 2. Competition of strain 987 from intestinal epithelial cells by purified pili: 987P (0), K99 (0), type I (A). Bacterial cell concentration was 1O8/mL The percentage of bacteria remaining on epithelial cells was that of bacteria that attached in the absence of competitor pilL Each point represents the mean of three assays conducted on different days. 0

395

120

100

Z

z

Io

so

r

60

iJ

40

T5 2020 0

500

1000

1500

2000

CMETITOR PILI (p&g) FIG. 3. Competition of strain Be, from intestinal epithelial cells by purified pili: 987P (0), K99 (0), type 1 (A). Bacterial cell concentration was 2.5 x 109/ml. The percentage of bacteria remaining on epithelial cells was that of bacteria that attach in the absence of pili. Each point represents the mean of three assays conducted on different days.

cells, a result that was expected. However, when purified K99 was added as the competitor, results were variable. In some experiments, competition was almost complete (i.e., few bacteria were attached to epithelial cells at the highest K99 concentration). In other experiments, no competition occurred, and in yet other experiments, there was an enhancement in bacterial attachment at the highest K99 concentration. Thus, specific effects sometimes result from the addition of purified homologous pili (K99), although these effects are either enhancement or competition and are variable from experiment to experiment. Effect of Fab fragments on in vitro attachment. Fab fragments were prepared from gamma globulins from rabbits that had been immunized with either K99, 987P, or type 1 pili or strain 1475. Fab fragments were then tested for the ability to inhibit bacterial attachment to epithelial cells. Fab fragments were selected for these experiments instead of whole antibodies to avoid bacterial agglutination. The results are ummarized in Table 4. Fab fragments inhibited attachment of bacteria possessing the cognate pilus only. That is, Fab-K99 inhibited 1474 attachment, Fab-987P inhibited 987 attachment, and Fab-type 1 pill inhibited BAm attachment. Fab fragments specific for the E. coli K-12 envelope (Fab-1475) did not inhibit attachment by strain 1474. No other combinations of Fab fragments and bacteria resulted in inhibition of attachment. Role of the K103 capsule in adhesion. Previously, we showed that an acapsular, piliated mutant (I40) of strain 987 failed to colonize

ISAACSON, BRINTON, AND MOON

396

TABLE 4. Effect of Fab fragments on pilusmediated in vitro adhesion of E. coli strains to intestinal epithelial cells Pilus type

No. of bacterial cells

Strain

FabFab (2 (2mg mg added)

attached/epithelial

1474

None 1475 K99

39.5 39.2 4.3

Type 1 987P

40.6 40.3

None K99 Type 1 987P

40.5

987

cellara

39.3 36.6 4.1

33.3 None 35.1 K99 1.2 Type 1 33.4 987P a Each number is the arithmetic mean of three

BA.m

determinations.

the small intestine of neonatal pigs, and we concluded that the K103 capsule on strain 987 was also a colonization factor (10). In an attempt to determine the role of the capsule in adhesion, the capsulated, piliated parent 987 and the acapsular, piliated mutant I40 were used in vitro adhesion experiments. Both strains were used in the assays at the same approximate concentration (8 x 10i bacteria per ml). At this cell concentration, both strains adhered equally well to intestinal epithelial cells. In duplicate experiments run on separate days, the mean number of attached bacteria per epithelial cell was 31.9 for strain 987 and 32.1 for the mutant strain I40. DISCUSSION

In several reports, in vitro adhesion of K88+ porcine ETEC strains to either isolated porcine intestinal epithelial cells (20) or purified porcine intestinal epithelial brush borders has been described (12, 19). K88-negative ETEC, even those with the potential to produce K99, failed to adhere in both assays. Recently, we reported that one K88-negative porcine ETEC, strain 987, does in fact adhere in vitro to epithelial cells if the cultures are grown in a manner that helps to maximize expression of pilus production (15). Presumably, the attachment was dependent upon pili because the stock culture of 987, which is currently mostly nonpiliated, failed to adhere in vitro, whereas cultures prepared from heavily piliated clones did adhere. The data presented in this paper confirm the observation that 987 adheres to intestinal epithelial cells in vitro. In addition, data show that the K99+ strains and a type 1 pilus+ strain, when grown in a manner

INFECT. IMMUN.

that mximizes pilus production, also adhere in the same in vitro system. The production of K99 in vitro requires specific cultivation procedures for expression (5, 6; Isaacson et al., Am J. Vet. Res., in press); therefore, it is probable that the reason why K99' bacteria previously tested did not adhere in vitro was that they were not grown in a manner that promoted K99 production. Piliated strain I40, a mutant of strain 987 that lacks a capsule, adheres in vitro to intestinal epithelial cells. Previously, we showed that the capsule (K103 antigen) of strain 987 also acts as an in vivo colonization factor (10, 14). At that time we proposed that the 987 pilus and K103 capsule may act synergistically (10). Thus, an effect against the action of one trait impairs the action of the other strain. However, this is unlikely, because the acapsular, piliated mutant of 987, strain 140, adheres in vitro as well as the parent strain. Another explanation is that the capsule also is an adhesive factor. If the capsule is involved in attachment to epithelial cells, purified pili should not compete against bacteria because the bacteria have an alternate means of adhering. Alternatively, the data in Fig. 3 show that at the highest competitor concentration of 987 pili, some bacteria still adhere to epithelial cells. This residual adherence could result from capsule attachment activity. Residual adherence could also reflect an insufficient amount of competitor pili added to the reaction mixture or nonspecific attachment. Further experiments are needed to clarify this point. A major hypothesis invoked to explain the in vitro adhesion results is that adhesion is mediated by pili. Data from in vivo colonization experiments are consistent with this hypothesis but cannot be taken as a rigorous proof (10). The data presented here demonstrate an intrinsic role for pili in in vitro adhesion. Bacterial cultures that were producing or had produced pili at the time ofthe assay attached to epithelial cells, but nonpiliated bacteria did not. It is possible that the selection of nonpiliated clones also selects for other phenotypic differences that are also vital for attachment. If this is so, then the experiments with the isogenic strains 1474 and 1475 indicate that at least for K99 the other differences are also mediated by the K99 plasmid. More convincing evidence in support of a role of pili in adhesion comes from the pilus competition and Fab inhibition studies. Competition against 987 bacterial cell adhesion by purified 987P and against BAm bacterial cell adhesion by purified type 1 pili indicates that these pili each interact at unique sites on the epithelial cell surface. If these pili were not involved in adhesion, it would be expected that no competition would occur. Furthermore, the

VOL. 21, 1978

competition experiments also demonstrate a specificity of 987 cell-987P and BAM cell-type 1 pilus attachment to epithelial cells. Not all pili are capable of competing. Thus, neither type 1 pili nor K99 act as competitors against 987 adherence, and 987P and K99 do not compete against BAM adherence. An alternate method of showing the adhesive property of 987P and type 1 pili comes from the Fab inhibition studies. Fab fragments directed against the two pili inhibited adhesion of the strain carrying the cognate pilus. Therefore, coating of pili with Fab fragments results in a neutralization of adhesiveness of the bacterial strain. This indicates a direct role of these pili in adhesion. Even though K99 does not consistently act as a competitive inhibitor of K99' bacteria, K99 specific effects do sometimes occur. It is probable that the inability to obtain a consistent effect from K99 reflects uncontrolled aggregation of K99. Electron micrographs of purified K99 show a strong tendency for self-aggregation at higher protein concentrations (8). However, the role of K99 in attachment was amenable to study by using various antigen specific Fab fragments. Fab fragments specific for K99 effectively inhibited adherence of K99' bacteria to epithelial cells. On the other hand, Fab fragments against type 1 pili or 987P did not inhibit adhesion. Fab fragments directed against the bacterial envelope minus K99 (1475 Fab fragments) also had no inhibitory effect. Thus, Fab fragments must be strategically placed on the bacterial surface, namely on K99, to prevent bacterial attachment. These data strongly suggest that K99 is an adhesive factor in vitro. The data also demonstrate an interesting property of porcine small intestinal epithelial cells with regard to E. coli receptors in that the surfaces of these epithelial cells have at least three E. coli pilus-specific receptors. ACKNOWLEDGMENTS We thank R. A. Schneider and S. M. Skartvedt for excellent technical assistance. This work was supported by the Agricultural Research Service, the U.S. Army Medical Research and Development Command (grants 17-75-C-5014 and 17-75-C-5005), and Bactex Inc. LITERATURE CITED 1. Arbuckle, J. B. R. 1970. The location of Escherichia coli in the pig intestine. J. Med. Microbiol. 3:333-340. 2. Bertschinger, H. U., H. W. Moon, and S. C. Whipp. 1972. Association of Escherichia coli with the small intestinal epithelium. I. Comparison of enteropathogenic and nonenteropathogenic porcine strains in pigs. Infect. Immun. 5:595-605. 3. Brinton, C. C. 1965. The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 4. Burrows, M. R., R. Sellwood, and R. A. Gibbons. 1976. Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Esch-

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erichia coli. J. Gen. Microbiol. 96: 269-275. 4a.Fusco, P. A., A. To, S. To, and C. Brinton, Jr. 1978.

Characterization of four types of E. coli pili. Proceedings of the XIIIth Joint U.S.-Japan Conference on Cholera, Atlanta, Ga., September 1977, p. 60-70. DHEW publication no. (NIH) 78-1590. National Institutes of Health, Bethesda, Md. 5. Guinee, P. A. M., W. H. Jansen, and C. M. Agterberg. 1976. Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli 6.

7. 8. 9.

10.

11.

12.

isolates from calves and its correlation with enterotoxigenicity. Infect. Immun. 13:1369-1377. Guinee, P. A. M., J. Veldkamp, and W. H. Jansen. 1977. Improved Minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infect. Immun. 15:676-678. Hohmann, A., and M. R. Wilson. 1975. Adherence of enteropathogenic Escherichia coli to intestinal epithelium in vivo. Infect. Immun. 12:866-880. Isaacson, R. E. 1977. K99 surface antigen of Escherichia coli: purification and partial characterization. Infect. Immun. 15:272-279. Isaacson, R. E., and J. Konisky. 1974. Studies on the regulation of colicin Ib synthesis. II. The synthesis of Col lb-P9 specific RNA in vivo. Mol. Gen. Genet. 132:223-232. Isaacson, R. E., B. Nagy, and H. W. Moon. 1977. Colonization of porcine small intestine by Escherichia coli: colonization and adhesion factors of pig enteropathogens that lack K88. J. Infect. Dis. 135:531-539. Jones, G. W., and J. M. Rutter. 1972. Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by Escherichia coli in piglets. Infect. Immun. 6:918-927. Jones, G. W., and J. M. Rutter. 1974. Contribution of the K88 antigen of Escherichia coli to enteropathogenicity; protection against disease by neutralizing the adhesive properties of K88 antigen. Am. J. Clin. Nutr. 27:1414-1449.

Moon, H. W., B. Nagy, R. E. Isaacson, and I. 0rskov. 1977. Occurrence of K99 antigen on Escherichia coli isolated from pigs and colonization of pig ileum by K99' enterotoxigenic E. coli from calves and pigs. Infect. Immun. 15:614-620. 14. Nagy, B., H. W. Moon, and R. E. Isaacson. 1976. Colonization of porcine small intestine by Escherichia coli: ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants. Infect. Immun. 13:1214-1220. 15. Nagy, B., H. W. Moon, and R. E. Isaacson. 1977.

13.

Colonization of porcine small intestine by enterotoxigenic Escherichia coli: selection of piliated forms in vivo, adhesion of piliated forms to epithelial cells in vitro, and incidence of a pilus antigen among porcine enteropathogenic E. coli. Infect. Immun. 6:344-352. 16. 0rskov, I., F. 0rskov, W. J. Sojka, and J. M. Leach. 1961. Simultaneous occurrence of E. coli B and L antigens in strains from diseased swine. Acta Pathol. Microbiol. Sand. 53:404-422. 17. Porter, R. R. 1959. The hydrolysis of rabbit gamma-

globulin and antibodies with crystalline papain. Biochem. J. 73:119-127. 18. Sellwood, R., R. A. Gibbons, G. W. Jones, and J. M. Rutter. 1975. Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: the existence of two pig phenotypes. J. Med. Microbiol. 8:405-411. 19. Swaney, L. M., V.-P. Lui, C.-M. To, C.-C. To, K. IppenIhler, and C. C. Brinton, Jr. 1977. Isolation and

characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production. J. Bacteriol. 130:495-505. 20. Wilson, M. RI, and A. W. Hohmann. 1974. Immunity to Escherichia coli in pigs: adhesion of enteropathogenic Escherichia coli to isolated intestinal epithelial cells. Infect. Immun. 10:776-782.

In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

Vol. 21, No. 2 INFECTION AND IMMUNITY, Aug. 1978, P. 392-397 0019-9567/78/0021-0392$02.00$0 Copyright i 1978 American Society for Microbiology Print...
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