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Serotypes, virulence genes, and PFGE profiles of Escherichia coli isolated from pigs with postweaning diarrhoea in Slovakia

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Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods. These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected. Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.
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BMC Veterinary Research
Open Access
Research article
Serotypes, virulence genes, and PFGE profiles of Escherichia coli
isolated from pigs with postweaning diarrhoea in Slovakia
Hung Vu Khac
1,3
, Emil Holoda
1
, Emil Pilipcinec
1
, Miguel Blanco
2
,
Jesús E Blanco
2
, Azucena Mora
2
, Ghizlane Dahbi
2
, Cecilia López
2
,
Enrique A González and Jorge Blanco*
2
Address:
1
Institute of Microbiology and Immunology, Department of Food Hygiene and Technology, University of Veterinary Medicine,
Komenskeho 73, Slovakia,
2
Laboratorio de Referencia de E. coli, Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria,
Universidade de Santiago de Compostela, 27002 Lugo, Spain and
3
Department of Bacteriology, Central Vietnam Veterinary Institute, km4 Dong
De street, NhaTrang, Vietnam
Email: Hung Vu Khac - vukhac68@hotmail.com; Emil Holoda - holoda@uvm.sk; Emil Pilipcinec - pilipcin@uvm.sk;
Miguel Blanco - mba@lugo.usc.es; Jesús E Blanco - jeba@lugo.usc.es; Azucena Mora - amg@lugo.usc.es; Ghizlane Dahbi - gdahbi@lugo.usc.es;
Cecilia López - clopezca@lugo.usc.es; Enrique A González - ecoli@lugo.usc.es; Jorge Blanco* - jba@lugo.usc.es
* Corresponding author
Abstract
Background: Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of
large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly
by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of
101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and
genotypic methods.
Results: These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged
to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including
two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC
isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%,
46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for
F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them
were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins
were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of
genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36
distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed
together in the dendrogram, but high degree of polymorphism among certain serotypes was
detected.
Conclusion: Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly
detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five
isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have
emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic
variation mainly among ETEC of O149:H10 serotype.
Published: 20 March 2006
BMC Veterinary Research 2006, 2:10 doi:10.1186/1746-6148-2-10
Received: 06 December 2005
Accepted: 20 March 2006
This article is available from: http://www.biomedcentral.com/1746-6148/2/10
© 2006 Vu Khac et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Background
Postweaning diarrhoea (PWD) is usually the main infec-
tious problem of large-scale farms and is responsible for
significant losses worldwide [1,2]. The disease is caused
mainly by enterotoxigenic E. coli (ETEC), and Shiga-toxin
producing E. coli (STEC), also called verotoxin-producing
E. coli (VTEC) [2-7]. Porcine pathogenic E. coli involved in
PWD typically belong to serogroups O8, O138, O139,
O141, O147, O149 and O157, of which O149 seems to
be the predominant serogroup in most countries
[1,5,7,8]. ETEC can cause severe diarrhoea in newborn
and weaned piglets by the production of heat-labile enter-
otoxin (LT) and/or heat-stable enterotoxins (STa or STb).
These enterotoxins are extracellular proteins or peptides,
which are able to cause diarrhoea by changing the water
and electrolyte balance of the small intestine [5]. Porcine
STEC produce the edema verotoxin (VTe), also named
Shiga toxin 2e (Stx2e), which damages the vascular
endothelium of the small intestine, subcutis and brain
and ultimately leads to subcutanneous edema and neuro-
logical disorders [9]. ETEC and STEC implicated in PWD
in pigs most frequently produce either the F4 (K88) or F18
fimbrial adhesins [10,11]. Two variants of the F18 fim-
briae exist: F18ab (F107) and F18ac (2134P) [11,12].
F18ac is associated with diarrhoea while F18ab is
involved in edema disease [11]. In addition to F4 (K88)
and F18, other fimbrial colonization antigens such as F5
(K99), F6 (P987), and F41 have also been associated with
postweaning diarrhoea, but less frequently [4,13-15].
Porcine attaching and effacing E. coli (AEEC) induce intes-
tinal lesions similar to those produced by enteropatho-
genic E. coli (EPEC) in humans. These E. coli carry eae gene
encoding a 94 kDa outer membrane protein (intimin)
which is responsible for intimate attachment to epithelial
cells. However, the pathogenic significance of porcine eae-
positive isolates in weaned pigs is unclear [16,17]. A new
category of the diarrhoeagenic E. coli family, named enter-
oaggregative E. coli (EAEC), has been recognized. EAEC
elaborate a low-molecular-weight, partially heat-stable,
plasmid-encoded enterotoxin named enteroaggregative E.
coli heat-stable enterotoxin 1 (EAST1). The gene (astA)
encoding the production of EAST1 has been detected in
several groups of diarrhoeagenic E. coli (EAEC, EPEC,
ETEC, and STEC) isolated from humans and from pigs.
The pathogenic significance of EAST1 in diarrhoea in pigs
is not known [7,8,18-20].
Although, PWD is frequently observed in Slovakia, there
is a lack of information about the prevalence of sero-
groups, serotypes, and virulence factors of porcine patho-
genic E. coli. Thus, the aim of this study was to determine
the distribution of serogroups, serotypes, and virulence
genes, and to study the genetic relatedness among E. coli
isolated from pigs with PWD. This is the first study in Slo-
vak Republic of a large collection of pathogenic E. coli iso-
lated from PWD.
Results
Serogroups and serotypes
The 101 porcine isolates belonged to 24 O serogroups and
40 O:H serotypes. However, 54% were of one of these
eight serogroups (O8, O9, O45, O54, O141, O147, O149,
and O163) and 57% of the isolates belonged to only six
serotypes, including two new (O163:H- and ONT:H4)
serotypes not previously found among porcine patho-
genic E. coli. The most common serotypes were:
O149:H10 (16 isolates), ONT:H- (13 isolates), O163:H-
(11 isolates), O9:H51 (nine isolates), ONT:H4 (five iso-
lates), and O147:H- (four isolates) (Table 1).
Toxin genes
Amplification of the toxin genes by PCR showed that 77%
of isolates possessed genes for production of five types of
toxins: LT, STa, STb, Stx2e, and EAST1. The gene encoding
for EAST1 toxin (65 isolates) was the most prevalent, fol-
lowed by the STb (47 isolates), STa (27 isolates), and LT
(20 isolates) genes. The Stx2e gene was detected in five
isolates, which also carried genes for STa and STb (Table
1). Genes encoding Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2d,
and Stx2g toxins were not detected in any of the 101 por-
cine isolates studied.
Adhesin genes
The PCR analysis of all 101 isolates of E. coli showed that
61 (60%) carried at least one fimbrial or intimin gene. The
most prevalent fimbrial adhesin was F18, detected in 35
isolates. Analysis by restriction endonucleases of PCR
F18-positive products revealed that all 35 F18-positive
isolates showed the F18ac variant. Three of these 35 F18
isolates were also positive for either F4 or F17 genes. The
gene encoding F4 was identified in 19 isolates. F6, F5,
F41, and F17 genes were detected in five, one, one, and
three isolates, respectively. The eae gene (intimin type β1)
was detected in only one isolate (0.9%) of serotype
O45:H- (Table 1).
Of the 19 E. coli F4-positive, 15 isolates belonged to
O149:H10 serotype. The remaining four isolates
belonged to O8:H19, O8:HNT, O118:H9, and ONT:H19.
The F18 isolates were distributed in a wide range of sero-
types, however, 29 of 35 isolates belonged to four pre-
dominant including O163:H- (11 isolates), ONT:H- (nine
isolates), ONT:H4 (five isolates), and O147:H- (four iso-
lates). Also F6 isolates were widespread among serotypes.
Isolates carrying F17 gene belonged to O141:H25,
O147:H-, and ONT:H- serotypes, and the isolate carrying
both F5 and F41 genes belonged to ONT:H9 serotype.
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Most isolates showing genes for fimbrial adhesins also
possessed genes for toxin production, and the most com-
mon associations were: LT/STb/EAST1/F4 (18 isolates),
EAST1/F18 (13 isolates), STa/STb/F18 (seven isolates),
STa/STb/EAST1/F18 (five isolates), STa/STb/Stx2e/F18
(five isolates), STa/STb/EAST1/F6 (five isolates), and STa/
STb/EAST1/F17/F18 (two isolates).
Seropathotypes
Although the 101 porcine E. coli isolates belonged to 57
different seropathotypes (association between serotypes
and virulence genes), only seven accounted for 39% of
isolates. Seropathotype O149:H10 LT/STb/EAST1/F4 (14
isolates) was the most common, followed by O163:H-
EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18
(five isolates) (Table 1).
Haemolytic activity
Haemolytic activity on blood agar plates was detected in
72 (71%) of the 101 E. coli isolates. All F4 and F18 isolates
were haemolytic. Regarding the serotypes, all isolates of
O149:H10, O163:H- and ONT:H4 serotypes were haemo-
lytic (Table 1).
Macrorestriction fragment analysis by pulsed-field gel
electrophoresis (PFGE)
A representative group of 46 isolates (45 ETEC and/or
STEC) were selected to be analyzed by PFGE: O149:H10
(15 isolates), O163:H- (11 isolates), O147:H- (four iso-
lates), ONT:H- (eight isolates), ONT:H4 (five isolates),
O141 (two isolates), and O60:H- (one isolate). The study
revealed 36 distinct restriction profiles, considering as sig-
nificative a difference of a single band (Fig. 1). In the den-
drogram produced by the UPGMA algorithm, the isolates
were clustered in eight groups (I to VIII; 1 to 13 isolates
per group) of 70% similarity according to the Dice simi-
larity index, with 35 isolates clustering in nine subgroups
of closely related (similarity > 85%) PFGE profiles. E. coli
isolates of the same serotype were placed together in the
dendrogram, but high degree of polymorphism among
certain serotypes was detected. Thus, the 15 O149:H10
isolates were clustered in three groups (I-III, 70% similar-
ity) with only three small subgroups of closely related pro-
files (similarity > 85%; five, two and two isolates, each).
Genetic distance among O149:H10 isolates was as consid-
erable as similarity < 66%. Group V clustered 13 isolates
(all 11 O163:H- and two ONT:H- isolates; similarity >
71%) with three subgroups (two of them clustering five
isolates each, similarity > 85%). Curiously, the highest
homogeneity (similarity > 92%) was observed among a
group of 10 isolates (group VI) belonging to serotypes
O147:H- (four isolates) and ONT:H- (six isolates). E. coli
isolates of serotypes O141:H- and O141:H34 were clus-
tered in group VII (similarity > 97%). And all five isolates
of serotype ONT:H4 clustered in group VIII showing a
similarity > 81%.
Discussion
It is widely accepted that specific serotypes and patho-
types of ETEC and STEC are responsible for the major part
Table 1: Serotypes, virulence genes, and hemolytic activity of 101 porcine E. coli isolates
Virulence genes Total no. of isolates Serotypes (No. of haemolytic isolates/Total no. of isolates)
LT STb EAST1 F4 F18 1 O149:H10 (1/1)
LT STb EAST1 F4 18 O8:H19 (1/1), O8:HNT (1/1), O118:H9 (1/1), O149:H10 (14/14), ONT:H19
(1/1)
LT STb EAST1 1 O8:H14 (0/1)
STa STb EAST1 F17 F18 2 O147:H- (1/1), ONT:H- (1/1)
STa STb EAST1 F18 5 O147:H- (3/3), O163:H- (2/2)
STa STb EAST1 F6 5 O2:H- (1/1), O9:H51 (2/2), O52:H45 (0/1), O64:H- (1/1)
STa STb 1 ONT:H- (0/1)
STa STb F18 7 O141:H- (1/1), O141:H34 (1/1), O163:H- (3/3), ONT:H- (2/2)
STa STb Stx2 F18 5 ONT:H4 (5/5)
STa EAST1 F18 1 ONT:H- (1/1)
STa EAST1 F5 F41 1 ONT:H9 (0/1)
STb EAST1 F18 1 ONT:H- (1/1)
STb EAST1 1 O141:H7 (0/1)
EAST1 F18 13 O9:H- (1/1), O60:H- (1/1), O163:H- (6/6), O175:H- (1/1), ONT:H- (4/4)
EAST1 16 O1:H34 (0/1), O9:H51 (4/5), O23:H15 (1/1), O45:H2 (0/2), O54:H- (3/3),
O106:H15 (0/1), O123:H40 (0/1), O128:H28 (0/1), ONT:H- (1/1)
F17 1 O141:H25 (0/1)
eae (intimin type β1) 1 O45:H- (0/1)
None 21 O2:H1 (0/1), O3:H21 (1/1), O9:H51 (2/2), O20:H- (0/2), O23:H- (0/2),
O23:H15 (1/1), O26:H28 (0/3), O71:H12 (0/1), O78:H11 (0/1), O84:H- (0/1),
O141:H8 (0/1), O149:H10 (1/1), ONT:H- (0/2), ONT:H10 (0/1), ONT:HNT
(0/1).
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Table 2: Primer sequences and predicted size of amplification products
Target gene coding for virulence factors Oligonucleotide sequences of primers Amplified Products (bp) Primer Coordinates Accession number Reference
LT 5'-ATT TAC GGC GTT ACT ATC CTC-3'
5'-TTT TGG TCT CGG TCA GAT ATG-3'
281 27–47 287–307 S60731 Osek et al. [35]
STa
a
5'-TCC GTG AAA CAA CAT GAC GG-3' 5'-
ATA ACA TCC AGC ACA GGC AG-3'
244 267–286
j
492–510 M58746 Ojeniyi et al. [34]
STb 5'-GCC TAT GCA TCT ACA CAA TC-3' 5'-
TGA GAA ATG GAC AAT GTC CG-3'
279 515–534 773–793 AY028790 Ojeniyi et al. [34]
Stx1all
b
5'-CGC TGA ATG TCA TTC GCT CTG C-3'
5'-CGT GGT ATA GCT ACT GTC ACC-3'
302 113–134 394–414 M17358 Blanco et al. [32]
Stx2all
c
5'-CTT CGG TAT CCT ATT CCC GG-3' 5'-
CTG CTG TGA CAG TGA CAA AAC GC-3'
516 50–69 543–565 M59432 Blanco et al. [32]
Stx2e 5-ATG AAG AAG ATG TTT ATA GCG-3'
5'-TCA GTT AAA CTT CAC CTG GGC-3'
264 1176–1196 1419–1439 M36727 Osek et al. [35]
EAST1 5'-CCA TCA ACA CAG TAT ATC CGA-3'
5'-GGT CGC GAG TGA CGG CTT TGT-3'
111 2–24 94–114 S81691 Yamamoto and
Nakazawa [18]
F4 (K88)
d
5'-GCT GCA TCT GCT GCA TCT GGT
ATG G-3' 5'-CCA CTG AGT GCT GGT
AGT TAC AGC C-3'
792 31–54 798–822 M29374 This study
F5 (K99) 5'-TGC GAC TAC CAA TGC TTC TG-3' 5'-
TAT CCA CCA TTA GAC GGA GC-3'
450 45–64 475–494 M35282 Ojeniyi et al. [34]
F6 (P987) 5'-TCT GCT CTT AAA GCT ACT GG-3' 5'-
AAC TCC ACC GTT TGT ATC AG-3'
333 193–212 506–525 M35257 Ojeniyi et al. [34]
F17
e
5'-GGG CTG ACA GAG GAG GTG GGG
C-3' 5'-CCC GGC GAC AAC TTC ATC
ACC GG-3'
411 289–310 677–699 AF055313 This study
F18
f
5'-GTG AAA AGA CTA GTG TTT ATT TC-
3' 5'-CTT GTA AGT AAC CGC GTA AGC-
3'
510 1–23 490–510 M61713 Imberechts et al. [33]
F41 5'-GAG GGA CTT TCA TCT TTT AG-3' 5'-
AGT CCA TTC CAT TTA TAG GC-3'
431 154–173 565–584 X14354 Ojeniyi et al. [34]
eae
g
5'-GGA ACG GCA GAG GTT AAT CTG
CAG-3' 5'-GGC GCT CAT CAT AGT CTT
TC-3'
775 1441–1460 2193–2215 AF022236 Blanco et al. [32]
a
Detects STaP and STaH variants.
b
Detects Stx1 and the variants Stx1c and Stx1d.
c
Detects Stx2 and the variants Stx2c, Stx2d, Stx2e, and Stx2g.
d
Detects K88ab, K88ac, and K88ad.
e
Detects F17a, F17b, F17c and F17d.
f
Detects F18ab (F107) and F18ac (2134P).
g
Detects all types of eae variants (Primer pair with homology to the 5' conserved region of eae).
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of PWD in piglets. However, the distribution and frequen-
cies of serotypes and pathotypes can vary considerably
from region to region and over time in a given region. The
majority of the virulence factors are controlled by transfer-
able genetic elements (plasmids and trasposons) and
thus, common pathogenic seropathotypes may be
replaced by previously uncommon types emerging as new
pig pathogenic E. coli.
This is the first study in Slovak Republic of a large collec-
tion of pathogenic E. coli isolated from PWD. In the
present study, although 101 isolates belonged to 40 differ-
ent O:H serotypes, more than a half of ETEC and STEC
belonged to only five serotypes: O147:H-, O149:H10,
O163:H-, ONT:H-, and ONT:H4. Most isolates of these
five serotypes possessed either the F4 or the F18 genes.
ETEC of O147:H-, O149:H10 and ONT:H- serotypes are
also frequently detected in pigs from other countries,
especially O149:H10 [5,8]. The seropathotype O149:H10
LT/STb/EAST1/F4 (14 isolates) was the most prevalent in
the present study, and the reason for its predominance is
not known. A possible explanation could be that the viru-
lence factor association of these isolates makes them espe-
cially adapted to propagation in swine populations and
their enviroment [8].
The main discovery of this study was the identification of
two new serotypes (O163:H- and ONT:H4) not previ-
ously detected among porcine ETEC and STEC isolated in
other geographical zones. All 11 isolates of O163: H- sero-
type harbored the F18ac fimbriae gene and five of them
were positives for STa and STb enterotoxins. Interestingly,
all five Stx2e-positive isolates identified in the current
study belonged to the new serotype ONT:H4. In previous
studies the Stx2e production was associated mainly with
O138:H14, O138:H-, O139:H1, O141:H4, O147:H6 and
O157:H19 serotypes [5,12,21]. Thus, this study reports
two new serotypes (O163:H- and ONT:H4) emerging as
pig pathogens in Slovakia.
Although haemolysin does not seem to play an essential
role in the virulence of porcine ETEC and STEC, most of
the typical PWD E. coli are haemolytic [22]. In this study,
71% of isolates were haemolytic, and 66 isolates of those
72 possessed other virulence genes. Furthermore, all 32
isolates belonging to the three seroypes (O149:H10,
O163:H- and ONT:H-) most frequently detected in this
study showed haemolytic activity. Thus, the haemolytic
activity is a very good marker for pig pathogenic E. coli.
F4 and F18 are the most important fimbrial adhesins of
ETEC and STEC causing PWD [10,11]. In the present
study using PCR analysis, it was shown that 35% of E. coli
isolated from PWD carried genes for F18 colonization
antigen. Our findings are in accordance with those of oth-
ers [1,7,10,23,24]. Based on the studies of Rippinger et al.
[25], the F18-family of fimbriae were divided into two var-
iants F18ab and F18ac. The E. coli expressing F18ab cause
edema disease, whereas the isolates with F18ac cause
PWD [11]. In the present study, after digestion of PCR
products of F18 isolates, all 35 F18-positive showed the
F18ac variant, and more than a half of these 35 isolates
belonged to O141, O147 and O163 serogroups. We
found that 19 (19%) of 101 isolates from pigs with PWD
carried the F4 gene. Several studies have demonstrated
that the O serogroups associated with the fimbria F4 are
mainly: O8, O149, and O157 [1,5,7,13,15]. Our results
confirm these findings as 17 of 19 of Slovak F4 isolates
belonged to O8 and O149 serogroups.
Zhu et al. [16] demonstrated that the eae gene is associated
with A/E activity of O45 E. coli isolated from swine PWD.
However, the AEEC are less commonly associated with
PWD than ETEC. In the present study, we found only one
Dendrogram generated by Bionumeric software, showing distance calculated by the Dice similarity index of PFGE XbaI profiles among 46 strains isolated from PWD pigsFigure 1
Dendrogram generated by Bionumeric software, showing
distance calculated by the Dice similarity index of PFGE XbaI
profiles among 46 strains isolated from PWD pigs. The
degree of similarity (%) is shown on the scale.
Dendrogram PFGE XbaI profiles
Strain
code
Serotype Pathotype PFGE
Group
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O45:H- isolate (0.9%) positive for the eae gene. In accord-
ance with our results, Frydendahl [7] and Osek [26] also
found only 1% (3 of 219) and 3% (6 of 224) of Danish
and Polish PWD isolates carrying the eae gene, respec-
tively. F17-producing E. coli are commonly isolated from
calves with or without diarrhoea [27]. In this study, the
F17 gene was detected in three E. coli, two of which were
also positive for F18, enterotoxins, and EAST1 toxin. Sim-
ilarly, Osek [6] found that only four (1%) of 372 isolates
from PWD in Poland were positive for F17.
The role of EAST1 toxin in swine colibacillosis has not
been demonstrated, however, the gene encoding EAST1
toxin is commonly found in isolates associated with PWD
[7,8,19,20]. Our results confirm these observations as we
found that 65 of 101 isolates harbored astA gene and all
F4 isolates were astA positive. The high frequency of the
astA gene suggests the necessity of further studies to inves-
tigate the significance of this toxin in porcine PWD.
Genotyping methods such as multilocus enzyme electro-
phoresis (MLEE) and pulsed-field gel electrophoresis
(PFGE) have been used for differentiation and epidemio-
logical characterization of E. coli isolated from pigs with
PWD and edema disease. PFGE is a powerfull tool to
reveal inter- and intra-serotype specific genetic differences
among porcine pathogenic E. coli [12,28,29]. However,
there are few studies reporting genetic relatedness of E. coli
isolated from diarrhoea in pigs. Osek [29] used the PFGE
technique to analyze 82 E. coli from pigs with PWD, iso-
lated from geographically separated farms in the western
part of Poland. The 82 isolates belonging to four sero-
groups (O138, O139, O141, and O149) showed 13 differ-
ent PFGE profiles and although a high degree of
polymorphism among different serotypes was observed,
isolates belonging to the same serological group showed a
close relationship. Thus, the 25 isolates of serotype
O149:K91 generated only two PFGE types. In our study, a
representative group of 46 ETEC and STEC isolates
revealed 36 distinct restriction profiles. Although isolates
of the same serotype were placed together in the dendro-
gram, high degree of polymorphism among certain sero-
types was detected. Thus, 13 distinct PFGE profiles
resulted from 15 O149:H10 isolates analyzed, in spite of
the fact that 14 E. coli of those 15 carried the same viru-
lence genes (LT/ STb/EAST1/F4). Similarly results have
been found in Spain among isolates of the most prevalent
serotype (O157:H- LT/STb/F4) nowadays (unpublished
data). Further studies are necessary to know if some of
these clusters have appeared recently, and if so, analyze its
evolution, as well as if there is any relationship with path-
ogenicity in farms.
Conclusion
Our results indicate that in Slovakia, as described in other
countries, pathogenic E. coli isolates from PWD belong to
a restricted number of serotypes and pathotypes. The
ETEC serotype O149:H10 seems to be predominant, but
also two new serotypes (O163:H- and ONT:H4) not pre-
viously described in porcine ETEC and STEC isolated in
other countries are common. The F18ac and F4 fimbriae
were the most prevalent colonization factors detected in
postweaning E. coli in Slovakia. Macrorestriction analysis
showed that, although isolates of the same serotype and
virulence markers mainly share the same PFGE group,
there is a high genetic variation, especially among ETEC of
O149:H10 serotype.
Methods
E. coli isolates
One hundred and one E. coli isolated from the same
number of pigs with PWD were investigated in this study.
The 101 post-weaning isolates came from 20 farms
located in different parts of Slovakia. Five isolates were
selected randomly from each farm, except one farm from
Zemplinska Teplica, from which six isolates were col-
lected. Of these 20 farms, nine farms were located in East
part of Slovakia, seven farms in Central part of Slovakia,
and the remaining four farms were in West part of Slova-
kia. The E. coli were isolated from the intestinal contents
of carcasses of postweaning pigs with diarrhoea at the
Department of Bacteriology (State Veterinary Institutes in
Bratislava, Nitra, and Zvolen, Slovakia) and the Depart-
ment of Food Hygiene and Technology (Institute of
Microbiology and Immunology, University of Veterinary
Medicine, Kosice, Slovakia) between 2001 and 2003. The
fecal samples were plated onto MacConkey agar (Oxoid,
UK) and the E. coli isolates were identified by standard
biochemical procedures. After isolation, the E. coli were
stored in Luria-Bertani broth containing 20% glycerol at -
70°C for further characterization studies.
Reference strains
The E. coli strains used as a control were: 298 (F4/K88),
329 (F5/K99), 318 (F6/987P), 320 (F41), 216 (F18 and
Stx2e), 281 (LT), 256 (STa and STb), EDL933 (Stx1, Stx2
and eae-γ1), G491(F4/K88ac), P201 (F4/K88ad), 5138
(F18ab), 8813 (F18ac), 253KH85 (F17), 226KH85 (F17),
960205 (EAST1), 022206 (EAST1), and E. coli C600 (as
negative control). Some of the control strains were kindly
supplied by Dr. J. Osek (National Veterinary Research
Institute, Pulawy, Poland), Dr. P. Alexa (Veterinary
Research Institute, Brno, Czech Republic), Dr. P.F. Linter-
mans (Institut National de Recherches Veterinaires, Brux-
elles, Belgium), and Dr. C. Chae (Department of
Veterinary Pathology, College of Veterinary Medicine,
Seoul National University, Republic of Korea).
BMC Veterinary Research 2006, 2:10 http://www.biomedcentral.com/1746-6148/2/10
Page 7 of 8
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Serotyping
The determination of O and H antigens was carried out by
the method described by Guinée et al. [30] employing all
available O (O1–O181) and H (H1–H56) antisera in the
LREC (Lugo). All antisera were obtained and absorbed
with the corresponding cross-reacting antigens to remove
the nonspecific agglutinins. The O antisera were produced
in the LREC (Lugo, Spain) and the H antisera were
obtained from the Statens Serum Institut (Copenhagen,
Denmark).
Haemolysin activity
The isolates were inoculated on blood agar base supple-
mented with 5% sheep blood (Oxoid, UK) and incubated
at 37°C for 18h. β-Haemolysis was evident as a zone of
lysis surrounding the bacterial growth.
Detection of virulence genes by PCR
The polymerase chain reaction (PCR) for detection of tox-
ins (LT, STa, STb, Stx1, Stx2, Stx2e, and EAST1) and adhes-
ins (F4, F5, F6, F17, F18, F41, and eae) was carried out as
described by Vu-Khac et al. [31] and Blanco et al. [32].
Base sequences and predicted sizes of the amplified prod-
ucts for the specific oligonucleotide primers used in this
study are shown in Table 2[31-35]. Typing of eae
(intimin) gene detected in one positive isolate identified
in this study was carried out by PCR as described else-
where [32].
Digestion of PCR products with restriction endonuclease
After amplification, the PCR products of F18-positive iso-
lates were digested with restriction enzyme NgoMIV (for-
merly NgoMI) to distinguish genes encoding F18ab and
F18ac [33]. The master mix was prepared with a total of
15µ l volume containing 5µ l of PCR products (after puri-
fying with Wizard PCR Preps [Promega]); 10µ l 1x MULTI-
CORE™ buffer (Promega); and 1 unit of enzyme NgoMIV.
After incubation at 37°C for one hour, the DNA digestion
was analyzed by electrophoresis in 2 % agarose gel.
Pulsed-field gel electrophoresis
PFGE was performed in a CHEF MAPPER system (Bio-
Rad, Hemel Hempstead, United Kingdom) at 14°C in
0.5XTBE by the Enternet proposed standard-protocol for
PFGE [36]. Cleavage of the agarose-embedded DNA was
achieved with 0.2–0.8 U/µ l Xbal (Roche) according to
instructions of the manufacturer. Run times and pulse
times were 2.20 to 54.0s for 22 h with linear ramping.
PFGE was used to establish clonal relatedness and diver-
sity among a representative group of 46 isolates. To per-
form the comparison of the PFGE pulsotypes, TIFF files
were analyzed with BioNumerics software (Applied
Maths, Sint-Martns-Latem, Belgium). Cluster analysis of
the Dice similarity indices based on the unweighted pair
group method using aritmetic averages (UPGMA) was
done to generate a dendrogram describing the relation-
ship among EPEC pulsotypes. A difference of at least one
restriction fragment in the profiles was considered the cri-
terion for discriminating between clones.
Authors' contributions
H. Vu-Khac, E. Holoda, and E. Pilipcinec isolated the E.
coli and performed the detection of virulence genes by
PCR, M. Blanco, G. Dahbi and E. A. González also partic-
ipated in the PCR study, J.E. Blanco did the serotyping of
the isolates, A. Mora and C. López were responsible for the
study of genetic diversity carried out by PFGE, and H. Vu-
Khac, and J. Blanco designed the study and drafted the
manuscript. All authors read, commented on and
approved the final manuscript.
Acknowledgements
This paper is dedicated to the memory of Dr. Enrique A. González, an emi-
nent scientist, an excellent Professor of Microbiology and a very good
friend. We thank to Dr. Majerèiak (State Veterinary Institute, Nitra, Slova-
kia), MVDr. Gašpar (State Veterinary Institute, Bratislava, Slovakia), and
MVDr. Novák (State Veterinary Institute, Zvolen, Slovakia) for providing E.
coli strains, and Monserrat Lamela for her skillful technical assistance. This
work was supported by VEGA grant 1/1352/04 of the Slovak Grant Agency,
an internal grant of the University of Veterinary Medicine in Košice (Slova-
kia), a grant from the the Spanish Fondo de Investigación Sanitaria (FIS G03-
025-COLIRED-O157), and three grants from the Xunta de Galicia
(PGIDIT02BTF26101PR, PGIDIT04RAG261014PR, and
PGIDIT05BTF26101PR).
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... The boiling method was used to extract total bacterial DNA of each E. coli strain (Levesque, Piche, Larose, & Roy, 1995). As previously described (Bertin, Martin, Girardeau, Pohl, & Contrepois, 1998;Cheng et al., 2006;Liu et al., 2014;Osman, Mustafa, Aly, & AbdElhamed, 2012;Paton & Paton, 1998Vu-Khac et al., 2007;Zhang et al., 2007), multiplex polymerase chain reaction (mPCR) was used to determine 21 virulence genes encoding enterotoxins (LT-I, LT-II, STa, STb, and EAST1), fimbriae (K88, K99, 987P, F41, and F18), non-fimbrial adhesins (AIDA-I, paa, eae, and saa), Shiga toxins (Stx1, Stx2, and Stx2e), HPI and α-haemolysin (hlyA). The reaction volume (20μL) comprised 2μM of each primer (Table 1), 2 × Master Mix (Takara, Dalian, China) and 3μl of DNA template. ...
... During the past decades, the occurrence and spread of the PWD in piglets have posed an enormous economic loss to the development of swine breeding industry in mainland China (Cheng et al., 2006;Liu et al., 2014). Numerous studies have showed that ETEC are the most important agents of enteric colibacillosis in pig, and fimbrial adhesins are necessary in the pathogenetic mechanism (Do, Byun, & Lee, 2018;Toledo et al., 2012;Vu-Khac et al., 2007;Zhang et al., 2007). Of note, E. coli with fimbrial adhesins can be also detected in pigs without diarrhoea. ...
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