Enterotoxigenic Escherichia coli 08:KX105 Strains - NCBI

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CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli 08:KX105 strains lacking the colonization factor antigens K88,. K99, 987P and F41 was ...
Requirement for Capsular Antigen KX105 and Fimbrial Antigen CS 1541 in the Pathogenicity of Porcine Enterotoxigenic Escherichia coli 08:KX105 Strains Andre Broes, John M. Fairbrother, Mario Jacques and Serge Lariviere

ABSTRACT The requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli 08:KX105 strains lacking the colonization factor antigens K88, K99, 987P and F41 was investigated using two encapsulated strains and their acapsular variants, one of which produced the fimbrial antigen CS1541 in vitro. None of the strains adhered in vitro to enterocytes isolated from newborn colostrum-deprived piglets. All of the strains caused diarrhea in orally infected, hysterotomy-derived, colostrum-deprived piglets although a great variability in the clinical response of the piglets was observed. Colonization of the small intestine of infected piglets by these strains was only moderate and no differences in the ability to colonize the small intestine was noted between the strains. All of the strains reacted in the indirect fluorescent antibody test with both CS1541 and 987P antisera when applied to organisms in the intestines of infected piglets. A control strain expressing the 987P fimbrial adhesin also reacted with the CS1541 antiserum applied to organisms in the intestines of an infected piglet. It was concluded that capsular antigen KX105 was not essential for intestinal colonization and production of diarrhea in hysterotomy-derived colostrum-deprived pigs, and that fimbrial antigen CS1541 does not

promote in vitro adherence to enterocyte brush borders but could be

porcelets experimentaux. Une souche temoin qui produisait l'adhesine des important in bacterial colonization in franges reagit aussi avec l'antiserum vivo. contre l'antigiene CS1541, lorsqu'on l'appliqua aux collibacilles de lintestin grele d'un porcelet experimental. Les auteurs conclurent que l'antigene RESUME capsulaire KX105 n'etait pas essentiel Cette experience consistait a etudier pour la proliferation des colibacilles le role de l'antigiene capsulaire KX105 dans l'intestin grele et la production de et de l'antigene des franges CS1541, diarrhee, chez des porcelets obtenus dans la pathogenicite des souches par cesarienne et prives de colostrum, d'Escherichia coli 08:KX105 entero- et que l'antigene CS1541 des franges toxinogenes, depourvues des anti- ne favorise pas, in vitro, I'adherence a genes K88, K99, 987P et F41, respon- la bordure en brosse des enterocytes, sables de la proliferation, a l'aide de bien qu'il puisse s'averer important deux souches encapsulees et de leurs dans la proliffration des colibacilles, variantes, depourvues de capsule, in vivo. dont l'une produisait, in vitro, I'antigene CS1541 des franges. Aucune de ces souches n'adherait, in INTRODUCTION vitro, aux enterocytes isoles de procelets nouveau-nes, prives de Enterotoxigenic Escherichia coli colostrum, mais elles causerent toutes (ETEC) strains cause watery diarrhea de la diarrhee chez des porcelets in the piglet by colonizing the small obtenus par cesarienne, prives de intestine and producing enterotoxins colostrum et infectes par la voie (1). Colonization of the intestine by buccale, meme s'ils manifesterent une most ETEC strains results from their grande variabilite dans leur reaction adhesion to the intestinal mucosa by clinique. La proliferation de ces means of one or more of the fimbrial souches dans l'intestin grele des adhesins K88 (F4) (2), K99 (F5) (3), porcelets experimentaux ne se revela 987P (F6) (4) and F41 (5) (see que moderee et elles n'afficherent reference 6 for a discussion of fimbrial aucune difference dans leur habilete nomenclature). Most strains also individuelle a y proliferer. Toutes les express type 1 (F1) fimbriae whose souches reagirent a l'epreuve indirecte role in the pathogenesis of ETEC d'immunofluorescence, avec les infections is controversial (7,8). antiserums contre les antigenes Several other adhesins have been CS1541 et 987P, lorsqu'on l'appliqua recently demonstrated in ETEC aux colibacilles de lintestin grele des strains from pigs (9,10,11,12) but their

Departement de Pathologie et Microbiologie, Faculte de Medecine veterinaire, Universite de Montreal, Case postale 5000, Saint-Hyacinthe (Quebec) J2S 7C6. This work was supported by grant MMV-85-1150 from the Conseil des Recherches et Services Agricoles du Quebec (CRSAQ). -Submitted May 10, 1988.

Can J Vet Res 1989; 53: 43-47

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1385A (08:K-:H9) are spontaneous four animals, strain 1592 was inocuacapsular variants of the latter strains lated into one piglet, and strain 88-628 produced in our laboratory as des- was inoculated into two animals. cribed previously (18). They had been When 4 to 6 h old, piglets were found to be similar to the parent inoculated intragastrically with about strains with respect to most of the 109 colony forming units (CFU). They characters examined, i.e. biotype, were kept in isolators at 30 to 350C, antibiotic resistance, colicin produc- and monitored for diarrhea. Piglets tion and resistance, and enterotoxin which did not exhibit diarrhea within production. However, strain 1541P 24 h postinoculation were fed Lifeexpresses the fimbrial antigen CS 1541 Guard (Norden Laboratories, Inc., in culture whereas the other 08 strains Lincoln, Nebraska) to avoid dehydrado not (17, unpublished data). Strain tion and hypoglycemia. When diar1592 (09:K103:NM:987P), isolated rheic, or 40 h postinoculation, piglets from a newborn piglet with diarrhea were euthanized and necropsied and provided by H.W. Moon immediately. Ileal segments 10 cm in (National Animal Disease Center, length were aseptically removed, Ames, Iowa), produces the fimbrial homogenized in an Omnimixer (Ivan adhesin 987P and the enterotoxins Sorval Inc., Norwalk, Connecticut), STa and STb. Strain 88-628 serially diluted tenfold, and plated (08:K+:NM), originally isolated at the onto tryptic soy agar (Difco LaboratoFaculty of Veterinary Medicine, ries, Detroit, Michigan) + 0.1% Saint-Hyacinthe, from the intestinal glucose (16). After overnight incubacontent of a piglet, is nonenterotoxi- tion at 370 C, the colonies were genic and does not express the fimbrial identified using the slide agglutination antigens K88, K99, F41 and 987P test (SAT) and the number of CFU of the infecting strain was determined as (unpublished data). described previously (16). Adjacent ENTEROCYTE ADHESION ASSAY intestinal segments were examined by Adhesion assays were carried out the IFAT in order to evaluate the with enterocytes and enterocyte brush association of the bacteria with the borders isolated from colostrum- intestinal mucosa and the expression deprived newborn piglets as pre- of the fimbrial antigens CS1541 and viously described (15). Enterocytes 987P (16). Briefly, frozen intestinal were suspended in Krebs-Henseleit sections were incubated with antibuffer (0.12 M NaCl, 0.014 M KCI, 08:KX105, 987P, or CS1541 sera 0.025 M NaHCO3, 0.001 M KH2PO4) raised in rabbits as previously des(pH 7.4) with 1% (w/v) D-mannose cribed (16,17) and then with and used on the day of preparation. fluorescein-labeled goat antirabbit Bacteria were grown on blood agar immunoglobulin G (Bio/Can Scienovernight at 370 C, harvested, and tific Inc., Mississauga, Ontario). The suspended in Krebs-Henseleit buffer. association of the bacteria with the Equal volumes of enterocytes and intestinal mucosa was estimated by bacterial suspensions were mixed and using an association index of 0 to 5 as incubated at room temperature with previously described (16). Data were MATERIALS AND METHODS shaking for 1 h. After two washes, the analyzed with the Student's t and chicell suspensions were examined by square tests. BACTERIAL STRAINS phase-contrast microscopy at a magEnterotoxigenic Escherichia coli nification of 500 X. The number of strains 1541, 1385, 1541P, 1385A and bacteria adhering to 20 enterocytes or RESULTS 1592 and non-ETEC strain 88-628 brush borders was counted for each were used. Strains 1541 and 1385 suspension. Each strain was tested None of the 08 strains adhered to (08: KX 105: H9) were originally iso- against enterocytes from ten animals enterocytes isolated from newborn lated at the Faculty of Veterinary of ten unrelated litters. piglets (less than 1 bacterial cell per Medicine, Saint-Hyacinthe from the enterocyte) whereas strain 1592 intestines of newborn piglets with EXPERIMENTAL INFECTIONS adhered strongly (more than 15 A total of 19 hysterotomy-derived, bacterial cells per enterocyte) (Fig. 1). diarrhea (16). These strains have been extensively characterized and found to colostrum-deprived newborn piglets The 08 strains caused acute watery be identical with respect to all of the from two unrelated litters were used as diarrhea and severe dehydration in 9 characters examined (Broes et al, described previously (16). Each of the of the 16 inoculated piglets (Table I). unpublished data). Strains 1541 P and four 08 strains was inoculated into All of the piglets inoculated with strain role in colonization has not been established. In addition to fimbrial adhesins, certain porcine ETEC strains produce heat-stable (type A) capsular antigens which enhance their ability to colonize the small intestine and increase their virulence (4,13,14). On the other hand, certain capsular antigens reduce the ability of the organisms to adhere to isolated porcine enterocytes (14,15). We previously reported that ETEC strains of the "nonclassical" serogroup 08:KX105 cause acute watery diarrhea in experimentally infected, colostrum-deprived newborn piglets (16). These strains were K88-, K99-, F41- and 987P-negative in culture but they reacted with an anti-987P serum when examined by the indirect fluorescent antibody test (IFAT) in the intestines of infected piglets (16, unpublished data). However, we subsequently demonstrated by colony hybridization that these strains did not possess the genes coding for the K88, K99 and 987P structural subunits (Broes et al, unpublished data). Recently, we found that an acapsular variant of one of these strains expressed a new fimbrial antigen (CS 1541) which demonstrated serological cross-reactions with 987P (17). The objectives of the present study were to investigate the requirement for fimbrial antigen CS 1541 and capsular antigen KX105 in the adhesion to intestinal epithelial cells, colonization of the small intestinal mucosa, and production of diarrhea in hysterotomy-derived colostrumdeprived piglets by ETEC 08:KX105 strains.

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DISCUSSION

Fig. 1. Interaction of strains 1592 (09:K103:NM:987P) and 1541P (08:K-:CS1541:H9) with enterocytes isolated from the small intestine of a newborn, colostrum-deprived piglet. Strain 1592 strongly adheres (A) whereas strain 1541P does not (B). (Magnification X20,000).

1385 developed diarrhea whereas only 2/ 4, 2/4 and I/4 of the piglets infected with strains 1385A, 1541 and 1541P respectively became diarrheic. Diarrhea usually occurred within 16 h postinoculation although one piglet inoculated with strain 1385 developed diarrhea only at 36 h postinoculation. The other piglets remained healthy until they were euthanized at 40 h postinoculation. There was no significant difference between the encapsulated and noncapsulated 08 strains in their ability to cause diarrhea (p > 0.1). In addition, no difference in the degree of intestinal colonization was noted between the 08 strains (p > 0.1) nor between diarrheic and nondiarrheic piglets (p > 0.3). On the other hand, the control piglet inoculated with strain 1592 developed severe diarrhea within 18 h postinoculation and had a bacterial count of 6.0 x 109, whereas the two control piglets inoculated with non-ETEC strain 88-628 remained healthy until at 40 h postinoculation and had ileal bacterial counts < 106 (Table I). On examination of IFAT stained ileal sections from piglets infected with the 08 strains, bacteria were observed in significant numbers (association index > 1) only in the piglets with bacterial counts > 108 CFU/ 10 cm of ileum. Bacteria were closely associated with the intestinal villi or randomly distributed in the intestinal lumen (Fig. 2). In contrast, on ileal sections of the piglet inoculated with strain 1592, continuous layers of bacteria

several cells thick covered the intestinal villi (Fig. 3). Bacteria reacting with the CS1541 and 987P antisera in the IFAT were observed in the intestines of all piglets infected with the 08 strains (Fig. 2). On examination with the SAT and the IFAT, freshly isolated bacteria from the piglets infected with strain 1541P were positive with the CS1541 antiserum but negative with the 987P antiserum. In contrast, bacteria isolated from the piglets infected with strains 1541, 1385, or 1385A were negative with both antisera. Strains 1385A and 1541 P remained noncapsulated on reisolation from piglets. Strain 1592 reacted with the CS1541 and 987P antisera in the intestines of the infected piglet (Fig. 3) and bacteria isolated from this piglet reacted with both antisera in the SAT but with only the 987P antiserum in the IFAT.

The 08 strains caused diarrhea in a variable proportion of piglets challenged. Such variability in the development of diarrhea was surprising with regard to strains 1541 and 1385, as these strains, as other 08:KX105 strains examined, had caused diarrhea within 18 to 24 h in all previously inoculated piglets (30/30 inoculated piglets) (16). Variability in response to inoculation with ETEC strains is a common feature of strains expressing the K88 antigen (2,19; personal observations). The resistance of certain piglets may be explained by the absence of intestinal colonization due to a genetic lack of intestinal receptors for the K88 adhesin in these animals (19). However, such piglets are fully susceptible to ileal colonization and diarrhea caused by ETEC strains expressing the K99 or 987P adhesins (13,20). In contrast, in the present study, all of the 08 strains colonized the intestines of both diarrheic and nondiarrheic piglets to the same degree. Moreover, the intensity of colonization resembled that observed in a previous study with other pathogenic 08:KX 105 strains and was inferior to that demonstrated by classical ETEC strains (13,16,20). Moon et al (20) observed that ETEC strains which lacked the antigens K88, K99, 987P, and F41 (4P-) but produced either LT or STb enterotoxin moderately colonized the ileum but failed to cause diarrhea in orally infected newborn piglets. However, the piglets were euthanized at 18 h postinoculation, possibly before they

TABLE I. Response of Hysterotomy-derived, Colostrum-deprived, Newborn Piglets to Inoculation with Enterotoxigenic Escherichia coli

Infecting Strain

Ileal Colonization in Piglets Without With Diarrhea Diarrhea NAc 1.4 x 108 ± 0.7b 5.0 x 108 ± 4.2 3.5 x 108 ± 1.8 4.5 x 108 ± 3.5 4.0 x 108 ± 5.6 1.9 x 108 + 1.5 2.0 x 108 NA 6.0x 109 -.

---A=

Fig. 2. Sections of the ileum in a piglet experimentally infected with enterotoxigenic E. coli strain 1541P (08:K-:CS1541) stained by IFAT with CS1541 (A) or 987P (B) antiserum: few fluorescing coccobacilli are closely associated with the villi. (Magnification X12,500).

of the intestinal mucosa has been demonstrated for strains expressing the fimbrial adhesins K88, K99, 987P and F41 (2,5,13,24,26). However, intestinal colonization is a complex process of which adherence to enterocyte brush borders represents only one aspect. Certain capsular antigens reduce the ability of ETEC strains to adhere to isolated enterocytes but nevertheless enhance their ability to colonize the intestines (13,14,15,24). We observed that strain 1541P had strong tendency to autoagglutinate and using the "salting out" test (27), we demonstrated that CS 1541 antigen conferred high surface hydrophobicity to the bacterial cells (unpublished data). Thus, it is possible that this antigen contributes to the colonizing ability of 08:KX105 strains by stabilizing microcolonies associated with the intestinal mucosa, as has been proposed for certain capsular antigens (15). Although the 08 strains in the present study did not have the gene coding for the 987P structural subunit and thus did not express the 987P fimbrial antigen, they were found to react with the 987P antiserum when they were examined in the intestines of infected piglets, confirming previous observations with other 08:KX105 strains (16). In addition, the 987Ppositive strain 1592 reacted with the CS1541 antiserum in the intestines. We have shown that the 987P and CS 1541 antigens demonstrate antigenic cross-reactions which are more

were able to develop diarrhea. In the The role of the CS 1541 fimbrial case of moderately colonizing ETEC antigen in colonization is not clear. All strains, such as the 4P- strains of the 08 strains reacted with CS 1541 examined by Moon et al (20) and the antiserum in the intestines of infected 08 strains in the present study, it is piglets although only strain 1541 P possible that the development of produced this antigen in culture. Thus, diarrhea depends on the production of strains 1541, 1385 and 1385A probahigh levels of enterotoxin(s) by the bly produce the CS 1541 antigen in the strains in the intestines or on the intestines but rapidly shift to a susceptibility of the piglets to the CS 1541-negative phase after being action of the enterotoxin(s). This cultured in vitro. Such a phenomenon, hypothesis may be supported by the commonly observed with the 987P variability observed between different antigen (19,24,25), may reflect the ETEC strains in the production of importance of the CS 1541 antigen in enterotoxin(s) in vitro (21) as well as the piglet intestines. On the other between different ETEC strains and hand, strain 1541 P was heavily animals in the gut loop test for fimbriated in vitro but did not adhere detection of enterotoxigenic strains to isolated enterocytes. A high correlation between adherence to (16,22; personal observations). Several studies have emphasized the isolated enterocytes and colonization role of certain capsular antigens in the ability of porcine and bovine K99- or 987P-positive ETEC to colonize the intestine and cause diarrhea (4,13,14,23), although conflicting results have been observed depending on the acapsular variants examined (4,14). Furthermore, capsular antigen appeared to play an important role in the intestinal colonization and production of diarrhea in piglets due to a calf ETEC K99-negative strain (13). In contrast, the results of the present study indicate that the capsular antigen KX105 does not play an essential role in the colonization of the pig intestine by the 08:KX105 ETEC strains. Thus, these strains probably produce another factor enabling them Fig. 3. Sections of the ileum in a piglet experimentally infected with enterotoxigenic E. coli strain to colonize the small intestine of 1592 (09:K103:987P) stained by IFAT with 987P (A) or CS1541 (B) antiserum: numerous fluorescing coccobacilli are attached to the villi. (Magnification X12,500). newborn piglets.

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evident in the SAT, immunoelectronmicroscopy and immunoblotting (17). The present results indicate that the cross-reactions observed in the IFAT are more easily demonstrated in the pig intestine than in culture. ACKNOWLEDGMENTS We thank Bernadette Foiry, Clarisse Desautels, Richard Bourassa, Yvon Couture and Gilles Fecteau for their technical assistance. We are grateful to Wendy M. Johnson (Laboratory Centre for Disease Control, Ottawa, Canada) for LT testing, Ida and Frits Orskov (International Escherichia and Klebsiella Center, Copenhagen, Denmark) for serotyping, and Harley W. Moon (National Animal Disease Center, Ames, Iowa, U.S.A.) for providing strain 1592. We are greatly indebted to Jacques Mainil (Faculty of Veterinary Medicine, University of Liege, Brussels, Belgium) for his assistance in the genetic characterization of the strains.

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