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Journal of Medical Microbiology (2005), 54, 333346

DOI 10.1099/jmm.0.45690-0

Amplified fragment length polymorphism (AFLP)


versus randomly amplified polymorphic DNA (RAPD)
as new tools for inter- and intra-species
differentiation within Bordetella
Anna Gzyl,1 Ewa Augustynowicz,1 Ewa Mosiej,2 Monika Zawadka,1
Grzegorz Gniadek,1 Aneta Nowaczek1 and Janusz Slusarczyk1
Correspondence

Department of Sera and Vaccine Evaluation, National Institute of Hygiene, 24 Chocimska Str.,
00-791 Warsaw, Poland

Interfaculty Studies of Biotechnology, Warsaw Agricultural University, 159 Nowoursynowska Str.,


00-776 Warsaw, Poland

Anna Gzyl
agzyl@pzh.gov.pl

Received 5 April 2004


Accepted 19 November 2004

Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic
DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among
isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of
these species from international and Polish bacterial collections were genotyped according to
RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed
SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was
concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intraand inter-species differentiation of isolates of the Bordetella genus. The most precise level of interspecies discrimination and the highest level of intra-species discrimination of the Bordetella isolates
of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both
techniques might provide alternative tools for the identification of Bordetella at the genomic species
and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in
epidemiology. By applying the AFLP technique presented in this article, more precise data on the
emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the
Bordetella genus in the human and animal environments might be obtained.

INTRODUCTION
Currently, three of the eight species of the Bordetella genus
that have been described, Bordetella pertussis, Bordetella
parapertussis and Bordetella bronchiseptica, present the highest level of genetic relatedness and are associated with
infections of the upper respiratory tract of many mammals
(Gerlach et al., 2001; Khattak & Matthews, 1993; Musser et
al., 1986; van der Zee et al., 1997). B. pertussis is an obligate
pathogen of humans and in susceptible individuals induces
highly contagious disease so-called whooping cough that
still causes over 250 000 deaths around the world annually
(World Health Organization, 2002). B. parapertussis is
capable of inducing typical and mild forms of pertussis in

Abbreviations: AFLP, amplified fragment length polymorphism; IS,


insertion sequence; MLEE, multilocus enzyme electrophoresis; MLST,
multilocus sequence typing; RAPD, randomly amplified polymorphic DNA;
REA, restriction enzyme analysis; UPGMA, unweighted pair group method
using arithmetic averages.

humans and has also been recognized as a source of


asymptomatic and symptomatic infections of the respiratory
tract in ovine animals (Gerlach et al., 2001; Porter et al.,
1996). B. bronchiseptica is a common commensal or pathogen in the respiratory tract of several mammalian species.
Respiratory or systemic B. bronchiseptica infections in humans have occasionally been described (Dworkin et al., 1999;
Woolfrey & Moody, 1991). Most of the B. bronchiseptica
human infections described in the literature (septicaemia,
pneumonia, pertussis-like illness, tracheobronchitis, sinusitis, peritonitis and meningitis) occurred in immunosuppressed patients (Dworkin et al., 1999) or in individuals with
other underlying conditions (Sacco et al., 2000a). Zoonoses
from pets might be involved, since B. bronchiseptica is known
to produce infection and disease in many mammals (e.g.
kennel cough in dogs and atrophic rhinitis in pigs) including
those kept in the home (Dworkin et al., 1999; Woolfrey &
Moody, 1991).
These three species can be differentiated by phenotype.
However, the genetic diversity shown with many techniques

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45690 & 2005 SGM Printed in Great Britain

333

A. Gzyl and others

[DNADNA hybridization, multilocus enzyme electrophoresis (MLEE), and 16S and 23S rRNA gene typing] has been
found to be limited (Gerlach et al., 2001; Musser et al., 1986;
van der Zee et al., 1997). Thus they are commonly considered
as members of a single B. bronchiseptica cluster (Gerlach et al.,
2001).
Bordetella avium colonizes mainly the respiratory tract of
birds, inducing bird bordetellosis and turkey coryza (Gerlach
et al., 2001). In poultry, B. avium infections result in coryza or
rhinotracheitis, highly contagious diseases resulting in economic losses to the turkey/poultry industry (Register et al.,
2003). Bordetella hinzii found in birds has recently been also
associated with human infections seen in patients with AIDS
or cystic fibrosis (Cookson et al., 1994; Dworkin et al., 1999;
Funke et al., 1996; Kattar et al., 2000; Vandamme et al., 1995).
Bordetella holmesii has been recovered from patients with
underlying disorders, including Hodgkin lymphoma, sicklecell anaemia, pulmonary disease and asplenia, septicaemia
and endocarditis (Russell et al., 2001; Tang et al., 1998; Yih et
al., 1999). Surprisingly, in the last few years it has also been
isolated from the nasopharynx of patients with pertussis-like
symptoms (Mazengia et al., 2000).
Bordetella trematum, commonly isolated in humans from
wounds or ears but not from the respiratory tract, is a species
of unknown pathogenic potential (Gerlach et al., 2001).
Bordetella petrii, the first anaerobic environmental isolate of
the Bordetella genus, was described in 2001 and still only a
single strain is available (von Wintzingerode et al., 2001).
Tracking epidemiological routes is extremely important in
the case of B. pertussis since it still affects large proportions of
individuals and infection rates are rising unexpectedly in
highly vaccinated populations (Mooi et al., 2000; van der Zee
et al., 1996a). Similarly, studies of B. parapertussis transmission and co-infection with B. pertussis might elucidate many
aspects of the epidemiology of whooping cough. Tracking
differences among strains of B. bronchiseptica, B. avium, B.
hinzii or B. holmesii as opportunistic infections of humans,
together with differences found in strains isolated from
natural animal hosts, might be important for human and
animal microbiology.
Several studies have proved the usefulness of molecular tools
in differentiating particular species of the Bordetella genus.
Some of them were able to track genetic differences among/
within two to three species simultaneously. For several years,
amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) have been
found to be very useful in studying the epidemiology of
many pathogens, e.g. in order to follow transmission routes
(van der Zwet et al., 1999) or to study phylogenetic relationships between organisms (Torriani et al., 2001). In this study
we believe we present the first report on the application of
AFLP for genotyping isolates within the Bordetella genus. The
discriminatory power of AFLP fingerprinting has been
compared with RAPD performed with protocols previously
applied by Yuk et al. (1998) for B. parapertussis. In addition,
334

we describe a newly designed SpeI/ApaI restriction enzymes/


adapters/primers protocol not used previously for AFLP
fingerprinting. Our preliminary study was aimed at investigating the possibility of Bordetella inter- and intra-species
genotyping and evaluating the taxonomic potential of AFLP
and RAPD in the delineation of species-specific identification.

METHODS
Bacterial strains and culture media. A total of 81 representative

isolates belonging to the Bordetella genus were investigated. The


number, origin and host species of all the isolates tested are presented
in Table 1. Thirty-five B. pertussis strains collected by the Department of
Sera and Vaccine Evaluation of the National Institute of Hygiene,
Warsaw, Poland were analysed. Among them, 27 strains were isolated
from non-epidemiologically related cases of pertussis that occurred in
19602000 in Poland. The other isolates of B. pertussis used in the study
originated from Hungary (two), the former Soviet Union (two), the
former Yugoslavia (one) and Finland (one). Three international strains
Tohama I and W28 (both received from Dr N. Guiso, Institut Pasteur,
Paris) and 18323 (Kendrick) were also studied.
Isolates of B. avium (nine), B. hinzii (four) and B. trematum (five) came
from the BCCG/LMG Collection, Gent, Belgium. Among the 13 isolates
of B. bronchiseptica, nine came from the BCCG/LMG Collection and one
from the Institute of Immunology and Experimental Therapy in
Wroclaw, Poland. Two B. bronchiseptica strains were obtained from
Dr N. Guiso (Institut Pasteur, Paris). Regarding the B. holmesii strains
analysed, three isolates came from the BCCG/LMG Collection and two
were kindly obtained again from Dr N. Guiso (Institut Pasteur, Paris).
Five and four isolates of B. parapertussis originated from the BCGG/
LMG Collection and the parental Bacterial Institute Collection, NIH,
Warsaw, Poland. B. petrii was kindly received from Professor R. Gross
(Universitat Wurzburg, Germany).
B. pertussis strains were cultured on BordetGengou agar (Difco)
supplemented with 15 % sheep blood and incubated at 35 8C for 23
days. The B. petrii strain was grown anaerobically on LB agar at 30 8C for
48 h (von Wintzingerode et al., 2001).
Isolation of total DNA. DNA from 81 strains belonging to the

Bordetella genus was isolated by means of a commercially available


Qiagen DNA isolation kit. The concentration of the DNA was measured
by electrophoresis of samples on 1 % agarose gel against diluted
preparations of phage DNA of known concentrations.
Typing by the RAPD method. Several different short primers designed

by Williams et al. (1990) were screened for fingerprinting efficiency of


different isolates of the eight Bordetella species. For screening, five
different strains of each species were used where possible. The two
primers with the best discriminatory potential were chosen to study all
of the strains collected: 1247, 59-AAGAGCCCGT-39, and 1254, 59CCCGTCAGCA-39. Forty-two cycles of denaturation at 94 8C for
1 min, annealing at 36 8C for 1 min and extension at 72 8C for 1 min
were applied in the Biometra thermal cycler. Aliquots of amplified PCR
products were electrophoresed in 1.5 % agarose gels stained with
ethidium bromide. Electrophoresis was carried out in TAE buffer
(40 mM Tris/acetate, 1 mM EDTA).
Gels were photographed with the Gel Doc 1000 Gel Documentation
System (Bio-Rad). Stored patterns were analysed with the GelCompar
Software version 3.1 (Applied Maths) after conversion of the data into
TIF format. Electrophoresis gels were normalized according to the
external standards contained in each fifth lane (1 kb ladder, Gibco).
Dendrograms for cluster analysis were based on similarity matrices

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Journal of Medical Microbiology 54

AFLP in genotyping of isolates of Bordetella genus

Table 1. Designation, origin and RAPD and AFLP profile types of the Bordetella strains in the study.
No.

Species

Strain/origin

Host

Host collection

Cluster
RAPD RAPD AFLP AFLP
1247 1254 EcoRI/ SpeI/
MseI ApaI

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. pertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. parapertussis
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica

324, Hungary
2896, Hungary
353, former Soviet Union
5375, former Soviet Union
Tohama I, Japan
60623/67, former Yugoslavia
W 28, England
18530, Finland
3/60, Poland
7/60, Poland
12/60, Poland
2/62, Poland
6/62, Poland
A/63, Poland
253/67, Poland
2/68, Poland
9/68, Poland
45/68, Poland
1253/73, Poland
6635/73, Poland
6770/73, Poland
6901/73, Poland
2955/74, Poland
3835/74, Poland
7097/74, Poland
2066/95, Poland
2214/95, Poland
2245/95, Poland
3599/97, Poland
1038/99, Poland
785/00, Poland
871/00, Poland
1468/00, Poland
2632/00, Poland
18323 (Kendrick)
1816
1820
1821
1823
1829
1251/70, Poland
1356/75, Poland
762/00, Poland
46/00, Poland
3525, Denmark
3530
3533, Sweden
3534, Sweden
3536
3537

http://jmm.sgmjournals.org

Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Rabbit
Monkey
Horse
Human
Rat
Piglet

NIH, Warsaw, Poland


NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
CIP, The Institut Pasteur, Paris, France
NIH, Warsaw, Poland
CIP, The Institut Pasteur, Paris, France
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
NIH, Warsaw, Poland
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium

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RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RC
RC
RC
RC
RC
RC

AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AIIa
AII
AII
AII
AII
AI
AI
AI
AI
AIIb
AI

E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5c
E5b
E5b
E5b
E5b
E5b
E5d
E5d
E5d
E5d
E5b
E5b
E5b
E5b
E5b
E5a

S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8
S8a
S8
S8
S8
S8
S8
S8
S4b
S4b
S4b
S4b
S4b
S4b
S4b
S4b
S4b
S4a
S4a
S4a
S4b
S4c
S4a

335

A. Gzyl and others

Table 1. cont.
No.

Species

Strain/origin

Host

Host collection

Cluster
RAPD RAPD AFLP AFLP
1247 1254 EcoRI/ SpeI/
MseI ApaI

51
52
53
54
55
56
57

B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica
B. bronchiseptica

3538
3540
3543
1033, Czech Republic
107597
107599
1943

Dog
Cat
Guinea pig
Unknown
Unknown
Unknown
Unknown

58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81

B. avium
B. avium
B. avium
B. avium
B. avium
B. avium
B. avium
B. avium
B. avium
B. hinzii
B. hinzii
B. hinzii
B. hinzii
B. holmesii
B. holmesii
B. holmesii
B. holmesii
B. holmesii
B. trematum
B. trematum
B. trematum
B. trematum
B. trematum
B. petrii

1851, Germany
1854, USA
1855, Spain
1856, Israel
3522, Germany
3549, UK
3556, Germany
3557, Germany
10973, Belgium
10980, Belgium
13494, Australia
14052, USA
16211, Belgium
15047, USA
15945, USA
15946, USA
104394
104395
5894
14993
15543
16652, Switzerland
16877
4/03

Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Goose
Duck
Broiler
Chicken
Chicken
Human
Human
Human
Human
Human
Unknown
Unknown
Human
Human
Human
Human
Human
Isolated from an
anaerobic
bioreactor

calculated from the Pearson product-moment correlation coefficient


and the unweighted pair group method using arithmetic averages
(UPGMA) algorithm (Everitt, 1993).
Typing by the AFLP method. In the preliminary screening DNA from,
where possible, five isolates from each of the Bordetella species was used
to evaluate the potential to obtain distinctive AFLP patterns. Six
different previously described AFLP protocols, using HindIII/TagI,
ApaI/TagI, MfeI/BglII, HindIII, PstI/TaqI and EcoRI/MseI, based on
different enzyme restriction/specific adapter ligation and primerspecific amplification with/without selective bases complementary to
nucleotides flanking the restriction sites, were tested (Grady et al., 1999;
Huys et al., 1996; Kokotovic et al., 1999; McLauchlin et al., 2000; van
Elderle et al., 1999; Vos & Kuiper, 1998). Additionally, new AFLP sets
with SpeI restriction and the frequent-cutter enzymes BglII, HindIII,

336

BCCM/LMG, Gent, Belgium


BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
NIH, Warsaw, Poland
CIP, The Institut Pasteur, Paris, France
CIP, The Institut Pasteur, Paris, France
The Institute of Immunology and
Experimental Therapy, Wroclaw,
Poland
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
CIP, The Institut Pasteur, Paris, France
CIP, The Institut Pasteur, Paris, France
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
BCCM/LMG, Gent, Belgium
Universitat Wurzburg, Germany

RC
RA
RA
RA
RA
RA
RA

AIIc
AI
AI
AI
AI
AI
AI

E5b
E5a
E5a
E5b
E5b
E5b
E5b

S8a
S4b
S4c
S4a
S4a
S4a
S4c

RBa
RBa
RBa
RBa
RBa
RBa
RBa
RBa
RBa
RBb
RBd
RBd
RBd
RBc
RBc
RBc
RBc
RBc
RBb
RBb
RBe
RBe
RBe
RBa

B
B
B
B
B
B
B
B
B
AIIc
AIIc
AIIa
AIIc
AIIa
AIIa
AIIa
AIIa
AIIa
AI
AIII
AIIb
AI
AI
AIId

E1
E1
E1
E1
E1
E1
E1
E1
E1
E4
E4
E4
E4
E3
E3
E3
E3
E3
E2
E2
E2
E2
E2
E6

S1
S1
S1
S1
S1
S1
S1
S1
S1
S2
S6
S2
S2
S5
S5
S5
S5
S5
S3
S3
S6
S6
S6
S7

PstI, ApaI and EcoRI, coupled with ligation by newly designed SpeI
adapters and primers, were tested (Table 2).
Briefly, in all tested AFLP sets, 200 ng of Bordetella DNA was digested
with the following restriction enzymes according to manufacturers
recommendations: HindIII (BioLabs)/TagI (Gibco-BRL); ApaI (BioLabs)/TagI; HindIII; MfeI (BioLabs)/BglII (BioLabs); PstI (Eurogentec)/
TagI; EcoRI (Gibco-BRL)/MseI (BioLabs); SpeI (BioLabs)/BglII; SpeI/
HindIII; SpeI/PstI; SpeI/ApaI; and SpeI/EcoRI. Samples were heattreated to inactivate restriction enzymes. Next, adapters, synthesized
by Invitrogen, were ligated to the restriction DNA fragments by T4 DNA
ligase (BioLabs) according to manufacturers recommendations. For
rare-cutting enzymes, adapters were used at a concentration of
5 pmol l 1 , and for frequent-cutting enzymes, at 50 pmol l 1 . After
completion of ligation, samples were heat-inactivated, 10-fold diluted
in distilled water and refrigerated at 20 8C until the start of PCR.

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Journal of Medical Microbiology 54

AFLP in genotyping of isolates of Bordetella genus

Table 2. Characteristics of the AFLP sets


AFLP set

HindIII/TagI
ApaI/TagI
ApaI/TagI
HindIII
MfeI/BgllI
PstI/TagI
EcoRI/MseI

Strains
studied*

Reference or adaptor/primer sequence

5
5
5
5
5
5
81

van Elderle et al. (1999)


Grady et al. (1999)
Huys et al. (1996)
McLauchlin et al. (2000)
Kokotovic et al. (1999)
Vos & Kuiper (1998)
Vos et al. (1998)
Adaptor sequence

SpeI/ApaI

SpeI/BglII

SpeI/HindIII

SpeI/PstI

SpeI/EcoRI

81

AD1 SpeI
59-CTCGTAGACTGCGTACC-39
AD2 SpeI
59-CTAGGGTACGCAGTCTAC-39
AD1 ApaI
59-CATCTGACGCATGT-39
AD2 ApaI
59-TCGTAGACTGCGTACAGGCC-39
AD1 SpeI
59-CTCGTAGACTGCGTACC-39
AD2 SpeI
59-CTAGGGTACGCAGTCTAC-39
AD1 BglII
59-CGGACTAGAGTACACTGTC-39
AD2 BglII
59-GATCGACAGTGTACTCTAGTC-39
AD1 SpeI
59-CTCGTAGACTGCGTACC-39
AD2 SpeI
59-CTAGGGTACGCAGTCTAC-39
AD1 HindIII
59-CTCGTAGACTGCGTACC-39
AD2 HindIII
59-AGCTGGTACGCAGTC-39
AD1 SpeI
59-CTCGTAGACTGCGTACC-39
AD2 SpeI
59-CTAGGGTACGCAGTCTAC-39
AD1 PstI
59-CTCGTAGACTGCGTACATGCA-39
AD2 PstI
59-TGTACGCAGTCTAC-39
AD1 SpeI
59-CTCGTAGACTGCGTACC-39
AD2 SpeI
59-CTAGGGTACGCAGTCTAC-39
AD1 EcoRI
59-CTCGTAGACTGCGTACC-39
AD2 EcoRI
59-AATTGGTACGCAGTCTAC-39

Cy5-labelled primer

Unlabelled primer (5939)

ApaI+0

SpeI+0

59-GACTGCGTACAGGCCC-39

59-GACTGCGTACCCTAGT-39

BglII+0
59-GAGTACACTGTCGATCT-39

HindIII+1
59-GACTGCGTACCAGCTTA-39

PstI+0
59-GACTGCGTACATGCAG-39

EcoRI+0
59-GACTGCGTACCAATT C-39

*5 indicates tested on five strains of each species of Bordetella (except a single isolate of B. petrii); 81 indicates tested on all 81 Bordetella strains in the
study.

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337

A. Gzyl and others

Restriction fragments tagged with specific adapters were selectively


amplified with relevant primers. PCR was performed in a 20 l volume
in a Biometra thermal cycler. Each amplification reaction mixture
contained 5 l of a 10-fold diluted ligation reaction sample, 12 ng Cy5labelled primer, 60 ng non-labelled primer, 120 M each of dATP,
dGTP, dCTP and dTTP, 1.0 U AccuTaq-LA DNA polymerase (Sigma),
3.0 mM MgCl2 , 50 mM Tris/HCl, 15 mM ammonium sulphate and
0.1 % Tween 20. Amplification for previously described sets was
performed according to recommended conditions (Table 2). In the
AFLP amplification step with SpeI primers tested with BglII, HindIII,
PstI, ApaI or EcoRI ones, the following conditions were used: cycle 1,
94 8C for 30 s, 65 8C for 30 s, 72 8C for 60 s; cycles 213, the same PCR
profile as seen in the first cycle except for a stepwise 0.7 8C decrease in the
annealing temperature in each subsequent cycle of the 12 cycles; cycles
1436, 94 8C for 30 s, 56 8C for 30 s, 72 8C for 60 s.
After completion of the PCR, 5 l of each reaction tube mixture was
mixed with 3 l loading dye (Amersham Pharmacia), denatured at
90 8C for 3 min and applied to the gel. Selectively amplified fragments
were separated through a ReproGel High Resolution (Amersham
Pharmacia) in 0.53 TBE buffer on an ALFexpress DNA sequencer.
Separation was done at 1500 V, 60 mA, 25 W for 420 min at 55 8C. To
evaluate intra- and inter-gel differences and identity levels, a fluorescein-labelled molecular size marker (ALFexpress Sizer 50500) and
an external reference strain were used as external size markers. Stored
fluorograms were analysed with the GelCompar Software version 3.1
(Applied Maths) after conversion into TIF format. The track resolution
was reduced, excluding the primer front and fragments higher than
500 bp. Dendrograms for cluster analysis were based on similarity
matrices calculated from the Pearson product-moment correlation
coefficient and the UPGMA algorithm (Everitt, 1993).

RESULTS
RAPD analysis of isolates at the Bordetella genus
level
For RAPD fingerprinting, six short (10 bp) primers previously described by Williams et al. (1990) were used. Of all
the primers tested, two (1254 and 1247) were chosen to
screen all of the collected isolates, since they enabled the most
informative DNA profiles to be observed. The Bordetella
strain-specific DNA arrays consisted of approximately 212
bands with a fragment-length distribution in the 0.13.0 kb
range. The level of similarity for reference strain patterns was
at least 94 % within the same gel and at least 90 % between
gels. The number of RAPD patterns was evaluated after
consideration of the identity level of 90 %.
The overall genetic similarities of all the RAPD patterns
obtained using primers 1254 and 1247 within the dendrograms, as defined by the Pearson product-moment correlation coefficient, were 53.9 % and 64.0 %, respectively. RAPD
arrays classified Bordetella isolates into several clusters. Yet,
exact species-specific differentiation of patterns was not
found, however B. avium isolates were found exclusively in
single clusters in both of the dendrograms.
Two clusters were found for the RAPD protocol using the
1254 primer: A, containing 72 non-Bordetella avium isolates,
and B, containing nine B. avium isolates. Cluster A, at the
63 % overall similarity level, consisted of two subclusters, AI
and AII, and a single pattern that was specific for B. trematum
338

no. 14993, defined as AIII. Eleven B. bronchiseptica and four


B. trematum isolates clustered at a linkage level of 77 %
within the AI subcluster. Subcluster AII patterns clustered at
a level of 76 % and contained four clades AIIad. The AIIa
clade contained groups that were related to each other,
including patterns specific for B. pertussis and B. holmesii
strains, and a group with five B. parapertussis and a single B.
hinzii isolate (no. 14052). Polish isolates of B. parapertussis,
B. trematum no. 15543, and B. bronchiseptica no. 3536
formed the AIIb clade. Clade AIIc contained strains of B.
hinzii (nos 10980, 16211 and 13494) and B. bronchiseptica no.
3538. The AIId pattern was characteristic of B. petrii.
In the 1247 RAPD set, at the 72 % similarity level, three main
clusters were identified. Cluster RA contained patterns of all
of the B. pertussis and B. parapertussis strains, and six isolates
of B. bronchiseptica (nos 3540, 3543, 1033, 107597, 1943 and
107599). Cluster RB, contained groups of related patterns for
nine B. avium isolates and B. petrii (RBa), five B. holmesii
isolates (RBc), B. hinzii nos 16211, 13494 and 14052 (RBd),
B. trematum nos 5894 and 14993, and B. hinzii no. 10980
(RBb), and B. trematum nos 16877, 15543 and 16652 (RBe).
Cluster RC contained seven B. bronchiseptica isolates presenting almost 100 % as the similarity value. The B. petriispecific pattern has been classified as directly adjacent to the
B. avium cluster (RBa). Within the B. bronchiseptica cluster,
four non-species-specific pattern types were observed. Similarly, for B. hinzii and B. trematum three non-species-specific
pattern types were found. For B. holmesii and for B. avium
isolates single clusters have been generated (however, the RBa
B. avium-like cluster contained B. petrii).
No band variation was observed in RAPD band patterns
obtained for DNA isolated from seven passages of a single
strain or with DNA content in the range 10200 ng. Data
obtained show that typing with the RAPD 1254 primer can
easily identify B. avium from other Bordetella species, as
species-specific patterns classified them into a separate
branch of the dendrogram. Similarly, typing with 1247
primer may be of value for confirming B. avium and B.
holmesii species.
Analysis of RAPD profiling within species
We found that primers used in RAPD typing hardly
differentiated B. pertussis, B. parapertussis, B. avium and B.
holmesii isolates (Table 3). Relevant isolates of these four
species tested with RAPD using primer 1254 or 1247
presented similarity levels near identity value: 87.9/86.7 %,
85.6/91.5 %, 95.5/94.8 % and 95.0/97.4 %, respectively. High
homogeneity of patterns was also seen in the case of B. hinzii
in RAPD fingerprinting with primer 1247 (91.6 %). Typing
allowed differentiation of B. bronchiseptica and B. trematum
isolates at the level of 64.3/49 % and 82.2/66.8 % in RAPD
profiling using the of 1247/1254 primers, respectively. Some
potential for differentiation using RAPD with primer 1254
was seen in the case of B. hinzii (80.8 %). The greatest value
for intra-species RAPD typing using these primers was for B.

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Journal of Medical Microbiology 54

AFLP in genotyping of isolates of Bordetella genus

Table 3. Overall genetic similarity values as defined by the Pearson product-moment correlation
coefficient and the UPGMA algorithm for Bordetella patterns obtained with RAPD or AFLP at the genus
and species levels
Species

Bordetella species
B. pertussis
B. parapertussis
B. bronchiseptica
B. avium
B. hinzii
B. holmesii
B. trematum
B. petrii

No. of
isolates

81
35
9
13
9
4
5
5
1

Overall genetic similarity (%)/number of patterns differentiated


after consideration of the relevant identity levels obtained under
study conditions
RAPD 1254

RAPD 1247

AFLP
EcoRI/MseI

AFLP
SpeI/ApaI

53.9/19
87.9/2
85.6/2
49.0/7
95.5/1
80.8/3
95.0/1
66.8/3

64.0/11
86.7/2
91.5/1
64.3/3
94.8/1
91.6/1
97.4/1
82.2/2

23.6/45
77.8/6
77.1/5
70.9/12
56.6/9
78.3/4
72.8/4
71.4/5

4.3/51
19.1/16
69.4/6
30.1/10
47.6/8
42.1/4
76.1/4
10.7/4

bronchiseptica and B. trematum, since for 13 and five strains


tested, seven and three RAPD patterns, respectively, could be
differentiated.
Screening of AFLP sets
Preliminary screening of 12 different AFLP schemes revealed
that all of them except the HindIII AFLP set were able to
fingerprint Bordetella isolates efficiently (Table 2). Profiles
obtained for screened isolates visually appeared to differentiate particular species. For genotyping of all the strains in
the study, the EcoRI/MseI and SpeI/ApaI AFLP sets were
chosen. The profiles of these sets presented two options of
band distribution: higher (EcoRI/MseI, 1639) and lower
(SpeI/ApaI, 625) numbers of bands within the profiles.
Overall intra-/inter-gel similarity levels for the external
reference strain were 97/92 %, respectively, and thus the
number of patterns obtained relates to the identity level
evaluated at 92 %. Under the conditions tested, the method
was able to discriminate the analysed strains at both the
species and the inter-species level. Overall genetic similarities
defined by Pearson product-moment correlation coefficient
for banding patterns of Bordetella isolates, reached values of
23.6 % and 4.3 % for the EcoRI/MseI and SpeI/ApaI AFLP
sets, respectively.
Cluster analysis of the AFLP EcoRI/MseI patterns identified
five pattern groups, at the 50 % overall similarity level, each
consisting of strains belonging to a single species with the
exception of B. pertussis, B. parapertussis and B. bronchiseptica, which all clustered together (Fig. 1). The clusters
identified were: E1, nine isolates of B. avium; E2, five isolates
of B. trematum; E3, five isolates of B. holmesii; E4, four
isolates of B. hinzii; E5, 57 isolates of B. bronchiseptica, B.
pertussis and B. parapertussis; and a singly classified B. petriispecific pattern (E6).
http://jmm.sgmjournals.org

The closest relationship and lowest diversity were found for


patterns clustering at the 67 % level of similarity within E5.
Four subclusters were found within E5: E5a, three isolates of
B. bronchiseptica; E5b, 10 isolates of B. bronchiseptica and five
B. parapertussis; E5c, containing exclusively B. pertussis
strains; and E5d, four Polish isolates of B. parapertussis.
The subcluster specific for B. pertussis showed a 79 %
similarity value. The nine isolates of B. avium showed nine
AFLP patterns, which clustered at a linkage level of 74 % into
the E1 cluster. The E2 cluster contained five different AFLP
patterns for the five B. trematum strains, which associated
together at a linkage level of 72 %. The five isolates of B.
holmesii, presenting four different patterns, clustered into E3
at a linkage level of 73 %. The four analysed B. hinzii isolates
revealed four AFLP patterns which grouped at a linkage level
of 78 %. The B. pertussis, B. parapertussis and B. bronchiseptica isolates showed six, five and 12 different AFLP patterns,
respectively, and clustered at a linkage level of 69 %.
Dendrograms constructed separately for isolates of eight
particular species from the EcoRI/MseI AFLP patterns presented similarity values in the range of 70.978.3 % except
for B. avium (56.6 %) (Table 3).
The highest number of different patterns was observed
within the SpeI/ApaI AFLP set evaluated as the most
discriminatory (Table 2, Fig. 2). At the 30 % similarity level,
eight clusters (S1S8) were differentiated. Within the S1
cluster, nine B. avium isolates clustered at a linkage level of
46 %. Two clusters S2 (three isolates of B. hinzii, nos 10980,
16211 and 14052) and S3 (B. trematum 14993 and 5894)
clustered together at a linkage level of 20 %. S6 (three isolates
of B. trematum nos 15543, 16877 and 16652 and B. hinzii no.
13494) and S5 (all B. holmesii isolates in the study) clustered
at linkage levels of 69 % and 78 %, respectively. S8 contained
B. pertussis strains that were highly related to each other and
clustered at a linkage level of 59 %. Within S8 also, isolates of

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339

A. Gzyl and others


Similarity (%)
25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Species

Strain
No.

Cluster

B.avium

1855

E1

B.avium

10973

E1

B.avium

3557

E1

B.avium

1854

E1

B.avium

3556

E1

B.avium

1851

E1

B.avium

3522

E1

B.avium

1856

E1

B.avium

3549

E1

B.trematum

5894

E2

B.trematum

16877

E2

B.trematum

16652

E2

B.trematum

15543

E2

B.trematum

14993

E2

B.holmesii

15047

E3

B.holmesii

15945

E3

B.holmesii

104394

E3

B.holmesii

15946

E3

B.holmesii

104395

E3

B.hinzii

13494

E4

B.hinzii

16211

E4

B.hinzii

10980

E4

B.hinzii

14052

E4

B.bronchiseptica

3540

E5a

B.bronchiseptica

3543

E5a

B.bronchiseptica

3537

E5a

B.bronchiseptica

1033

E5b

B.bronchiseptica

107597

E5b

B.bronchiseptica

3525

E5b

B.bronchiseptica

107599

E5b

B.bronchiseptica

1943

E5b

B.parapertussis

1820

E5b

B.parapertussis

1821

E5b

B.bronchiseptica

3533

E5b

B.parapertussis

1823

E5b

B.parapertussis

1829

E5b

B.bronchiseptica

3534

E5b

B.parapertussis

1816

E5b

B.bronchiseptica

3536

E5b

B.bronchiseptica

3530

E5b

B.bronchiseptica

3538

E5b

B.pertussis

7097/74

E5c

B.pertussis

1253/73

E5c

B.pertussis

2066/95

E5c

B.pertussis

2896

E5c

B.pertussis

5375

E5c

B.pertussis

3/60

E5c

B.pertussis

7/60

E5c

B.pertussis

6901/73

E5c

B.pertussis

60623/67

E5c

B.pertussis

3835/74

E5c

B.pertussis

W 28

E5c

B.pertussis

3599/97

E5c

B.pertussis

785/00

E5c

B.pertussis

6770/73

E5c

B.pertussis

18530

E5c

B.pertussis

324

E5c

B.pertussis

6/62

E5c

B.pertussis

6635/73

E5c

B.pertussis

253/67

E5c

B.pertussis

353

E5c

B.pertussis

2/68

E5c

B.pertussis

A/63

E5c

B.pertussis

45/68

E5c

B.pertussis

2/62

E5c

B.pertussis

Tohama I

E5c

B.pertussis

9/68

E5c

B.pertussis

12/60

E5c

B.pertussis

1468/00

E5c

B.pertussis

2214/95

E5c

B.pertussis

2245/95

E5c

B.pertussis

1038/99

E5c

B.pertussis

2955/74

E5c

B.pertussis

871/00

E5c

B.pertussis

2632/00

E5c

B.pertussis

18323

E5c

B.parapertussis

762/00

E5d

B.parapertussis

46/00

E5d

B.parapertussis

1251/70

E5d

B.parapertussis

1356/75

E5d

B.petrii

4/03

E6

Fig. 1. Genotypic relationships among AFLP patterns obtained with EcoRI/MseI set for strains of Bordetella genus. The dendrogram
was generated with similarity matrices calculated from the Pearson product-moment correlation coefficient and the UPGMA
algorithm.
340

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Journal of Medical Microbiology 54

AFLP in genotyping of isolates of Bordetella genus


Similarity (%)
5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95 100

Species

Strain
No.

Cluster

B.bronchiseptica

3538

S8a

B.pertussis

3599/97

S8a

B.pertussis

3835/74

S8

B.pertussis

6635/73

S8

B.pertussis

6901/73

S8

B.pertussis

3/60

S8

B.pertussis

2/62

S8

B.pertussis

6770/73

S8

B.pertussis

2632/00

S8

B.pertussis

2214/95

S8

B.pertussis

1468/00

S8

B.pertussis

9/68

S8

B.pertussis

2/68

S8

B.pertussis

W 28

S8

B.pertussis

2955/74

S8

B.pertussis

12/60

S8

B.pertussis

7/60

S8

B.pertussis

7097/74

S8

B.pertussis

353

S8

B.pertussis

253/67

S8

B.pertussis

2066/95

S8

B.pertussis

1253/73

S8

B.pertussis

A/63

S8

B.pertussis

785/00

S8

B.pertussis

18530

S8

B.pertussis

2245/95

S8

B.pertussis

2896

S8

B.pertussis

45/68

S8

B.pertussis

1038/99

S8

B.pertussis

6/62

S8

B.pertussis

Tohama I

S8

B.pertussis

18323

S8

B.pertussis

871/00

S8

B.pertussis

5375

S8

B.pertussis

324

S8

B.pertussis

60623/67

S8

B.petrii

4/03

S7

B.trematum

15543

S6

B.trematum

16877

S6

B.trematum

16652

S6

B.hinzii

13494

S6

B.holmesii

15047

S5

B.holmesii

15946

S5

B.holmesii

15945

S5

B.holmesii

104394

S5

B.holmesii

104395

S5

B.bronchiseptica

107597

S4a

B.bronchiseptica

107599

S4a

B.bronchiseptica

1033

S4a

B.bronchiseptica

3525

S4a

B.bronchiseptica

3537

S4a

B.bronchiseptica

3533

S4a

B.bronchiseptica

3530

S4a

B.bronchiseptica

3534

S4b

B.bronchiseptica

3540

S4b

B.parapertussis

1251/70

S4b

B.parapertussis

1356/75

S4b

B.parapertussis

1829

S4b

B.parapertussis

1821

S4b

B.parapertussis

1823

S4b

B.parapertussis

1816

S4b

B.parapertussis

1820

S4b

B.parapertussis

762/00

S4b

B.parapertussis

46/00

S4b

B.bronchiseptica

1943

S4c

B.bronchiseptica

3543

S4c

B.bronchiseptica

3536

S4c

B.trematum

14993

S4c

B.trematum

5894

S4c

B.hinzii

10980

S2

B.hinzii

16211

S2

B.hinzii

14052

S2

B.avium

3557

S1

B.avium

3522

S1

B.avium

1855

S1

B.avium

10973

S1

B.avium

1851

S1

B.avium

1854

S1

B.avium

3549

S1

B.avium

3556

S1

B.avium

1856

S1

Fig. 2. Genotypic relationships among AFLP patterns obtained with SpeI/ApaI set for strains of Bordetella genus. The dendrogram
was generated with similarity matrices calculated from the Pearson product-moment correlation coefficient and the UPGMA
algorithm
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341

A. Gzyl and others

B. bronchiseptica (no. 3538) and B. pertussis (no. 3599/97)


were classified as clade S8a and clustered with the B. pertussis
clade at a linkage level of of 32 %. B. petrii was classified as a
single type pattern (S7) related to the S8 cluster. Cluster S4,
with the overall similarity value of 35 %, formed three
subclusters: S4a, seven isolates of B. bronchiseptica; S4b, nine
B. parapertussis isolates and two B. bronchiseptica, nos 3540
and 3534; and S4c, B. bronchiseptica nos 1943, 3543 and 3536.
Within the SpeI/ApaI AFLP set, 16, 10 and six different
patterns for the 35, 13 and nine isolates of B. pertussis, B.
bronchiseptica and B. parapertussis were found, respectively.
For B. trematum, B. hinzii, B. holmesii and B. avium, five,
four, four and eight patterns were identified, respectively.
DNA isolates obtained from seven subcultures of two different strains repeated over time were used to compare specific
pattern reproducibility. The band patterns did not change
after seven passages in any of the strains tested (data not
shown). Similarly, differing quantities of DNA in the range of
100500 ng did not induce band variation in the AFLP
profile of a single strain.

DISCUSSION
In this study, we describe AFLP as a new tool for generating
the detailed genotyping data of isolates belonging to the
Bordetella genus. The purpose of the study was to evaluate the
discrimination level of intra- and inter-species fingerprinting
by AFLP/RAPD methods for use in epidemiology and in
searching for real associations between Bordetella species in
terms of previously described genetic relationships. For
RAPD fingerprinting we have chosen two primers previously
found by Yuk et al. (1998) to be useful in the study of
genotypes of B. parapertussis. From six previously described
(Grady et al., 1999; Huys et al., 1996; Kokotovic et al., 1999;
McLauchlin et al., 2000; van Elderle et al., 1999; Vos &
Kuiper, 1998), and five newly designed and applied AFLP
protocols, two were chosen to test discrimination ability of
representative isolates of the Bordetella genus.
In order to overcome the liability of the RAPD results
according to the procedures and reagents used (Heersma et
al., 2001; Olive & Bean, 1999) and to achieve higher levels of
AFLP reproducibility, a standardized methodology, including commercially available kits for DNA isolation and gels,
was applied. General approaches for reliable and accurate
intra- and inter-gel comparison of banding patterns were
included (Desai et al., 1998; Janssen, 2001; Savelkoul et al.,
1999). The reproducibility was tested by generating multiple
subcultures on which DNA extraction, RAPD and AFLP
procedures were performed.
Intra-/inter-gel reproducibility found identity levels for the
RAPD and AFLP protocols as 94/90 % and 97/91 %, respectively. The overall genetic similarities, defined by the Pearson
product-moment correlation coefficient, were 53.9 % and
64 % for RAPD using primers 1254 and 1247, respectively,
and 23.6 % and 4.4 % for AFLP with EcoRI/MseI and SpeI/
ApaI protocols, respectively. Although RAPD can provide
342

analysis data very rapidly, the discriminatory power in


comparison to AFLP, as predicted, was much lower.
Although the limitations of the RAPD method are wellknown, it seems that it has the potential to be a quick tool for
intra-species differentiation of some species of Bordetella.
For B. bronchiseptica, RAPD was found to be highly accurate
in tracking differences among isolates coming from different
mammalian species; thus in this case it might well provide an
additional tool to study genetic diversity. In addition, RAPD
can also be useful for confirming B. avium, B. parapertussis
and B. holmesii identification.
Previously, AFLP has been found to be useful for both
identifying and typing many micro-organisms after defining
windows of similarity (Savelkoul et al., 1999). The definition
of the similarity value ranges in AFLP genotyping was found
to be useful in the taxonomic studies of for example
Klebsiella, Mycoplasma, Acinetobacter, Aeromonas and
Xanthomonas species (Huys et al., 1996, Kokotovic et al.,
1999). Moreover, in some cases, the discriminatory power
has been evaluated as equal to or higher than that obtained
with the gold standard method of PFGE (van Elderle et al.,
1999), and congruent with similar grouping through multilocus sequence typing (MLST; Schouls et al., 2003). AFLP has
been widely accepted as an excellent genotyping tool because
of its typability and discriminatory power, and good reproducibility; likewise, it is easy to perform and to draw
interpretations from (van Belkum et al., 2001).
AFLP fingerprints of the isolates of the Bordetella genus, as
revealed by cluster analysis, have retained attributes of
species specificity, leading to the conclusion that the method
may be used as an additional tool for species-specific
identification. In our study, four B. parapertussis isolates
from Poland were first incorrectly identified as B. pertussis in
the diagnostic laboratory. The AFLP technique clustered
them not within the highly related group of B. pertussis but
within the B. bronchiseptica cluster. By applying PCR (van
der Zee et al., 1996a), their identification as B. parapertussis
was confirmed. Additionally, because of high reproducibility
and easy performance AFLP has value in epidemiological
studies of the Bordetella. Slightly different classifications of
Bordetella isolates in the two AFLP sets screened might result
from the different levels of polymorphism detected. Greater
levels of discrimination of fingerprinting in the case of AFLP
with the SpeI/ApaI protocol might allow for a more in-depth
examination of polymorphism compared to AFLP with the
EcoRI/MseI set.
The AFLP set with the EcoRI/MseI protocol seems to be a
potential alternative for studying genetic relationships among
Bordetella species. The DNADNA hybridization of nucleotide sequences of the pertussis toxin operon and MLEE found
B. pertussis, B. parapertussis and B. bronchiseptica to be highly
related and with limited diversity (Arico et al., 1987; Khattak &
Matthews, 1993; Muller & Hildebrandt, 1993; Musser et al.,
1986; van der Zee et al., 1997). Genetic differences within the
B. bronchiseptica cluster were suggested to be difficult to
detect because the mechanism of regulation of expression

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Journal of Medical Microbiology 54

AFLP in genotyping of isolates of Bordetella genus

seems to be species-specific. Different strategies in regulating


gene expression have been suggested to be more important for
the adaptation of particular lineages of the B. bronchiseptica
cluster to the particular host than horizontal gene transfer
(Gerlach et al., 2001). On the other hand, adaptation to
different hosts has been suggested to be a force capable of
inducing differences, which could be observed by some tools
(Gerlach et al., 2001). Insertion sequence (IS)-dependent
rearrangements responsible for different genome organization and size were assumed to be the most important changes
possible to observe (Cummings et al., 2004). As for the B.
bronchiseptica cluster and other Bordetella isolates, the
genetic differences generated could be observed through
AFLP and the method can be considered as an additional tool
to study their relationships.
The different extents of intra-species polymorphism detected
seem to confirm the varying degrees of genetic diversity
among analysed species (Kokotovic et al., 1999). For B.
pertussis, substantial homogeneity was found both in RAPD
and AFLP genotyping compared with B. bronchiseptica. As
such, in MLEE in the case of B. bronchiseptica as many as 38
polymorphic types have been identified in comparison to the
four to five found for B. pertussis and B. parapertussis (van der
Zee et al., 1997). Higher genetic variation seen in B.
bronchiseptica versus B. pertussis and B. parapertussis has also
been found in other studies (Boursaux-Eude & Guiso, 2000;
van der Zee et al., 1996b).
The AFLP fingerprinting data are in agreement with previously published data on the close relationship of the B.
bronchiseptica cluster, since B. pertussis, B. parapertussis and
B. bronchiseptica were strictly classified as a single branch
during the clusterization process. Moreover, fingerprints of
B. parapertussis and B. bronchiseptica isolates were mixed
together in the clade, unlike the highly related group of B.
pertussis isolates. Comparative analyses of the genome
sequences revealed that B. parapertussis and B. bronchiseptica
are more similar in overall genome organization than B.
bronchiseptica and B. pertussis (Cummings et al., 2004;
Parkhill et al., 2003). Phenotype analysis by in vitro and in
vivo assays also has shown that B. parapertussis is more
similar to B. bronchiseptica than to B. pertussis, thus the
phenotype is related to the genetic species-specific differences (Heininger et al., 2002).
B. pertussis isolates were found to be hardly differentiated by
RAPD and AFLP using the EcoRI/MseI protocol compared
with AFLP using the SpeI/ApaI protocol. MLEE, PFGE and
IS-RFLP studies have shown the population of B. pertussis to
be homogeneous or clonal, resulting most probably from
high adaptation to humans and limited opportunity for
horizontal genetic exchange (Musser et al., 1986; van Loo et
al., 1999; van der Zee et al., 1996a). IS sequences are
considered to be reasons for genome plasticity of B. pertussis
coupled with high-genetic-relatedness (Gerlach et al., 2001;
Stibitz & Yang, 1997).
PFGE as a gold standard has been successfully used to
study B. pertussis challenges with time, identify outbreakhttp://jmm.sgmjournals.org

associated isolates, monitor transmission and evaluate genetic


relatedness (Hardwick et al., 2002a; Khattak et al., 1992; Mooi
et al., 2000; Peppler et al., 2003; Weber et al., 2001). For strains
belonging to the B. bronchiseptica cluster, using PFGE it was
possible to differentiate isolates between and within species
(Khattak & Matthews, 1993). Moreover, it has been successfully applied for differentiation of isolates of B. parapertussis,
B. bronchiseptica and B. holmesii (Hardwick et al., 2002a,
2002b; Khattak etal., 1992; Makinen et al.,2003; Mastrantonio
et al., 1998; Mazengia et al., 2000; Peppler et al., 2003; Weber et
al., 2001). Although PFGE is the most reproducible method, it
is time-consuming and labour-intensive. MLST studies (van
Loo et al., 2002), although highly useful for tracing the
movements of B. pertussis strains within and between individuals and communities, need sophistically equipped
laboratories and highly experienced personnel. We postulate
that AFLP using the SpeI/ApaI protocol may be a new, rapid
and reproducible option for the genotyping of B. pertussis.
The epidemiology of B. bronchiseptica is still not well enough
elucidated, especially in terms of the possibility of host interspecies transmission. Some studies have documented cospecies transmission between dogs and cats (Binns et al.,
1998; Dawson et al., 2000; Foley et al., 2002), from rabbits to
humans (Gueirard et al., 1995) and from rats/cats to pigs
(Switzer et al., 1966). Available genotyping tools involve
restriction enzyme analysis (REA) of chromosomal DNA,
ribotyping, PFGE and RAPD (described for canine isolates)
(Keil & Fenwick, 1999; Sacco et al., 2000b). Genetic diversity
of B. bronchiseptica isolates from 12 different host animals
measured by REA was considerable in the study by Sacco et
al. (2000b), with similarity ranges of 6897 % for HinfI and
4696 % for AluI. Thus AFLP using SpeI/ApeI might present
a better alternative because of its higher discrimination level
(30.1 %). In our study, we found similar heterogeneity of B.
bronchiseptica strains isolated from different host animals
using the AFLP SpeI/ApaI protocol and even using as simple
an approach as RAPD. Further studies are expected to show a
degree of variation detected by AFLP in a higher number of B.
bronchiseptica strains exclusively isolated from particular
hosts, e.g. swine and dogs. In addition, AFLP SpeI/ApaI
fingerprinting might be a new option to the previously
described alternatives to follow the genotype trends for
vaccine and clinical strains in the population of B. bronchiseptica in dogs, similarly as it is possible for B. pertussis in
humans (Keil & Fenwick, 1999; Mooi et al., 1998, 1999; van
der Zee et al., 1996a; Fry et al., 2001; Gzyl et al., 2001).
Among nine B. parapertussis isolates, four and six different
patterns in the AFLP EcoRI/MseI and SpeI/ApaI sets were
observed, respectively, proving its high capability for differentiation. A higher number of B. parapertussis isolates is
expected to be studied and compared with AFLP results with
previously described data on limited genetic variability
(Heininger et al., 2002; Khattak & Matthews, 1993; Makinen
et al., 2003; Mastrantonio et al., 1998; Porter et al., 1996).
For 32 B. holmesii isolates recovered from nasopharyngeal
specimens up to five pulsofield types were identified, which

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343

A. Gzyl and others

supports restricted diversity of this species (Mazengia et al.,


2000). In our study, although only five isolates of B. holmesii
were available for testing, four different patterns in both
AFLP protocols were observed.

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ACKNOWLEDGEMENTS
This work was partly supported by a grant from the State Committee for
Scientific Research (2PO5D04726). We would like to express our
gratitude to Miss Marta Balinska for help with English version of the
manuscript and to Ms Barbara Husejnow for her excellent technical
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