Journal of Medical Microbiology (2007), 56, 323328

DOI 10.1099/jmm.0.46647-0

Quantitative detection of Aeromonas salmonicida

in fish tissue by real-time PCR using
self-quenched, fluorogenic primers
Jose Luis Balcazar, Daniel Vendrell, Ignacio de Blas, Imanol Ruiz-Zarzuela,
Olivia Girones and Jose Luis Muzquiz
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013
Zaragoza, Spain

Correspondence
Jose Luis Balcazar
balcazar@unizar.es

Received 27 March 2006

Accepted 6 November 2006

In this study a real-time PCR assay using self-quenched primers labelled with a single fluorophore
for the detection of Aeromonas salmonicida was developed. Probe specificity was confirmed
by amplification of 16 A. salmonicida strain templates and by the lack of a PCR product with 26
non-A. salmonicida strains. With a pure culture of A. salmonicida, the assay was linear over a
range of 0.5 pg to 50 ng and was able to detect 16 c.f.u. per reaction. A similar sensitivity was
observed in DNA extracted from a mixture of A. salmonicida and fish tissue. Results using artificially
inoculated tissues and diseased fish from outbreaks indicated that the assay can provide
sensitive species-specific detection and quantification of A. salmonicida in fish tissue.

INTRODUCTION
Aeromonas salmonicida is the aetiological agent of furunculosis, a disease that affects many species of fish and is a
significant cause of economic losses in the aquaculture of
trout and salmon (McCarthy & Roberts, 1980; OBrien et al.,
1994). The disease is characterized by the presence of general
septicaemia, lack of appetite, lethargy and haemorrhagic and
necrotic lesions in gills, gut and muscle (Hiney & Olivier,
1999). This bacterium has also been associated with ulcer
disease in carp and marine flatfish and erythrodermatitis in
carp (Austin & Austin, 1999).
Methods currently used to identify A. salmonicida within
infected fish can be time-consuming, labour-intensive and
hard to implement because of difficulties in distinguishing
the bacterium from other bacterial species given the wide
variety of existing biochemical profiles, the absence of an
efficient selective medium and the poor plating efficiency of
the bacterium in mixed cultures. For this reason, attention
has focused on the application of different serological
techniques (Adams & Thompson, 1990; Gilroy & Smith,
2003), and DNA sequence-based methods employing PCR
have been developed (Gustafson et al., 1992; Hiney et al.,
1992; Miyata et al., 1996).
Real-time PCR technology may be able to provide an
innovative method of detecting and enumerating several
Abbreviations: Ct, cycle threshold; FAM, 6-carboxyfluorescein; LUX,
Light Upon eXtension.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
gene sequences of the A. salmonicida strains determined in this study
are AM296501AM296510.

46647 G 2007 SGM

Printed in Great Britain

organisms in different environments (Sharkey et al., 2004).

With this method, PCR product accumulation is continuously monitored during cycle progression by means of
fluorescent detection. In addition, this technique is characterized by a large dynamic range of quantification,
sensitivity and throughput capacity and requires no postamplification manipulation, thus avoiding possible carryover contamination.
A fluorescence-based PCR technique that uses a fluorogenic
primer labelled with a single fluorophore was recently
developed as a cost-effective alternative to other fluorescence-based PCR techniques (Nazarenko et al., 2002a, b).
Light Upon eXtension (LUX) primers (Invitrogen) are
designed with the fluorophore attached near the 39 end and a
short complementary sequence at the 59 end that creates a
hairpin structure. This structure effectively quenches the
fluorophore so that a separate quenching moiety is not
required. When the primer is incorporated into a doublestranded PCR product and extended by DNA polymerase,
the fluorophore is dequenched, resulting in a significant
increase in fluorescent signal (Lowe et al., 2003).
The most frequent target of species-specific A. salmonicida
DNA probes and PCR assays is a 6.4 kb cryptic plasmid
known to occur in 90 % of A. salmonicida subsp. salmonicida
(Hiney et al., 1992; Srum et al., 1993; OBrien et al.,
1994; Mooney et al., 1995; Byers et al., 2002). This locus
is also found in all A. salmonicida subsp. achromogenes
strains tested and some A. salmonicida subsp. masoucida
strains. No reaction has been found with over 60 related
aeromonads and aquatic bacteria tested to date (Mooney
et al., 1995).
323

J. L. Balcazar and others

The present study employed this locus to develop a quantitative real-time PCR assay using fluorogenic primers. Assay
sensitivity and specificity were examined in pure or mixed
cultures, and enumeration of A. salmonicida by real-time
PCR in tissue samples from rainbow trout (Oncorhynchus
mykiss) was carried out.

METHODS
Bacterial strains and culture conditions. A total of 40 bacterial

strains comprising A. salmonicida (n=16) and non-A. salmonicida

strains (n=26) was employed to test the specificity of the PCR assay
(Table 1). Strains were obtained from the following sources: ATCC
(American Type Culture Collection, Manassas, VA, USA), NCIMB
(National Collections of Industrial and Marine Bacteria, Aberdeen,
UK) and LMG (Laboratory for Microbiology, Ghent University,
Belgium), as indicated in Table 1; strains CLFP 501505, isolated
from rainbow trout during a furunculosis outbreak at fish farms,
from the Autonomous Community of Aragon, Spain; strains 2320,
793 and 4814 from Dr D. Colquhoun (Department for Fish Health,
National Veterinary Institute, Oslo, Norway); strains 265/87,
MT-004, F-661-2/89, Fin 3, 870626-1/1C, S-228/91, S-123-91-1, Vib
293, Vib 305, Vib 281 and M 147/92 from Dr B. K. Gudmundsdottir
(Institute for Experimental Pathology, University of Iceland,
Reykjavk, Iceland); strains 146 and 147 from Dr Jose Guijarro
(Department of Functional Biology, University of Oviedo, Spain);
strains CECT 4342, CECT 4224, CECT 4245, CECT 4227, CECT
4257, CECT 4199, CECT 5761 and CECT 4237 from Dr Mara Jose
Figueras (Biology and Microbiology Research Unit, Rovira and
Virgili University, Spain); and strains Rs 146 and Rs 147 from Dr
Eva Jansson (Department of Wild Life, Fish and Environment,
National Veterinary Institute, Sweden). All strains were grown in
brain heart infusion broth (Scharlau Chemie) overnight at 22 uC,
with the exception of Flavobacterium psychrophilum, which was
grown in AnackelOrdal broth (0.5 % tryptone, 0.05 % yeast extract,
0.02 % sodium acetate, 0.02 % beef extract, adjusted to pH 7.4) for
2 days at 15 uC with agitation, and Renibacterium salmoninarun,
which was grown in modified KDM2 broth (1 % bactopeptone,
0.05 % yeast extract, 0.05 % L-cysteine, adjusted to pH 6.5) for
6 days at 15 uC with agitation.
16S rRNA gene sequencing. In order to ensure that strains were
A. salmonicida, the 16S rRNA gene sequence was PCR amplified in a
Perkin-Elmer GeneAmp PCR System 2400 thermocycler, as
described by Suzuki et al. (1996). The PCR products were sequenced
directly using the prokaryotic 16S rRNA gene universal primers 27F
(59-AGAGTTTGATCCTGGCTCAG-39) and 1492R (59-GGTTACCTTGTTACGACTT-39) on a MegaBACE 500 sequencer following the
manufacturers protocols (Amersham Biosciences). Sequences were
corrected manually and aligned with 16S rRNA gene sequences
obtained from the GenBank database using the BLAST algorithm
(Altschul et al., 1990). Subsequent multiple alignments were carried
out using CLUSTAL W (Thompson et al., 1994).
DNA extractions. Bacterial cultures (1.0 ml) were homogenized in
200 ml TE buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA], centri-

fuged at 12 000 g for 1 min and pellets were extracted using

InstaGene Matrix (Bio-Rad) following the manufacturers instructions. DNA yield and purity were determined spectrophotometrically
by measuring A260 : A280 ratios (Gene Quant pro RNADNA calculator; Amersham Pharmacia Biotech). Concentrated DNA was stored
at 220 uC.
Fluorogenic real-time PCR. Oligonucleotide sequences were

derived from a specific DNA probe for A. salmonicida (GenBank

accession no. X64214). The probe is specific for a region of the
324

Table 1. Bacterial strains detected by real-time PCR

Isolate

Detection

A. salmonicida subsp. salmonicida CLFP 501

A. salmonicida subsp. salmonicida CLFP 502
A. salmonicida subsp. salmonicida CLFP 503
A. salmonicida subsp. salmonicida CLFP 504
A. salmonicida subsp. salmonicida CLFP 505
A. salmonicida subsp. salmonicida 793
A. salmonicida subsp. salmonicida MT-004
A. salmonicida subsp. salmonicida NCIMB 1102
A. salmonicida subsp. salmonicida 4814
A. salmonicida subsp. achromogenes 2320
A. salmonicida subsp. achromogenes NCIMB 1110
A. salmonicida subsp. achromogenes 265/87
A. salmonicida subsp. achromogenes F-661-2/89
A. salmonicida subsp. achromogenes 870626-1/1C
A. salmonicida subsp. masoucida Fin 3
A. salmonicida subsp. masoucida NCIMB 2020
Aeromonas encheleia CECT 4342
Aeromonas eucrenophila CECT 4224
Aeromonas sobria CECT 4245
Aeromonas bestiarum CECT 4227
Aeromonas veronii CECT 4257
Aeromonas allosaccharophila CECT 4199
Aeromonas culicicola CECT 5761
Aeromonas media CECT 4237
Aeromonas hydrophila S-123-91-1
Aeromonas hydrophila S-228/91
Vibrio tubiashii LMG 10936
Vibrio fluvialis NCIMB 2249
Vibrio splendidus NCIMB 2251
Vibrio furnissii Vib 293
Vibrio pelagius Vib 305
Vibrio aestuarianus Vib 281
Vibrio anguillarum ATCC 19264
Moritella viscosa M 147/92
Photobacterium phosphoreum NCIMB 844
Yersinia ruckeri ATCC 29473
Y. ruckeri 146
Y. ruckeri 147
Flavobacterium psychrophilum NCIMB 1947
F. psychrophilum NCIMB 1826
Renibacterium salmoninarum Rs 146
R. salmoninarum Rs 147

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2

+, Fluorescent signal detected after 1540 cycles; 2, no fluorescent

signal detected after 40 cycles.

DNA fragment that has been used previously as a species-specific

genetic probe (Hiney et al., 1992). LUX designer software (www.
invitrogen.com/lux) was used to design the LUX fluorogenic primers
[59-CGGAACGTAATCTGAATTGTTCTTTTC(FAM)G-39] and (59ATTGCTTATCGAGGCAGCCAAC-39).
For this assay, each 50 ml PCR contained 25 ml Platinum Quantitative
PCR SuperMix-UDG (uracil DNA glycosylase) [60 U Platinum Taq
DNA polymerase ml21, 40 mM Tris/HCl (pH 8.4), 100 mM KCl,
Journal of Medical Microbiology 56

Real-time PCR detection of Aeromonas salmonicida

6 mM MgCl2, 400 mM each dGTP, dATP and dCTP, 800 mM dUTP,
40 U UDG ml21 and stabilizers], FAM-labelled LUX primer and
corresponding unlabelled primer (200 nM each final concentration),
1 ml ROX reference dye (Invitrogen) and 10 ml template. An ABI
PRISM 7000 was programmed as follows: 50 uC for 2 min and
denaturation at 95 uC for 2 min, followed by 40 cycles of 95 uC for 15 s,
55 uC for 30 s and 72 uC for 30 s. Data were analysed using ABI PRISM
7000 sequence detection software, version 1.2.3 (Applied Biosystems).
The cycle threshold (Ct) was calculated as the cycle number at which
the reaction became exponential. The Ct of each sample was then
compared with a standard curve and the result was expressed as
absolute amounts of genomic DNA. Standard curves for quantification
were plotted from triplicate samples by using Ct values of tenfold
dilutions of template extracted from 86106 c.f.u. A. salmonicida
NCIMB 1102 ml21. To confirm the results of the real-time PCR, 10 ml
aliquots of PCR products were analysed by electrophoresis on ethidium
bromide-stained 2 % agarose gels (Bio-Rad).
Sensitivity and specificity of the real-time PCR assay. The

specificity of the real-time PCR was determined by comparing PCR

products derived from A. salmonicida (n=16) and non-A. salmonicida strain (n=26) DNA templates. For sensitivity assays, DNA was
extracted from pure cultures of A. salmonicida NCIMB 1102
(86106 c.f.u. ml21) to generate a standard curve used for enumeration of unknown samples.
Detection of seed A. salmonicida in fish tissue homogenate.

Tissue samples (250 mg kidney, liver, spleen or intestine) from rainbow trout were homogenized in a 1 ml suspension of early stationary phase A. salmonicida NCIMB 1102 cells diluted in deionized
water to a final concentration of 32, 320 and 3200 c.f.u. (g tissue)21.
Homogenates were centrifuged at 12 000 g for 1 min and DNA was
extracted using InstaGene matrix following the manufacturers
instructions. Enumeration of A. salmonicida by real-time PCR was
compared with plate count values. Statistical analysis was based on
Students t-test using paired and equal variance of log c.f.u. g21. All
statistics were performed using SPSS for Windows version 11.5
(SPSS).
Detection of A. salmonicida in naturally infected fish. Tissue

samples (250 mg) of diseased fish associated with outbreaks on different fish farms were also tested. Samples were homogenized in
1 ml deionized water. Homogenates were centrifuged at 12 000 g for
1 min and DNA was extracted as described above.

RESULTS AND DISCUSSION

The fluorogenic primer method has several advantages over
other methods, including ease of design and synthesis of the
primers. The design of these primers is based on studies that
demonstrate the effects of the primary and secondary
structure of oligonucleotides on the emission properties of
a conjugated fluorophore (Nazarenko et al., 2002b). The
primers are chemically synthesized oligonucleotides
(2229 nt) with a fluorophore attached to the C5 position
of thymidine, which increases its fluorescence when incorporated into a double-stranded PCR product. This phenomenon results from (i) having the fluorophore close to the
39 end of an oligonucleotide with G or C at the 39 end, (ii)
the existence of a G within a few bases of the label and (iii)
the ability of the oligonucleotide to form a blunt-ended
hairpin at temperatures close to the annealing temperature
of the primer (Nazarenko et al., 2002a).
http://jmm.sgmjournals.org

Primer design
LUX fluorogenic primers were designed from a specific
DNA probe for A. salmonicida (Hiney et al., 1992), as 16S
rRNA gene sequence analysis did not provide sufficient
information for the delineation and identification of most
species of the genera Aeromonas. It has been reported that
A. salmonicida and Aeromonas bestiarum type strains
differ by only two nucleotides, at positions 1011 and 1018
(Martnez-Murcia et al., 2005).
A computer search using tBLASTX (Altschul et al., 1997) for
the matching sequences in GenBank including updates
against the DNA probe for A. salmonicida (GenBank
accession no. X64214) as a query sequence revealed more
than 97 % similarity with plasmids from A. salmonicida
subspecies listed in the NCBI genome database. The tBLASTX
search also found that the DNA probe for A. salmonicida
shared 47 % identity with the YopP gene from Yersinia
enterocolitica (GenBank accession no. AF023202), which
induces apoptosis in mouse macrophages (Mills et al.,
1997). Because of the similarity between the DNA probe for
A. salmonicida and the YopP gene from Y. enterocolitica, the
extreme 59-end region of the sequence was targeted, as this is
where the most sequence variation occurs between the two.
Specificity of detection
To improve the diagnosis capabilities for this bacterial
pathogen, a real-time PCR assay was developed. Species
specificity was confirmed by positive amplification of all 16
isolates of A. salmonicida and lack of a product from ten
other strains of Aeromonas species (Aeromonas encheleia,
Aeromonas eucrenophila, Aeromonas sobria, A. bestiarum,
Aeromonas veronii, Aeromonas allosaccharophila, Aeromonas
culicicola, Aeromonas media and Aeromonas hydrophila) and
16 strains of common bacterial fish pathogens (Flavobacterium psychrophilum, Moritella viscosa, Photobacterium phosphoreum, Renibacterium salmoninarum, Vibrio tubiashii,
Vibrio fluvialis, Vibrio splendidus, Vibrio furnissii, Vibrio
pelagius, Vibrio aestuarianus, Vibrio anguillarum and
Yersinia ruckeri) (Table 1). The size of the amplified PCR
product (131 bp) was verified by gel electrophoresis (not
shown).
Byers et al. (2002) showed that primers PAAS1 and PAAS2,
previously designed from the same sequence used in the
present study (Hiney et al., 1992), did not produce any falsepositive reactions with 27 non-target bacterial DNA extracts
and these primers were considered to be specific for A.
salmonicida. However, the PAAS primer set correctly
identified only 285/308 isolates as A. salmonicida and was
therefore determined to have an in vitro sensitivity of 93 %.
The failure of the PAAS primer set to identify 100 % of the A.
salmonicida isolates appeared to be related to the primer
target site, which had previously been shown to occur on a
6.4 kb cryptic plasmid (Srum et al., 1993) and to be present
in approximately 90 % of A. salmonicida isolates (Mooney
et al., 1995). It is therefore reasonable to speculate that the
325

J. L. Balcazar and others

for extracting genomic DNA. Based on this approach, a

correlation was observed between Ct and c.f.u. ml21 of
the starting quantity of A. salmonicida NCIMB 1102 DNA
(Fig. 1).

36
32

Ct

28
24
20
16
12

1.9 2.9 3.9 4.9 5.9 6.9

Bacterial concn (log c.f.u. ml1)

Fig. 1. Standard curve for quantification, plotted from triplicate

samples using Ct values of tenfold dilutions of template
extracted from 86106 c.f.u. A. salmonicida NCIMB 1102 ml1.

specificity of the reaction could be related to the virulence of

the strains, as all A. salmonicida strains used in the present
study were isolated from disease outbreaks in O. mykiss,
Salmo salar, Salvelinus alpinus and Zoarces viviparus. In
addition, the probe sequence homology with the YopP gene
from Y. enterocolitica could confirm this hypothesis.
Sensitivity of detection
The detection sensitivity of the real-time PCR assay was
determined by testing triplicate sets of genomic DNA
prepared from serial dilutions (86101 to 86106 c.f.u.
ml21) of A. salmonicida NCIMB 1102. The results were
reported as Ct values versus log starting quantities of DNA.
Positive signals were found in all dilutions except those
where the DNA concentration was below 561024 ng ml21
(80 c.f.u. ml21). The Ct values were also plotted against
c.f.u. ml21 (86101 to 86106 c.f.u. ml21) in tenfold serial
dilutions of the A. salmonicida NCIMB 1102 culture used

The standard curve demonstrated a Ct value of around 15

for 50 ng DNA (log 6.9 c.f.u. ml21) with a slope close to
23.53, indicating good sensitivity. The high coefficient of
determination (R2=0.99) obtained in the assay showed a
strong correlation between template DNA concentrations
ranging from 0.5 pg to 50 ng and the DRn signal and
indicated that the assay is useful for quantitative measurements of A. salmonicida.
The real-time PCR detection limit for A. salmonicida DNA
derived from pure culture was 0.5 pg PCR mixture ml21, or
the equivalent of 80 c.f.u. (ml culture)21, which would
extrapolate to 16 c.f.u. per PCR assay, assuming 100 %
extraction efficiency.

Detection of A. salmonicida in fish tissues

The Ct values obtained from the analysis of tissue samples
were extrapolated to the corresponding previously calculated standard curve. The presence of fish tissue did not
affect real-time PCR detection of A. salmonicida NCIMB
1102, and the results of plate counts yielded a good
correlation (r=0.85) between the two assays (Table 2). On
the other hand, amplification products were not detected
when tissues were inoculated independently with 32 c.f.u.
(g culture)21.
In order to check the potential application of this PCR in
natural infections, some samples from natural outbreaks
were processed (Table 3). These preliminary results showed

Table 2. Quantification of A. salmonicida from inoculated tissues

Sample
Kidney
Kidney
Spleen
Spleen
Liver
Liver
Intestine
Intestine
All tissuesd

Inoculum
(log c.f.u. g1)

Plate count*
(log c.f.u. g1SD)

3.5
2.5
3.5
2.5
3.5
2.5
3.5
2.5
1.5

3.640.14
2.570.12
3.560.06
2.480.12
3.550.08
2.490.12
3.530.14
2.480.08
1.120.25

Real-time PCRD
[log c.f.u. g1SD (CtSD)]
3.530.06
2.660.04
3.300.07
2.320.11
3.260.08
2.440.07
3.300.11
2.230.06

(29.10.56)
(32.60.42)
(30.10.78)
(34.10.77)
(30.30.08)
(33.40.10)
(30.10.70)
(34.50.07)

ND

ND, No amplification detected.

*A. salmonicida levels were determined by plate counts on tryptic soy agar spread plates.
DReal-time PCR determination of A. salmonicida concentrations was based on the mean of duplicate
samples. Concentrations were derived from a standard curve using the mean of triplicate Ct values for serial
tenfold dilutions of DNA extracted from known concentrations of bacteria.
dIndividual samples of kidney, spleen, liver and intestine.

326

Journal of Medical Microbiology 56

Real-time PCR detection of Aeromonas salmonicida

Table 3. Quantification of A. salmonicida from naturally

infected tissues
Sample

Real-time PCR* [log c.f.u. g1SD (CtSD)]

Kidney
Kidney
Liver
Liver
Spleen
Spleen

3.600.17
4.020.16
3.700.14
3.900.16

(29.80.22)
(28.20.42)
(29.10.10)
(28.60.08)

ND

2.710.12 (33.50.18)

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ACKNOWLEDGEMENTS
This study was supported by a grant from the National Adviser Body
of Continental Cultures (JACUCON). J. L. B. was supported by a
fellowship from the Spanish International Cooperation Agency
(AECI). We wish to express our gratitude to B. K. Gudmundsdottir,
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