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 BRIEF COMMUNICATION
Frequency of virulence factors in Escherichia coli
isolated from pigs with postweaning diarrhea and edema disease
in North Carolina
Karen W. Post DVM,MS; Brad T. Bosworth, DVM, PhD; Jenny L.
Knoth, BS
Post KW, Bosworth BT, Knoth JL. Frequency of virulence factors
in Escherichia coli isolated from pigs with postweaning
diarrhea and edema disease in North Carolina. Swine Health
Prod. 2000;8(3):119-120. This article is also available
in PDF format (420k).
KWP, JLK: North Carolina Department of Agriculture and Consumer
Services, Rollins Animal Disease Diagnostic Laboratory, 2101 Blue
Ridge Road, Raleigh, North Carolina, 27607; email: karen.post@ncmail.net;
BTB: PIC, Inc.
Summary
Polymerase chain reaction for five fimbriae (K88, K99, 987P,
F41, F18), two heat-stable enterotoxins (STa, STb), one heat-labile
enterotoxin (LT), and Shiga-like toxin type II (SLT) was performed
on Escherichia coli isolates to determine their genotype
among isolates from weaned pigs with diarrhea and/or edema disease
in North Carolina. A total of 175 isolates were tested. All but
two isolates possessed genes for at least one of the enterotoxins
or fimbrial adhesins. Nearly all (94.86%) of the isolates that
carried enterotoxin genes also carried genes for one of the fimbrial
adhesins. The two predominant genotypes were K88 LT STb and F18
STa STb SLT, respectively.
Keywords: swine, Escherichia
coli, fimbrial adhesins, genotypes
Received: September 2, 1999
Accepted: February 1, 2000
Postweaning diarrhea
and edema disease due to enterotoxigenic (ETEC) and verocytotoxigenic
(VTEC) strains of Escherichia coli are important causes
of morbidity and mortality in weaned pigs. Pathogenesis of ETEC
is mediated by enterotoxins (heat-labile toxin [LT] and heat-stable
toxins [STa and STb]), which act locally in the gut, stimulating
hypersecretion of water and electrolytes by enterocytes. ETEC
manifests clinically as diarrhea and dehydration.1
Edema disease, on the other hand, is an enterotoxemia, in which
strains of E. coli elaborate an exotoxin (verocytotoxin
or Shiga-like toxin type II), which is absorbed from the gut and
produces systemic lesions associated with vascular injury.2
Nonpathogenic strains of E. coli are commonly found
in the intestinal tracts of animals. It is important to identify
the virulence factors in order to establish the etiology of diarrhea.
Serologic tests have traditionally been used to identify virulence
factors associated with specific clinical syndromes. The application
of molecular methods, mainly the polymerase chain reaction (PCR)
assay, has increased both the rapidity of characterization and
the amount of information that can be gleaned from an individual
isolate.
The objective of the present work was to determine the frequency
of occurrence of selected virulence factors in strains of E.
coli isolated from pigs with postweaning diarrhea and/or edema
disease in North Carolina.
Materials and methods
Escherichia coli strains
We analyzed Escherichia coli isolates that were recovered
from 175 swine clinical specimens, either from feces or ileum,
and submitted to the Rollins Animal Disease Diagnostic Laboratory
between March 1998 and March 1999. Each isolate came from an individual
animal 21-42 days of age with postweaning diarrhea and/or edema
disease.
Genotyping
Analysis of the isolates was performed by a recently developed
multiplex PCR assay which detects genes for the enterotoxins LT,
STa, STb, Shiga-like toxin type II and fimbrial adhesins K88 ac,ad
(F4), K99 (F5), F41, 987P (F6), and F18 ab,ac -- common virulence
factors associated with swine enteric disease.3 Amplified
products were electrophoresed in 2% agarose gel, stained with
ethidium bromide, and examined under ultraviolet illumination.
DNA fragment lengths were verified by a digested £-DNA standard
run simultaneously. Control DNA from reference strains was included
in each reaction.
Results
Genotypes are listed for all 175 isolates in Table
1. All but two isolates possessed genes for at least one of
the enterotoxins or fimbrial adhesins. Isolates that carried fimbrial
adhesin genes also carried a gene for at least one of the enterotoxins.
Nearly all (94.86%) of the isolates that carried enterotoxin
genes also carried genes for one of the fimbrial adhesins. The
two predominant genotypes observed were K88 LT STb (72 of 175)
and F18 STa STb SLT (56 of 175). In total, these comprised 73.14%
(128 of 175) of the isolates.
Discussion
The most prevalent fimbrial adhesin identified in this study
was F18. K88 has previously been considered to be the predominant
fimbrial adhesin associated with postweaning disease in the pig.1,4-6
More recent surveys, however, have increasingly incriminated
F18 fimbriae in postweaning disease.7-12 F18 fimbriae
cannot always be reliably identified in vitro;13 therefore,
prior estimates of prevalence that were based upon immunologic
methods may have been lower than those based upon genetic analysis.
These data indicate a positive relationship between fimbrial
adhesin and enterotoxin genes. K88 positive E. coli usually
produced both LT and STb. This is in contrast to a report by Wilson
and Francis where K88-positive E. coli produced LT more
frequently in animals of postweaning age than LT and STb concurrently,1
but is in agreement with a more recent report.12
Historically, in the United States, a majority of edema disease
isolates have been F18 SLT or F18 SLT STb (Bosworth, unpublished
data, 1997). In our survey, F18 positive fimbriae were most often
found in association with SLT STa STb. The increase in this specific
genetic combination could be due to clonal expansion.
Nine isolates were negative for fimbrial genes. These could
be strains that either have lost the capability to produce fimbriae
due to loss of a plasmid during culture,13 or may suggest
a novel fimbrial type not currently characterized. Another reason
could be minor (even silent) changes in sequence, which cause
primers to fail to bind.
Results of this survey demonstrate the high frequency of strains
containing F18 fimbriae in North Carolina swine. These strains
typically are resistant in vitro to most of the currently approved
swine antimicrobials (Post, unpublished data, 1998). Since there
is no currently available commercial vaccine that contains this
fimbrial type, most control methods have involved dietary management,
sanitation, and the oral use of live, nontoxigenic strains to
impart localized immunity in at-risk animals.14 Nontoxigenic
strains, which may be naturally harbored in the intestines of
some pigs, have been theorized to be natural immunogens that may
protect against disease in herds.15
Implications
- There is a higher frequency of F18 strains than K88 strains
in North Carolina hogs during the postweaning period.
- The majority of these strains carry SLT STa STb and could
potentially cause both edema disease and diarrhea.
- The lack of commercially available vaccines make immunologic
prophlyaxis difficult.
- Additional research is needed to formulate adequate prevention
and control measures.
References--refereed
1. Wilson RA, and Francis DH. Fimbriae and enterotoxins associated
with Escherichia coli serogroups isolated from pigs with
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2. MacLeod DL, Gyles CL, Wilcock BP. Reproduction of edema
disease of swine with purified Shiga-like toxin-II variant. Vet
Pathol. 1991;28:66-73.
4. Nagy B, Casey TA, Moon HW. Phenotype and genotype of Escherichia
coli isolated from pigs with postweaning diarrhea in Hungary.
J Clin Microbiol. 1990;28:651-653.
5. Soderlind O, Thafvelin B, Mollby R. Virulence factors in
Escherichia coli strains isolated from Swedish piglets
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Molecular characterization of Escherichia coli strains
isolated from pigs with edema disease. J Clin Microbiol.
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12. Moon HW, Hoffman LJ, Cornick NA, Booher SL, Bosworth BT.
Prevalences of some virulence genes among Escherichia coli
isolates from swine presented to a diagnostic laboratory in Iowa.
J Vet Diagn Invest.1999;11:557-560.
13. Wittig W, Prager R, Stamm M, Streckel W, Tschape H. Expression
and plasmid transfer of genes coding for the fimbrial antigen
in porcine Escherichia coli strains. Zbl Bakt. 1994;281:130-139.
15. Sarrazin E, Bertschinger HU. Role of fimbriae F18 for actively
acquired immunity against porcine enterotoxigenic E. coli.
Vet Microbiol. 54:133-144.
References--nonrefereed
3. Bosworth BT, Casey TA. Identification of toxin and pilus
genes in porcine Escherichia coli using polymerase chain
reaction (PCR) with multiple primer pairs. Gen Meet Am Soc
Microbiol. 1997:B509.[Abstr.]
14. Gyles CG. Gut edema and the F18 pilus:pathogenesis and
laboratory findings. Proc AASP Ann Meet. 1999;457463.
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