The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain

Cornick, Nancy

Dean-Nystrom, Evelyn A.

Jordan, Dianna M.

Kaper, James B.

Moon, Harley W.

Torres, Alfredo G.

English

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 
10-2004 
Long Polar Fimbriae Contribute to Colonization by Escherichia coli 
O157:H7 In Vivo 
Dianna M. Jordan 
Iowa State University 
Nancy Cornick 
Iowa State University 
Alfredo G. Torres 
University of Maryland School of Medicine 
Evelyn A. Dean-Nystrom 
Pre-Harvest Food Safety and Enteric Diseases Research Unit, evelyn.nystrom@ars.usda.gov 
James B. Kaper 
University of Maryland School of Medicine 
See next page for additional authors 
Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub 
 Part of the Agricultural Science Commons 
Jordan, Dianna M.; Cornick, Nancy; Torres, Alfredo G.; Dean-Nystrom, Evelyn A.; Kaper, James B.; and 
Moon, Harley W., "Long Polar Fimbriae Contribute to Colonization by Escherichia coli O157:H7 In Vivo" 
(2004). Publications from USDA-ARS / UNL Faculty. 933. 
https://digitalcommons.unl.edu/usdaarsfacpub/933 
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research 
Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in 
Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of 
Nebraska - Lincoln. 
Authors 
Dianna M. Jordan, Nancy Cornick, Alfredo G. Torres, Evelyn A. Dean-Nystrom, James B. Kaper, and Harley 
W. Moon 
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
usdaarsfacpub/933 
INFECTION AND IMMUNITY, Oct. 2004, p. 6168–6171 Vol. 72, No. 10
0019-9567/04/$08.000 DOI: 10.1128/IAI.72.10.6168–6171.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Long Polar Fimbriae Contribute to Colonization by Escherichia coli
O157:H7 In Vivo
Dianna M. Jordan,1* Nancy Cornick,2 Alfredo G. Torres,3† Evelyn A. Dean-Nystrom,4
James B. Kaper,3 and Harley W. Moon1
Department of Veterinary Pathology1 and Department of Veterinary Microbiology and Preventive Medicine,2 Iowa State University,
and Preharvest Food Safety and Enteric Disease and Food Safety Research Unit, National Animal Disease Center, Agricultural
Research Service, U.S. Department of Agriculture,4 Ames, Iowa, and Center for Vaccine Development and Department of
Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland3
Received 20 April 2004/Returned for modification 1 June 2004/Accepted 2 July 2004
The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated
in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were
recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent
strain.
Escherichia coli O157:H7 is a cause of food-borne illness,
mostly due to consumption of undercooked contaminated
meat or contaminated vegetables (3, 14, 15, 19; J. Besser-Wiek,
D. Boxrud, J. Bender, M. Sullivan, L. Carroll, and F. Leano,
Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. C-364,
1996). E. coli O157:H7 can persist in experimentally infected
sheep and pigs for two months and in cattle for longer (2, 4, 6).
Intimin is imperative for the development of attachment and
effacement (A/E) lesions (9, 11, 12, 20) and important for the
colonization of cattle and sheep by E. coli O157:H7 (3, 5, 8).
While the initial bacterial interaction with enterocytes is poorly
understood, fimbriae may contribute early in the colonization
process.
Long polar (LP) fimbriae are important for the pathogenesis
and virulence of Salmonella enterica serovar Typhimurium
causing murine typhoid (1, 21). E. coli O157:H7 contains two
lpf operons homologous to lpf of serovar Typhimurium (16).
The sequences of E. coli O157:H7 lpfABCCDE operon 1 are
comparable and produce proteins with functions similar to
those of their serovar Typhimurium counterparts (19). LP fim-
briae associated with operon 1 are suggested to enhance at-
tachment to HeLa cells and formation of microcolonies by
transformed E. coli K-12 (19). LP fimbrial operon 2 in E. coli
O157:H7 has been recently characterized (10, 16; A. G. Torres,
K. J. Kanack, C. B. Tutt, V. Popov, and J. B. Kaper, submitted
for publication).
We hypothesized that LP fimbriae contribute to the coloni-
zation of sheep and pigs by E. coli O157:H7. Our approach
compared the ability to colonize of two lpf mutant strains to
that of the parent strain, E. coli O157:H7 86-24. Sheep were
used to evaluate the ability of the lpfA1 and lpfA1 lpfA2 mu-
tants to persist in a ruminant. Conventional pigs were dually
infected to determine if the lpfA1 lpfA2 mutant can be main-
tained as long as the parent strain. Neonatal gnotobiotic pigs
were used to evaluate the early phase of infection with all three
strains.
The inoculum strains were derivatives of E. coli O157:H7
strain 86-24 (18). The parent strain was a streptomycin- and
nalidixic acid-resistant derivative. A chloramphenicol resis-
tance cassette was inserted into lpfA1 of the parent strain to
create CVD468 (19). AGT210, the double mutant strain, was
derived from CVD468 by insertion of a tetracycline cassette
into lpfA2 (Torres et al., submitted). Nonpathogenic E. coli
strain 123 was used as a negative control (13). All bacteria were
grown as overnight cultures in tryptic soy broth with appropri-
ate antibiotics used at the following concentrations: 50 g of
streptomycin/ml, 10 g of nalidixic acid/ml, 30 g of chloram-
phenicol/ml, and 20 g of tetracycline/ml. The inocula were
prepared as previously described (2, 4, 17).
Prolonged colonization. Twenty-two 4- to 12-month-old
sheep and eight 10-week-old conventional pigs were obtained,
acclimated for 2 weeks, and screened to verify the lack of
sorbitol-negative, O157-positive E. coli and to characterize the
background intestinal flora. The sheep were divided into three
groups and inoculated with 1010 CFU of either the parent
strain (8 animals), CVD468 (6 animals), or AGT210 (8 ani-
mals). The pigs were dually inoculated with the parent and
AGT210 strains at a dosage of 1010 CFU per strain per pig.
The inoculum was delivered with feed; the animals were
housed in pairs. Fecal samples were collected on three consec-
utive days from each animal at designated time points postin-
oculation (PI). There were an initial collection and collections
at 2, 4, 6, and 8 weeks PI. The animals were necropsied on day
60 PI. The samples collected from the sheep were from the
rumen, ileum, Peyer’s patches, spiral colon, and distal colon.
The samples collected from the pigs included tonsil, cecum,
spiral colon, distal colon, and rectal content. Fecal and tissue
samples collected from the animals were processed for bacte-
riologic evaluation as previously described (2, 4–6; Besser-
Wiek et al., Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996).
A replica plating technique on additional selective media was
* Corresponding author. Present address: Veterinary Diagnostic
and Production Animal Medicine, Iowa State University, Ames, IA
50011. Phone: (515) 294-7792. Fax: (515) 294-3564. E-mail: dmjordan
@iastate.edu.
† Present address: Department of Microbiology and Immunology,
University of Texas Medical Branch, Galveston, TX 77555.
6168
used to separate the strains from pigs that were administered
the dual inoculum for quantification. Wilcoxon/Kruskal-Wallis
nonparameteric tests were done for the bacterial counts. Lo-
gistic regression was done to evaluate the decline in shedding.
JMP software (version 5.0.1a; SAS Institute, Inc.) was utilized
for analysis with P values of 0.05.
All three inoculum strains were recovered from the feces of
sheep throughout the study (Fig. 1). At 2 weeks PI, the parent
strain and CVD468 were recovered from the feces at signifi-
cantly (P  0.01) higher levels than AGT210. At necropsy, the
inoculum strains were recovered primarily from the lower gas-
trointestinal tract samples and only at enrichment levels (50
CFU/cm2). The proportion of sheep shedding the inoculum
strains was not different throughout the study.
Both inoculum strains were recovered from the feces of all
eight conventional pigs during the initial period (Fig. 2). Sig-
nificantly more (P  0.007) parent-strain than AGT210 bacte-
ria were recovered during the initial and 2-week periods. The
proportion of pigs shedding each strain declined significantly
(P  0.004) over the course of the study; however, both strains
were recovered intermittently from some pigs throughout the
study. The parent strain was recovered at necropsy from the
cecum, spiral colon, and distal colon of three pigs at levels of
100 CFU/g. One tonsil sample contained 7 104 CFU of the
parent strain/g. In contrast, even though two pigs were shed-
ding AGT210 in their feces at termination, AGT210 was not
recovered from the tissues of any pigs at necropsy and was
eliminated from significantly (P  0.003) more pigs than the
parent strain.
This study confirmed that E. coli O157:H7 can colonize
sheep and pigs and further indicated that long-term coloniza-
tion was facilitated by LP fimbriae. The effect of the double
mutation of the LP fimbrial genes was more pronounced in
pigs than in sheep. Significantly (P  0.03) more sheep than
pigs remained colonized by AGT210 during the study. In both
species, AGT210 tended to be recovered at lower levels than
the parent strain; this was demonstrated best at the early time
points (2 weeks PI). This has also been demonstrated in
rabbits infected with a rabbit enteropathogenic E. coli
(REPEC) strain with a mutation in lpf (15). Furthermore,
recovery of AGT210 from the pigs was intermittent and re-
duced compared to recovery of the parent strain, indicating
that LP fimbriae were facilitating its colonization. Intimin-
mediated adhesion is one possible mechanism contributing to
colonization by AGT210.
Early colonization. Germfree pigs were derived from five
litters and orally inoculated at 24 h after birth with 105 CFU of
bacteria. In aggregate, eleven pigs were inoculated with the
parent strain, eight were inoculated with strain CVD468, thir-
teen were inoculated with strain AGT210, and nine were in-
oculated with strain 123. Based on a pilot study of 7 days’
duration which indicated a possible effect of LP fimbriae early
in the period of colonization (24 h), the pigs were necropsied
at 24 h PI. The samples collected were from the ileum, cecum,
and spiral colon.
Colonization was defined by the presence of A/E lesions in
formalin-fixed tissues collected at necropsy. Tissues were sec-
tioned and stained with hematoxylin and eosin (H&E) and an
O157-specific horseradish peroxidase immunohistochemical
stain (7). Cross-sections of intestinal segments were evaluated
and scored using the criteria in Table 1. Contingency analysis
and pair-wise testing of the scored data were done.
The parent and mutant strains formed A/E lesions in cecum
and spiral colon. Pigs infected with the parent strain (median
lesion score  1; range, 0 to 2) or CVD468 (median lesion
score  1; range, 0 to 1) had significantly (P  0.02) higher
lesion scores for the spiral colon than those infected with
AGT210 (median  0.5; range, 0 to 2). There were no statis-
tical differences in the lesion scores for the cecum (median 
1) or the ileum (median  0) for any of the pathogenic strains.
FIG. 1. Recovery of E. coli O157:H7 from the feces of sheep inoculated with three isogenic strains. Each symbol represents the geometric mean
for one animal over each sampling period. The symbols below the dotted line represent samples that were positive only by enrichment culture.
Samples in which no growth was detected by direct plating or enrichment culture are plotted at zero.
VOL. 72, 2004 NOTES 6169
There were no lesions in pigs inoculated with E. coli strain 123.
The results were limited by the potential sampling error of
histopathology in that only a small portion of intestine can be
examined microscopically. Therefore, it is conceivable, but
seemingly unlikely, that lesions other than those examined
were dependent on lpf.
Sections of spiral colon from each group of inoculated pigs
were prepared and evaluated by transmission electron micros-
copy. Upon evaluation, classical A/E lesions (14) were dem-
onstrated in multiple sections of spiral colon from pigs infected
with the parent strain, CVD468, and AGT210 (in three of
three, one of one, and three of six pigs examined, respectively).
These findings support the hypothesis that LP fimbriae con-
tribute to colonization by E. coli O157:H7. Mutations in both
LP fimbrial operons diminished the ability of E. coli O157:H7
to establish and persist in sheep and pigs for 2 months and
reduced, but did not eliminate, A/E lesions. As established for
many bacterial species, redundant mechanisms enable bacteria
to thrive and survive within many different environments;
therefore, mutations in one system, i.e., the lpf operon, may not
reduce the survival of the bacterial population.
This work was supported in part by NIH grant 1R01DK58957-01 (to
J.B.K.) and a grant from the National Alliance for Food Safety through
the USDA (to N.C.).
We thank Lawayne Nusz and the NADC and ISU animal caretakers
for their assistance, ISU Histopathology Laboratory for slide prepara-
tions, Judy Stasko for transmission electron microscopy, Richard
Evans for statistical consultation, and especially Sheridan Booher and
Amy Helgerson for technical assistance in bacteriology and animal
handling.
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Description of lesion severity Lesionscore
No enterocytes had A/E lesions ...................................................... 0
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Editor: J. B. Bliska
VOL. 72, 2004 NOTES 6171


Long Polar Fimbriae Contribute to Colonization by \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 In Vivo

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 10-2004 Long Polar Fimbriae Contribute to Colonization by Escherichia coli O157:H7 In Vivo Dianna M. Jordan Iowa State University Nancy Cornick Iowa State University Alfredo G. Torres University of Maryland School of Medicine Evelyn A. Dean-Nystrom Pre-Harvest Food Safety and Enteric Diseases Research Unit, evelyn.nystrom@ars.usda.gov James B. Kaper University of Maryland School of Medicine See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub  Part of the Agricultural Science Commons Jordan, Dianna M.; Cornick, Nancy; Torres, Alfredo G.; Dean-Nystrom, Evelyn A.; Kaper, James B.; and Moon, Harley W., "Long Polar Fimbriae Contribute to Colonization by Escherichia coli O157:H7 In Vivo" (2004). Publications from USDA-ARS / UNL Faculty. 933. https://digitalcommons.unl.edu/usdaarsfacpub/933 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Dianna M. Jordan, Nancy Cornick, Alfredo G. Torres, Evelyn A. Dean-Nystrom, James B. Kaper, and Harley W. Moon This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/usdaarsfacpub/933 INFECTION AND IMMUNITY, Oct. 2004, p. 6168–6171 Vol. 72, No. 100019-9567/04/$08.000 DOI: 10.1128/IAI.72.10.6168–6171.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.Long Polar Fimbriae Contribute to Colonization by Escherichia coliO157:H7 In VivoDianna M. Jordan,1* Nancy Cornick,2 Alfredo G. Torres,3† Evelyn A. Dean-Nystrom,4James B. Kaper,3 and Harley W. Moon1Department of Veterinary Pathology1 and Department of Veterinary Microbiology and Preventive Medicine,2 Iowa State University,and Preharvest Food Safety and Enteric Disease and Food Safety Research Unit, National Animal Disease Center, AgriculturalResearch Service, U.S. Department of Agriculture,4 Ames, Iowa, and Center for Vaccine Development and Department ofMicrobiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland3Received 20 April 2004/Returned for modification 1 June 2004/Accepted 2 July 2004The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluatedin sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated wererecovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parentstrain.Escherichia coli O157:H7 is a cause of food-borne illness,mostly due to consumption of undercooked contaminatedmeat or contaminated vegetables (3, 14, 15, 19; J. Besser-Wiek,D. Boxrud, J. Bender, M. Sullivan, L. Carroll, and F. Leano,Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. C-364,1996). E. coli O157:H7 can persist in experimentally infectedsheep and pigs for two months and in cattle for longer (2, 4, 6).Intimin is imperative for the development of attachment andeffacement (A/E) lesions (9, 11, 12, 20) and important for thecolonization of cattle and sheep by E. coli O157:H7 (3, 5, 8).While the initial bacterial interaction with enterocytes is poorlyunderstood, fimbriae may contribute early in the colonizationprocess.Long polar (LP) fimbriae are important for the pathogenesisand virulence of Salmonella enterica serovar Typhimuriumcausing murine typhoid (1, 21). E. coli O157:H7 contains twolpf operons homologous to lpf of serovar Typhimurium (16).The sequences of E. coli O157:H7 lpfABCCDE operon 1 arecomparable and produce proteins with functions similar tothose of their serovar Typhimurium counterparts (19). LP fim-briae associated with operon 1 are suggested to enhance at-tachment to HeLa cells and formation of microcolonies bytransformed E. coli K-12 (19). LP fimbrial operon 2 in E. coliO157:H7 has been recently characterized (10, 16; A. G. Torres,K. J. Kanack, C. B. Tutt, V. Popov, and J. B. Kaper, submittedfor publication).We hypothesized that LP fimbriae contribute to the coloni-zation of sheep and pigs by E. coli O157:H7. Our approachcompared the ability to colonize of two lpf mutant strains tothat of the parent strain, E. coli O157:H7 86-24. Sheep wereused to evaluate the ability of the lpfA1 and lpfA1 lpfA2 mu-tants to persist in a ruminant. Conventional pigs were duallyinfected to determine if the lpfA1 lpfA2 mutant can be main-tained as long as the parent strain. Neonatal gnotobiotic pigswere used to evaluate the early phase of infection with all threestrains.The inoculum strains were derivatives of E. coli O157:H7strain 86-24 (18). The parent strain was a streptomycin- andnalidixic acid-resistant derivative. A chloramphenicol resis-tance cassette was inserted into lpfA1 of the parent strain tocreate CVD468 (19). AGT210, the double mutant strain, wasderived from CVD468 by insertion of a tetracycline cassetteinto lpfA2 (Torres et al., submitted). Nonpathogenic E. colistrain 123 was used as a negative control (13). All bacteria weregrown as overnight cultures in tryptic soy broth with appropri-ate antibiotics used at the following concentrations: 50 g ofstreptomycin/ml, 10 g of nalidixic acid/ml, 30 g of chloram-phenicol/ml, and 20 g of tetracycline/ml. The inocula wereprepared as previously described (2, 4, 17).Prolonged colonization. Twenty-two 4- to 12-month-oldsheep and eight 10-week-old conventional pigs were obtained,acclimated for 2 weeks, and screened to verify the lack ofsorbitol-negative, O157-positive E. coli and to characterize thebackground intestinal flora. The sheep were divided into threegroups and inoculated with 1010 CFU of either the parentstrain (8 animals), CVD468 (6 animals), or AGT210 (8 ani-mals). The pigs were dually inoculated with the parent andAGT210 strains at a dosage of 1010 CFU per strain per pig.The inoculum was delivered with feed; the animals werehoused in pairs. Fecal samples were collected on three consec-utive days from each animal at designated time points postin-oculation (PI). There were an initial collection and collectionsat 2, 4, 6, and 8 weeks PI. The animals were necropsied on day60 PI. The samples collected from the sheep were from therumen, ileum, Peyer’s patches, spiral colon, and distal colon.The samples collected from the pigs included tonsil, cecum,spiral colon, distal colon, and rectal content. Fecal and tissuesamples collected from the animals were processed for bacte-riologic evaluation as previously described (2, 4–6; Besser-Wiek et al., Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996).A replica plating technique on additional selective media was* Corresponding author. Present address: Veterinary Diagnosticand Production Animal Medicine, Iowa State University, Ames, IA50011. Phone: (515) 294-7792. Fax: (515) 294-3564. E-mail: dmjordan@iastate.edu.† Present address: Department of Microbiology and Immunology,University of Texas Medical Branch, Galveston, TX 77555.6168used to separate the strains from pigs that were administeredthe dual inoculum for quantification. Wilcoxon/Kruskal-Wallisnonparameteric tests were done for the bacterial counts. Lo-gistic regression was done to evaluate the decline in shedding.JMP software (version 5.0.1a; SAS Institute, Inc.) was utilizedfor analysis with P values of 0.05.All three inoculum strains were recovered from the feces ofsheep throughout the study (Fig. 1). At 2 weeks PI, the parentstrain and CVD468 were recovered from the feces at signifi-cantly (P  0.01) higher levels than AGT210. At necropsy, theinoculum strains were recovered primarily from the lower gas-trointestinal tract samples and only at enrichment levels (50CFU/cm2). The proportion of sheep shedding the inoculumstrains was not different throughout the study.Both inoculum strains were recovered from the feces of alleight conventional pigs during the initial period (Fig. 2). Sig-nificantly more (P  0.007) parent-strain than AGT210 bacte-ria were recovered during the initial and 2-week periods. Theproportion of pigs shedding each strain declined significantly(P  0.004) over the course of the study; however, both strainswere recovered intermittently from some pigs throughout thestudy. The parent strain was recovered at necropsy from thececum, spiral colon, and distal colon of three pigs at levels of100 CFU/g. One tonsil sample contained 7 104 CFU of theparent strain/g. In contrast, even though two pigs were shed-ding AGT210 in their feces at termination, AGT210 was notrecovered from the tissues of any pigs at necropsy and waseliminated from significantly (P  0.003) more pigs than theparent strain.This study confirmed that E. coli O157:H7 can colonizesheep and pigs and further indicated that long-term coloniza-tion was facilitated by LP fimbriae. The effect of the doublemutation of the LP fimbrial genes was more pronounced inpigs than in sheep. Significantly (P  0.03) more sheep thanpigs remained colonized by AGT210 during the study. In bothspecies, AGT210 tended to be recovered at lower levels thanthe parent strain; this was demonstrated best at the early timepoints (2 weeks PI). This has also been demonstrated inrabbits infected with a rabbit enteropathogenic E. coli(REPEC) strain with a mutation in lpf (15). Furthermore,recovery of AGT210 from the pigs was intermittent and re-duced compared to recovery of the parent strain, indicatingthat LP fimbriae were facilitating its colonization. Intimin-mediated adhesion is one possible mechanism contributing tocolonization by AGT210.Early colonization. Germfree pigs were derived from fivelitters and orally inoculated at 24 h after birth with 105 CFU ofbacteria. In aggregate, eleven pigs were inoculated with theparent strain, eight were inoculated with strain CVD468, thir-teen were inoculated with strain AGT210, and nine were in-oculated with strain 123. Based on a pilot study of 7 days’duration which indicated a possible effect of LP fimbriae earlyin the period of colonization (24 h), the pigs were necropsiedat 24 h PI. The samples collected were from the ileum, cecum,and spiral colon.Colonization was defined by the presence of A/E lesions informalin-fixed tissues collected at necropsy. Tissues were sec-tioned and stained with hematoxylin and eosin (H&E) and anO157-specific horseradish peroxidase immunohistochemicalstain (7). Cross-sections of intestinal segments were evaluatedand scored using the criteria in Table 1. Contingency analysisand pair-wise testing of the scored data were done.The parent and mutant strains formed A/E lesions in cecumand spiral colon. Pigs infected with the parent strain (medianlesion score  1; range, 0 to 2) or CVD468 (median lesionscore  1; range, 0 to 1) had significantly (P  0.02) higherlesion scores for the spiral colon than those infected withAGT210 (median  0.5; range, 0 to 2). There were no statis-tical differences in the lesion scores for the cecum (median 1) or the ileum (median  0) for any of the pathogenic strains.FIG. 1. Recovery of E. coli O157:H7 from the feces of sheep inoculated with three isogenic strains. Each symbol represents the geometric meanfor one animal over each sampling period. The symbols below the dotted line represent samples that were positive only by enrichment culture.Samples in which no growth was detected by direct plating or enrichment culture are plotted at zero.VOL. 72, 2004 NOTES 6169There were no lesions in pigs inoculated with E. coli strain 123.The results were limited by the potential sampling error ofhistopathology in that only a small portion of intestine can beexamined microscopically. Therefore, it is conceivable, butseemingly unlikely, that lesions other than those examinedwere dependent on lpf.Sections of spiral colon from each group of inoculated pigswere prepared and evaluated by transmission electron micros-copy. Upon evaluation, classical A/E lesions (14) were dem-onstrated in multiple sections of spiral colon from pigs infectedwith the parent strain, CVD468, and AGT210 (in three ofthree, one of one, and three of six pigs examined, respectively).These findings support the hypothesis that LP fimbriae con-tribute to colonization by E. coli O157:H7. Mutations in bothLP fimbrial operons diminished the ability of E. coli O157:H7to establish and persist in sheep and pigs for 2 months andreduced, but did not eliminate, A/E lesions. As established formany bacterial species, redundant mechanisms enable bacteriato thrive and survive within many different environments;therefore, mutations in one system, i.e., the lpf operon, may notreduce the survival of the bacterial population.This work was supported in part by NIH grant 1R01DK58957-01 (toJ.B.K.) and a grant from the National Alliance for Food Safety throughthe USDA (to N.C.).We thank Lawayne Nusz and the NADC and ISU animal caretakersfor their assistance, ISU Histopathology Laboratory for slide prepara-tions, Judy Stasko for transmission electron microscopy, RichardEvans for statistical consultation, and especially Sheridan Booher andAmy Helgerson for technical assistance in bacteriology and animalhandling.REFERENCES1. Baumler, A. J., R. M. Tsolis, and F. Heffron. 1996. The lpf fimbrial operonmediates adhesion of Salmonella typhimurium to murine Peyer’s patches.Proc. Natl. Acad. Sci. USA 93:279–283.2. Booher, S. L., N. A. Cornick, and H. W. Moon. 2002. Persistence of Esche-richia coli O157:H7 in experimentally infected swine. Vet. Microbiol. 89:69–81.3. Cookson, A. L., and M. J. Woodward. 2003. The role of intimin in theadherence of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 toHEp-2 tissue culture cells and to bovine gut explant tissues. Int. J. Med.Microbiol. 292:547–553.4. Cornick, N. A., S. L. Booher, T. A. Casey, and H. W. Moon. 2000. Persistentcolonization of sheep by Escherichia coli O157:H7 and other E. coli patho-types. Appl. Environ. Microbiol. 66:4926–4934.5. Cornick, N. A., S. L. Booher, and H. W. Moon. 2002. Intimin facilitatescolonization by Escherichia coli O157:H7 in adult ruminants. Infect. Immun.70:2704–2707.6. Cray, W. C., Jr., and H. W. Moon. 1995. Experimental infection of calves andadult cattle with Escherichia coli O157:H7. Appl. Environ. Microbiol. 61:1586–1590.7. Dean-Nystrom, E. A., B. T. Bosworth, W. C. Cray, Jr., and H. W. Moon. 1997.Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatalcalves. Infect. Immun. 65:1842–1848.8. Dean-Nystrom, E. A., B. T. Bosworth, H. W. Moon, and A. D. O’Brien. 1998.Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves.Infect. Immun. 66:4560–4563.9. Donnenberg, M. S., S. Tzipori, M. L. McKee, A. D. O’Brien, J. Alroy, andJ. B. Kaper. 1993. The role of the eae gene of enterohemorrhagic Escherichiacoli in intimate attachment in vitro and in a porcine model. J. Clin. Investig.92:1418–1424.10. Doughty, S., J. Sloan, V. Bennett-Wood, M. Robertson, R. M. Robins-Browne, and E. L. Hartland. 2002. Identification of a novel fimbrial genecluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli. Infect. Immun. 70:6761–6769.11. Fitzhenry, R. J., D. J. Pickard, E. L. Hartland, S. Reece, G. Dougan, A. D.FIG. 2. Recovery of E. coli O157:H7 from the feces of conventional pigs dually inoculated with two isogenic strains. Each symbol representsthe geometric mean for one animal over each sampling period. The symbols below the dotted line represent samples that were positive only byenrichment culture. Samples in which no growth was detected by direct plating or enrichment culture are plotted at zero.TABLE 1. Criteria for A/E lesion scoring of formalin-fixedintestinal sectionsDescription of lesion severity LesionscoreNo enterocytes had A/E lesions ...................................................... 0A/E lesions were detected only with immunohistochemicalstaining............................................................................................ 0.510% of the enterocytes in the section had A/E lesionsdetected with H&E staining ........................................................ 110–50% of the enterocytes in the section had A/E lesionsdetected with H&E staining ........................................................ 250% of the enterocytes in the section had A/E lesionsdetected with H&E staining ........................................................ 36170 NOTES INFECT. IMMUN.Phillips, and G. Frankel. 2002. Intimin type influences the site of humanintestinal mucosal colonisation by enterohaemorrhagic Escherichia coliO157:H7. Gut 50:180–185.12. McKee, M. L., A. R. Melton-Celsa, R. A. Moxley, D. H. Francis, and A. D.O’Brien. 1995. Enterohemorrhagic Escherichia coli O157:H7 requires in-timin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells.Infect. Immun. 63:3739–3744.13. Moon, H. W., and S. C. Whipp. 1971. Systems for testing the enteropatho-genicity of Escherichia coli. Ann. N. Y. Acad. Sci. 176:197–211.14. Moon, H. W., S. C. Whipp, R. A. Argenzio, M. M. Levine, and R. A. Gian-nella. 1983. Attaching and effacing activities of rabbit and human entero-pathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 41:1340–1351.15. Newton, H. J., J. Sloan, V. Bennett-Wood, L. M. Adams, R. M. Robins-Browne, and E. L. Hartland. 2004. Contribution of long polar fimbriae to thevirulence of rabbit-specific enteropathogenic Escherichia coli. Infect. Immun.72:1230–1239.16. Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose,G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J.Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis,A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Ananthara-man, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001.Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature409:529–533.17. Sarmiento, J. I., T. A. Casey, and H. W. Moon. 1988. Postweaning diarrheain swine: experimental model of enterotoxigenic Escherichia coli infection.Am. J. Vet. Res. 49:1154–1159.18. Tarr, P. I., M. A. Neill, C. R. Clausen, J. W. Newland, R. J. Neill, and S. L.Moseley. 1989. Genotypic variation in pathogenic Escherichia coli O157:H7isolated from patients in Washington, 1984–1987. J. Infect. Dis. 159:344–347.19. Torres, A. G., J. A. Giron, N. T. Perna, V. Burland, F. R. Blattner, F.Avelino-Flores, and J. B. Kaper. 2002. Identification and characterization oflpfABCC’DE, a fimbrial operon of enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 70:5416–5427.20. Tzipori, S., F. Gunzer, M. S. Donnenberg, L. de Montigny, J. B. Kaper, andA. Donohue-Rolfe. 1995. The role of the eaeA gene in diarrhea and neuro-logical complications in a gnotobiotic piglet model of enterohemorrhagicEscherichia coli infection. Infect. Immun. 63:3621–3627.21. van der Velden, A. W., A. J. Baumler, R. M. Tsolis, and F. Heffron. 1998.Multiple fimbrial adhesins are required for full virulence of Salmonellatyphimurium in mice. Infect. Immun. 66:2803–2808.Editor: J. B. BliskaVOL. 72, 2004 NOTES 6171

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Long Polar Fimbriae Contribute to Colonization by \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 In Vivo

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