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Applied and Environmental Microbiology, August 1998, p. 3066-3069, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Minimally Invasive Detection of
Piscirickettsia salmonis in Cultivated Salmonids via
the PCR
Sergio
Marshall,1
Sekou
Heath,2
Vitalia
Henríquez,1 and
Cristián
Orrego2,*
Instituto de Biología, Universidad
Católica de Valparaíso, Valparaíso,
Chile,1 and
Conservation Genetics
Laboratory, Department of Biology, San Francisco State University,
San Francisco, California 941322
Received 22 September 1997/Accepted 8 May 1998
 |
ABSTRACT |
The attributes of the PCR allowed implementation of an assay for
specific detection of Piscirickettsia salmonis from a few microliters of fish serum. This opens the way to less invasive modes of
sampling for this microbial pathogen in salmonids.
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TEXT |
Piscirickettsia salmonis
is a novel intracellular pathogen that has been identified as the
causative agent of an aggressive infectious disease affecting salmonid
mariculture in Chile (7). Although this disease has also
been reported in Canada (6), Norway (17), and
Ireland (18), its pathogenesis in fish farms in Chile seems
to be more severe than its pathogenesis elsewhere. Moreover,
rickettsialike pathogens may be involved in epizootics affecting other
cultured fish species (8). Similar agents have been found in
diverse fish species, including tilapias (Tilapia spp.)
(2), blue-eyed plecostomus (Panaque suttoni)
(10), and dragonet (Callionymus lyra)
(4). The results of in vitro studies in which nonsalmonid
cell lines were infected by P. salmonis (1)
seem to support the idea that the potential for infectivity of the
agent is wider than previously thought. The salmonid immune system does
not appear to exhibit a strong humoral response against the pathogen
(11), which may partially explain the virulence of
P. salmonis and suggests that the outlook for a
classical prophylactic approach to prevent outbreaks of the disease is
poor. Unfortunately, at present the biology of P. salmonis is almost completely unknown, as are its reservoir (if
any) and mode of transmission. The techniques available for detection
of this pathogen involve either sacrificing diseased specimens for
analysis or postmortem characterization of tissues. Thus, at present it
is not possible to survey for the presence and prevalence of
P. salmonis in susceptible populations, which might
help researchers understand P. salmonis behavior and design strategies for disease prevention.
Salmonid rearing in Chile has been seriously threatened by the
appearance of the fastidious intracellular gram-negative organism P. salmonis. One of the conspicuous features of this
agent is that it expresses itself 4 to 6 weeks after the fish have been transferred to seawater, thus spoiling the long and costly process of
rearing that begins with fertilized eggs. There are currently no
procedures that alert workers to the presence of the disease other than
the symptoms in its terminal stages. Therefore, a nonlethal and
minimally invasive alternative for early detection of P. salmonis would be useful for prevention and prophylactic
strategies. We developed a nonlethal screening procedure to detect this
pathogen in minute samples of fish serum (volume, <5 µl) by using
the PCR. Mauel and colleagues (14) have described a
PCR-based assay that they used to study internal tissues from infected
fishes. We extended the PCR-based approach to detect the presence of
amplifiable P. salmonis DNA in fish that do not display
signs of disease. Also, we were interested in using PCR primers
flanking a region of the ribosomal operon that is more variable than
the region exploited earlier (14).
Specimens and DNA extraction.
Tissues collected in HN buffer
(20 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid], 500 mM NaCl, 5 mM EDTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride), as well as sera, were obtained in 1996 and 1997 from farmed coho salmon (Oncorhynchus kisutch) and
rainbow trout (Oncorhynchus mykiss) from southern Chile
(Chiloé Island in Region Ten) and were stored at 
15°C. Tissue
from noninfected rainbow trout was received from a freshwater farm in
central Chile (located in Region Five) with no history of P. salmonis infestation. Tilapia (Oreochromis niloticus)
DNA was obtained from fresh muscle tissue. Initial optimization of the
PCR with nucleic acids from fish and from P. salmonis
EM-90 (originally isolated from Atlantic salmon [Salmo
salar]) grown in chinook salmon (Oncorhynchus
tshawytscha) embryo cell line CHSE-214 (12) was
performed with DNA purified by a slight modification of a previously
described protocol (7). For larger surveys, amplifiable DNA
was prepared by the Chelex procedure (20-22) by using <0.5
mg of tissue or 5 to 20 µl of serum. Each biological sample was
immersed in 100 µl of Instagene (6% [wt/vol] Chelex 100; catalog
no. 732-6030; Bio-Rad) in a 1.5-ml microcentrifuge tube (G tube,
catalog no. 4030; BIO PLAS Inc.) that is resistant to lid opening by
internal vapor pressure; the mixture was vortexed briefly at the
maximum speed and then incubated at 100°C for 15 min. Once cooled,
the tube was vortexed briefly and centrifuged for 1 min at the maximum
speed (16,000 × g) with a model 5415C Eppendorf
centrifuge to collect the Chelex beads at the bottom of the tube. The
supernatant (30 µl) was transferred to another tube for permanent
storage at 15°C. All extractions included at least two negative
controls that did not receive a biological sample and were taken
through the extraction procedure exactly like the samples. DNAs from
previously isolated or bacterial type species were obtained by the
Chelex procedure by using single colonies grown on suitable solid agar
media.
Primer design, amplification, and sequencing.
Primers for the
PCR (Fig. 1) were designed based on
alignments of sequences of the internal transcribed spacer (ITS) and
the flanking 23S rRNA gene of the ribosomal operon. Sequences of
P. salmonis LF-89 (= ATCC VR 1361) (type strain)
(GenBank accession no. U36943), EM-90 (GenBank accession no.
U36944), NOR-92 (GenBank accession no. U36946), ATL-4-91 (GenBank
accession no. U36945), SLGO-94 (GenBank accession no. U62104), and C1-95 (GenBank accession no. U62103) and of Escherichia coli (GenBank accession no. AE000474) were aligned with the Sequencer 3.0 software (Gene Codes Corporation) and confirmed by visual examination.
Primers RTS1 (5'-TGATTTTATTGTTTAGTGAGAATGA-3'; F-223), RTS2 (5'-AAATAACCCTAAATTAATCAAGGA-3'; R-266), and
RTS4 (5'-ATGCACTTATTCACTTGATCATA-3'; R-459)
(Fig. 1) were located at highly conserved sections of the P. salmonis ITS (the base at the 3' end of each primer
is indicated by F or R and the position in the LF-89 reference
sequence; the direction of extension of each primer is indicated on the
basis of whether extension produces the same strand as the LF-89
sequence [F, forward] or the other strand [R, reverse]). The
expected lengths of amplification fragments obtained with the RTS1-RTS4
and RTS1-RTS2 primer pairs are 283 and 91 bp, respectively. The
fragment flanked by primers RTS1 and RTS4 covers three-fourths of the
P. salmonis ITS. Pairwise comparisons among the
P. salmonis isolates listed above for this span of the
ITS revealed levels of sequence difference ranging from 0 to 6.4%
(including insertions and deletions), and this region therefore appears
to be a promising region for monitoring genetic variation in
P. salmonis. Amplification of nucleic acids of all
bacterial species was possible with two universal eubacterial primers
directed toward the 16S rRNA gene, with primers 358f and 517r of the
E. coli 16S rRNA sequence as described by Murray et al.
(15).
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FIG. 1.
Map of the prokaryotic ribosomal operon and positions of
primer pairs RTS1-RTS2 and RTS1-RTS4 for amplification by PCR of the
P. salmonis ITS. The map is drawn to scale for the ITS
section based on the P. salmonis LF-89 ITS sequence
(the first 18 nucleotides at the 5' end of primer RTS4 are located at
the beginning of the 23S rRNA gene). The rRNA genes are drawn to a
different scale, and the lengths are based on the lengths of these
genes in the E. coli rRNA operon.
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Each PCR was conducted in a total volume of 12.5 µl under a thin film
of mineral oil by using GeneAmp 1× PCR buffer (Perkin-Elmer),
which
consists of 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl
2, 50 mM
KCl, and 0.001% (wt/vol) gelatin, plus each of the four
deoxynucleoside
triphosphates at a concentration of 0.2 mM, both
oligonucleotide
primers at a concentration of 0.5 µM, and 0.025 U of
AmpliTaq
DNA polymerase (Perkin-Elmer) per µl. Template DNA (5 to 25 ng)
was delivered in a volume of 1 µl. A hot start (
3)
with a mixture
containing both primers and the four
deoxynucleoside triphosphates
added after the other components of
the reaction mixture reached
85°C was used to impede subsequent
unwanted primer extension.
An initial denaturation step (2 min at
94°C) preceded 36 to 39
cycles with the RTS1-RTS2 and RTS1-RTS4
primer pairs consisting
of 94°C for 30 s, 50°C for 30 s,
and 72°C for 30 s, with a final
extension step at 72°C for 7 min. Products of the PCR were analyzed
by electrophoresis in agarose
gels (2.5% Seakem GTG; FMC) in 1×
TBE buffer (
19). The
gels were stained with ethidium bromide
(0.5 µg/ml), and the
fluorescent bands visible with a UV transilluminator
were photographed.
Double-stranded PCR products were purified
with carboxylated magnetic
beads (
5) prior to the cycle sequencing
reaction, which was
carried out with an ABI PRISM Ready Reaction
dye terminator cycle
sequencing kit by using one-half the volume
recommended (AmpliTaq DNA
Polymerase FS; protocol P/N 402078,
revision A; Perkin-Elmer).
Then for electrophoresis and sequence
display we used an ABI
PRISM model 377 DNA sequencer (Perkin-Elmer)
with DNA Sequence Analysis
software, version 2.1.2.
PCR detection of P. salmonis.
Amplification of DNA
from P. salmonis EM-90 with the RTS1-RTS2 and RTS1-RTS4
primer pairs led to products of the expected lengths (Fig.
2, lanes 3 and 7, respectively), and no
amplification was evident from equivalent amounts of templates from
tilapia (Fig. 2, lanes 5 and 9) and a healthy trout (Fig. 2, lanes 4 and 8). Verification of the authenticity of the amplicon obtained with
primers RTS1 and RTS4 came from the following two lines of evidence
that were consistent with the previously reported sequence for
P. salmonis EM-90: digestion of the product with
restriction enzyme HinfI led to three fragments of the
predicted lengths, and the sequence of the amplicon matched the EM-90
sequence at positions 225 to 428 (data not shown).
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FIG. 2.
Specificity of the PCR for the P. salmonis ITS with primer pairs RTS1-RTS2 (lanes 2 through 5) and
RTS1-RTS4 (lanes 6 through 9), as determined with DNA (5 ng) from
P. salmonis EM-90 (lanes 3 and 7), rainbow trout (lanes
4 and 8), and tilapia (lanes 5 and 9). Amplicon sizes are indicated on
the right. Lanes 2 and 6 contained extraction controls, while lane 1 contained a 100-bp ladder; the 800-bp fragment is highlighted, and
sizes (in base pairs) are indicated on the left.
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The capabilities of the PCR assay were examined more thoroughly by
applying it to field-collected specimens. Animals displaying
visible
evidence of disease were PCR positive when a tissue sample
(from trout
R8) (Fig.
3A, lane 8) and serum samples
(Fig.
3A,
lane 9 and Fig.
3B, lane 7) were tested; however, there was
disparity
in the signal strengths of the two serum samples. The
presence
of
P. salmonis was detected in tissue from an
asymptomatic trout
(individual C3) (Fig.
3B, lane 2) collected from a
pen containing
fish displaying clinical symptoms of disease (the same
pen from
which trout R8 was obtained), and, most rewardingly, serum
from
the same individual was PCR positive (Fig.
3B, lane 3). The
sequences
of the amplification products from fish R8 and C3 were
identical
to each other and to the LF-89 sequence at positions 224 to
458
except for an additional T in an otherwise successive sequence
of
four Ts in the previously reported sequence (positions 448
to 451). The
results obtained with a dilution series of DNA obtained
from fluids of
a cell culture exhibiting full cytopathic effects
following infection
with EM-90 (
16) suggest that the PCR assay
used is capable
of revealing the presence of 10 to 100
P. salmonis cells (data not shown). The culture fluid was treated with an
excess of
DNase I prior to extraction of the nucleic acid from
the microorganism
in order to eliminate remnants of the host genome
that might contribute
to the final DNA fraction.
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FIG. 3.
PCR amplification with the RTS1-RTS4 primer pair and
DNA. (A) Amplification of DNA from tilapia (5 ng) (lane 5), cell line
CHSE-214 (25 ng) (lane 6), P. salmonis EM-90 propagated
in CHSE-214 (5 ng) (lane 7), tissue from trout R8 with visible signs of
piscirickettsiosis (lane 8), serum from trout R8 (lane 9), tissue from
trout Rb1 with no signs of disease (probably not infected) (lane 10),
and serum from trout Rb1 (lane 11). Lanes 1 and 12 contained a 100-bp
ladder, and lanes 2 to 4 contained negative extraction controls. (B)
Amplification of DNA from tissue from trout C3 with no signs of
piscirickettsiosis (taken from a netpen containing individuals with the
disease) (lane 2), serum from trout C3 (lane 3), tissue from coho
salmon CM13 with no signs of disease (lane 4), serum from coho salmon
CM13 (lane 5), serum from coho salmon CM6 with no signs of disease
(lane 6), and serum from coho salmon CE1 with visible
piscirickettsiosis (lane 7). Amplification reactions with coho
salmon and trout tissues and sera were performed with 25 ng of total
genomic DNA. Lane 1 contained a 100-bp ladder. Sizes (in base pairs)
are indicated on the left. Lane numbers are given only for lanes in
which the reactions produced the expected amplification product.
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We were interested in developing a simpler extraction procedure,
particularly in light of the earlier success in this regard
with
tissues (
14,
16). Treatment of 20 µl of serum by the
Chelex method (
21,
22) yielded amplifiable template from
infected
individuals (Fig.
4, lanes 2 and
4). Further optimization revealed
that 5 µl of serum was adequate for
detection of the pathogen
in infected animals and in asymptomatic
animals (Table
1), and
there was no
increase in detection obtained by including protease
K (
22)
during treatment with Chelex at 60°C (data not shown).
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FIG. 4.
Amplification with the RTS1-RTS4 primer pair and DNA.
Lane 2, DNA obtained by the Chelex method from serum from infected coho
salmon CC3; lane 3, extraction control; lane 4, 10 times less DNA from
infected coho salmon CC3 than was added to the PCR mixture loaded in
lane 2. Lane 1 contained a 100-bp ladder, and the 600-bp fragment is
highlighted. Sizes (in base pairs) are indicated on the left.
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TABLE 1.
Screening of adult coho serum and tissue samples for
P. salmonis by using PCR and DNA extracted by the
Chelex method
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Primer specificity studies.
There is no basis to expect
amplification of a fragment of the expected size from microorganisms
other than P. salmonis at the annealing temperature
employed in the PCR, as analysis of the available sequences from
microbes belonging to the gamma subdivision of the proteobacteria
(where P. salmonis is currently placed
[7]) revealed no discernible sequence similarity with,
for example, the site where primer RTS1 is located. Experimental
confirmation of this was obtained by using representative gram-negative
and gram-positive terrestrial microorganisms (E. coli [a
member of the gamma subdivision of the proteobacteria] and
Corynebacterium striatum) and aquatic microorganisms.
Several of the aquatic microorganisms used were originally isolated
from salmonids (Aeromonas salmonicida, Carnobacterium
piscicola, Renibacterium salmoninarum, Vibrio
anguillarum, and Yersinia ruckeri) and have been
implicated in fish diseases. While amplification of all DNA templates
with universal eubacterial primers 358f and 517r resulted in a fragment
of the expected length (193 bp for E. coli; insertions and
deletions are known for this section of the 16S rRNA gene) (Fig.
5A), only P. salmonis DNA was amplified with the RTS1-RTS4 primer pair (Fig. 5B). Discrimination of P. salmonis by the RTS1-RTS2 primer pair was also
observed (results not shown).
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FIG. 5.
Amplification of DNA obtained by the Chelex method. (A)
Amplification with primers 358f and 517r. Lane 2, Aeromonas
salmonicida ATCC 33658; lane 3, Aeromonas sp.; lane 4, Carnobacterium piscicola ATCC 35586; lane 5, Corynebacterium striatum; lane 6, Cytophaga sp.
strain DSM 3660 (Deutsche Sammlung von Mikroorganismen und
Zellkulturen); lane 7, E. coli; lane 8, P. salmonis LF-89; lane 9, P. salmonis EM-90; lane
10, Pseudoalteromonas antarctica CECT 4664 (Colección
Española de Cultivos Tipos); lane 11, Pseudoalteromonas atlantica ATCC 19262; lane 12, Renibacterium salmoninarum ATCC 33209; lane 13, Vibrio
anguillarum ATCC 43305; lane 14, Yersinia ruckeri ATCC
29473; lane 15, extraction control. Lane 1 contained a 100-bp ladder.
(B) Amplification with primers RTS1 and RTS4. In lanes 1 to 15, the
order of DNAs and the initial DNA concentration in each PCR are the
same as in panel A. Lanes 16 through 18 contained additional negative
controls, and lane 19 contained a 100-bp ladder.
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PCR in aquaculture.
Efficient screening procedures for
pathogens are valuable for ensuring good sanitation in aquaculture.
Assays of serum samples with PCR without terminally compromising the
fish open the way for more encompassing assessments of infections by
P. salmonis in aquaculture settings.
The PCR assay described here is directed toward a region of the rRNA
operon that is more variable than the region exploited
by Mauel et al.
(
14), which promises finer discrimination in
the description
of new isolates of
P. salmonis. In the present
study
the power of the PCR and the simplicity of direct fluorescent
sequencing allowed us to document quickly the existence of what
appeared to be a new strain of
P. salmonis in rainbow
trout in
Chile. This recent isolate differs from LF-89 only by one T in
a string of four Ts in the LF-89 sequence (positions 448 to 451;
GenBank accession no.
U36943). However, we also obtained the
sequence
of LF-89 by amplification and direct sequencing and found
that it
contains a sequence of five Ts at these positions, suggesting
that
recent isolates are identical to the type strain isolated
in 1989 (
7). The difference between our sequence and the previously
reported sequence (GenBank accession no.
U36943), although
unexplained,
does not deter us from speculating that strain LF-89
probably has been
prevalent in the many cycles of infection that
have occurred in the
ensuing time in the salmon farms of Region
Ten in southern Chile.
Otherwise, the five Ts which we observed
are shared with two previously
described
P. salmonis strains (C1-95
and SLGO-94).
However, the isolate from trout described here differs
at two positions
(positions 297 and 427) and three positions (positions
297, 412, and
427) from C1-95 and SLGO-94, respectively.
One apparent failure of the assay emerged: a PCR-negative serum sample
was obtained from an apparently healthy coho salmon,
salmon VH15-8
(obtained from a pen containing diseased individuals),
that was weakly
positive when its tissue was examined (Table
1).
This finding suggests
that at very early stages of the disease
during an evidently
asymptomatic period, there may be a paucity
of the pathogen in the
blood, and the initial focus of infection
may be the internal organs. A
similar scenario is suggested by
subsequent disease stages because of
the distribution of signal
between tissue and serum in trout R8 (Fig.
3A, lanes 8 and 9),
although such a picture is in contrast to the
similar signal strengths
obtained with serum and tissue samples from an
asymptomatic individual
(Fig.
3B, lanes 2 and 3). Estimates of initial
target sequences
(in this case
P. salmonis genomes) on
the basis of the plateau
of the PCR are qualitative at best
(
13). Therefore, sound conclusions
concerning the relative
burdens of the pathogen in different organs
of fish will require a
quantitative PCR approach (
9).
We envision changing the current gel-based assay into a colorimetric
format with microtiter plates for implementation of the
assay at
aquafarm sites as part of efforts to control the spread
of the disease
during artificial cultivation of salmonids. In
this regard, the
RTS1-RTS2 primer pair offers a very short amplicon
(Fig.
2, lane 3),
and increases in amplification efficiency are
expected relative to
generation of the threefold-longer fragment
obtained with the RTS1-RTS4
primer pair. The colorimetric format
coupled with miniaturization of
the Chelex extraction procedure
should encourage ambitious sampling
with the following three objectives
in mind: ascertaining the
reliability of the PCR assay under field
conditions; defining the
progression of disease within an animal;
and, in order to determine the
path of transmission of this novel
pathogen, pinpointing the presence
of the
P. salmonis genome both
in the environment and
in other organisms that coexist with salmonids
in greater or lesser
intimacy.
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ACKNOWLEDGMENTS |
S.M. and V.H. were supported by Fomento al Desarrollo
Científico y Tecnológico (FONDEF)/Comisión Nacional
de Investigación Científica y Tecnológica (CONICYT)
project 1038 and by the Dirección de Investigación y
Postgrado, Universidad Católica de Valparaíso. S.H. and
C.O. were supported by Minority Biomedical Research Support grant
GM52588 and Minority International Research Training grant 1T37TW00078-01 from the National Institutes of Health to C.O. C.O.
was also supported by National Science Foundation grant OCE-9315639 awarded to James T. Hollibaugh, San Francisco State University.
We are grateful to Enrique Madrid, Marine Harvest, Puerto Montt, Chile,
for P. salmonis EM-90 from cell culture; Ana
María Skármeta, Universidad Católica de
Valparaíso, for salmonid samples; Gloria León,
Universidad Austral de Chile, and Michael Mauel, University of Rhode
Island, for DNAs from microorganisms other than P. salmonis; Randy Zebell and Sunny Pak, San Francisco State
University, for assistance in sequencing and preparing figures; Ellen
Prager, San Francisco State University, for superb editorial work; and
two anonymous reviewers for helpful suggestions.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, San Francisco State University, 1600 Holloway Avenue, San
Francisco, CA 94132. Phone: (415) 338-3453. Fax: (415) 338-6245. E-mail: cob{at}sfsu.edu.
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Applied and Environmental Microbiology, August 1998, p. 3066-3069, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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