Development of a specific biotinylated DNA probe for the detection of ...

Development of a Specific Biotinylated DNA Probe for the Detection of Renibacterium salmoninarum Harry Hariharan, Biao Qian, Beatrice Despres, Frederick S. Kibenge, Susan B. Heaney, and Donald J. Rainnie

ABSTRACT

extrait de trois souches de R. salmoninarum et d'une souche de chacune des especes bacteriennes suivantes: Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordaUii et Yersinia ruckeri. Lors d'analyses par dot blot, une hydridation ne s'est produite qu'avec l'ADN provenant des souches de R. salmoninarum. Cette technique, lorsqu'utilisee sur des specimens de rein provenant de poissons inocule's expe'rimentalement avec R. salmoninarum, s'est averee aussi sensible que la culture sur gelose. A partir de specimens trouves negatifs a la culture ou lors de l'epreuve dot blot, pas plus d'une cellule fluorescente par 50 champs examines en microscopie n'a t observee lors d'un examen 'a l'aide d'anticorps fluorescents. Cette sonde possede donc le potentiel pour etre utilise comme outil de diagnostic pour la maladie bacterienne du rein chez le poisson.

A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot (Traduit par Dr Serge Messier) hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA INTRODUCTION probe, therefore, has the potential for use in the diagnosis of BKD of Renibacterium salmoninarum, the fish. causative agent of bacterial kidney disease (BKD) of salmonid fish, is the only species of a novel genus (1). RESUME Although it has been referred to as a Corynebacterium spp. (2), recent Afin d'identifier Renibacterium analysis of the phylogeny of R. salmosalmoninarum, I'agent de la maladie ninarum by 16S ribosomal RNA bacterienne du rein, une sonde ADN showed that it is more closely related a t developpee a partir d'un des to the genera Arthrobacter and Microtrois clones suivants pRS47, pRS49 coccus (3). Diagnosis of BKD by isoet pRS26 mesurant respectivement lation and identification of the agent 5,1 kb, 5,3 kb et 11,3 kb. Un insert is time consuming because R. salmobiotinyle de la sonde pRS47 a ete ninarum is very fastidious and slow teste avec trois dilutions d'ADN growing, and can take up to 6 wk for

visible growth on agar media. Several immunoassays have been developed for faster diagnosis but inconsistent results, and in some cases, low sensitivity, have been a concern (4). Fluorescent antibody tests (FAT) are fast and have found use for detecting asymptomatic cases of BKD (5,6). However, FAT has an additional problem of false-positive reactions (7). Nonspecific reactions may occur in immunodiagnostic procedures because of common antigens shared between R. salmoninarum and other bacteria of aquatic habitats, such as Carnobacterium piscicola (8). Recent developments in the diagnosis of BKD includes the use of DNA hybridization probes and the polymerase chain reaction (9-12). In this study, we report the development of a specific DNA probe for R. salmoninarum, which did not cross hybridize with 2 of its genetically related organisms, Micrococcus luteus and A rthrobacter protophormiae, and 7 other species of bacteria found in fish. The probe was also compared with culture and FAT on fish kidney samples.

MATERIALS AND METHODS BACTERIAL STRAINS AND CULTURE

Renibacterium salmoninarum strain FH106 (DFO No. 892 Y6-K) was obtained from Dr. Gilles Olivier, Department of Fisheries and Oceans, Halifax, Nova Scotia; and strains 33209 and 33739 were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland. The strains were grown in KDM-2 biphasic medium or on KDM-C agar (13,14) aerobically. Arthrobacter protophormiae 21348, Micrococcus luteus 23262, Carnobacterium piscicola 35586, Corynebacterium aquaticum

Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I. C IA 4P3, Canada. Submitted.September 21, 1994.

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14665, Listonella anguillarum 14181, Pseudomonas fluorescens 13525, and Vibrio ordalii 33509 from the ATCC and Yersinia ruckeri U9182-90 from the Fish Health Unit of Atlantic Veterinary College were cultured aerobically in brain heart infusion broth. BACTERIAL GENOMIC DNA EXTRACTION

For the extraction of genomic DNA from bacteria, we used previously reported methods (15) with modifications. Briefly, bacterial cells were lysed with TE buffer (10 mM Tris HCI, 1mM EDTA, pH 7.5) containing 4 mg/mL lysozyme at 37°C for 20 min. Then SDS and proteinase K were added to final concentrations of 0.5% and 100 jig/mL, respectively. The mixture was incubated at 37°C for 1 h. After phenol/chloroform extraction and ethanol precipitation, the pellet was redissolved in 0.3 M sodium acetate pH 5.0 and ethanol precipitated at -70°C. The genomic DNA was harvested and dissolved in TE buffer, pH 7.5. MOLECULAR CLONING OF

R. salmoninarum DNA Both R. salmoninarum genomic DNA and the vector pUC19 (GIBCOBRL Canada, Burlington, Ontario) were separately digested with EcoRI. The vector DNA was dephosphorylated

using calf intestinal phosphatase, and ligated with EcoRI-digested R. salmoninarum genomic DNA by T4 DNA ligase at 16°C for 4 h. The ligation mixtures were used to transform Escherichia coli DH5a competent cells (GIBCO-BRL Canada). The white colonies were picked from LuriaBertani agar plates containing ampicillin 50 pLg/mL and X-gal (5-bromo-4chloro-3-indolyl-,-D-galactoside) after incubation at 37°C overnight. PREPARATION OF PLASMID DNA

Mini-preparation - All the plasmid DNAs for probe screening were prepared from 5 mL of culture by using Magic Miniprep DNA Purification System (Promega Corporation, Madison, Wisconsin, USA). The DNA was finally eluted in 50 ,uL of TE buffer, pH 7.5. Midi-preparation - Plasmid DNA was extracted from 50 mL or 100 mL of culture by polyethylene glycol precipitation methods (16).

I kb

B E

E

PBE P

E

PBE

lir

B

PBE

E

A

BL E

CL

~iri

-if

E

BP

B

11

PE

11

Fig. 1. The restriction maps of pRS47, 5.1 kb(A); pRS49, 5.3 kb(B); and pRS26, 11.3 kb(C) deduced from the restriction analysis. Symbols: thick line - pUC19 vector sequences; thin line - R. salmoninarum DNA sequences; B, BamHI; E, EcoRI; P, PstI. PREPARATION OF BIOTINYLATED PROBES

The biotinylated DNA probes were prepared using photobiotin labelling system (GIBCO-BRL, Canada) following the manufacturer's instructions. Ten microliters of DNA (5-10 pug) was mixed with 10 puL (1 ,ug/mL) photobiotin acetate and the solution was irradiated for 20 min with a 400 W mercury lamp in a dark room. The DNA probe was precipitated by adding 0.5 vol. of 7.5 M ammonium acetate and 3 vol. ethanol at -20°C for 1 h. Finally, the biotinylated DNA was dissolved in TE buffer, pH 7.5.

0 54 -036.

t136 ..

18

SOUTHERN BLOT HYBRIDIZATION

Southern blot hybridization was conducted as previously described (16). All hybridizations were performed at 42°C for 16-20 h with the addition of the biotinylated DNA probe (100 ng/mL). The hybridized membranes were then processed for detection of the hybridized biotinylated probe.

Fig. 2. Electrophoresis in 1% agarose gel of 3 R. salmoninarum genomic DNA clones, pRS26 (lane 1), pRS47 (lane 2), and pRS49 (lane 3) digested with EcoRI. Lane M contains the lKb DNA ladder (GIBCO-BRL Canada) as molecular weight markers.

DOT BLOT HYBRIDIZATION

For dot blot hybridization assay, 10 pL of samples (extracted DNA) were denatured by heating at 100°C for 10 min and immediately chilled in ice before applying them to nitrocellulose filters using a Bio-Dot manifold (Bio-Rad Laboratories Ltd., Mississauga, Ontario) under vacuum. In the case of plasmid DNA dot blots, 2 pL of heat denatured DNA was directly spotted on the nitrocellulose filter. The filters were baked in a vacuum oven at 80°C for 1 h. The hybridization was performed as for

the Southern blot hybridization assay described above. COLORIMETRIC DETECTION OF BIOTINYLATED DNA PROBE

After Southern blot or dot blot hybridization, the biotinylated DNA probe was colorimetrically detected by using the Blu GENE nonradioactive DNA detection system (GIBCOBRL Canada) following the manufacturer's protocols. 307

1.0

0.5 0.1ug

Arthrobacter protophormiae 21348 Aeromonas salmonicida 33658

Corynebacterium aquaticum 14665 Carnobacterium piscicola 35586 Listonella anguaram 14181 Micrococcus luteus 23262

Pseudomonas fluorescens 13525 Renibacterium- salmoninamm 33209

Remibactenum salmoninarum 33739

J-enibaeterium salmoninanim FH106 ribo ordali 33509 Yersinia ruckeri U9182-90 pUC9 Fig. 3. Dot-blot hybridization of serially diluted DNA (1, 0.5, and 0.1 pg) of 12 bacterial strains with the biotinylated pRS47/BamHI insert probe. Details of the bacterial strains and detection procedures are described under materials and methods. USE OF BIOTINYLATED PROBES

The pRS47/BamHI probe was tested on DNA extracted from 3 strains of R. salmoninarum and 9 other bacterial species by dot blot hybridization method. This probe was also tested on DNA extracted from kidney specimens from fish. For kidney specimens, 12 Atlantic salmon (Saint John River strain from Brook Valley Marine Farm, Fortune, P.E.I.) of 25-50 g parr were used. All fish were kept in fresh water at 10°C. Ten of these in 1 tank received a suspension of R. salmoninarum, containing 10 colony forming units in 0.1 mL of peptone saline (0.1% peptone, 0.85% NaCI) intraperitoneally.

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TABLE 1. Comparison of dot blot hybridization using pRS47/BamHI insert probe, culture, and fluorescent antibody test in detecting R. salmoninarum in fish kidney samples Fish no. Dot blot Culture 2 + + 3 5 + + 7 8 + + 10 11 + + + + 12 13 14 15b 16b aOne positive cell/SO Fields bControl fish, not challenged with R. salmoninarum

F.A. test

1/SOF5 200/2F 200/6F 1/50F 0/SOF 200/6F 200/1F 200/7F 0/SOF 0/SOF 0/50F 0/SOF

Laboratories, Inc., Gaithersburg, Maryland) was used.

RESULTS SCREENING OF R. salmoninarum GENOMIC DNA CLONES

After cloning the EcoRI digested R. salmoninarum FH106 genomic DNA into the vector pUC 19, a total of 220 white colonies were picked from the X-gal agar plates. Among these

colonies, 56 recombinant plasmids were found to have the foreign DNA inserts after DNA extraction, EcoRI digestion, and electrophoresis in 1% agarose gel. Fifty-seven percent of the recombinant plasmids had small DNA inserts (100 to 500 bp), while 23% had large DNA inserts (>2000 bp). For screening, R. salmoninarum FH106 genomic DNA was digested by AluI and labelled with photobiotin acetate. Dot blot hybridization using the biotinylated genomic DNA detected 3 plasmids, designated pRS26, pRS47, and pRS49. On digestion of plasmid DNA with EcoRI, and electrophoresis in 1% agarose gel, it was found that plasmid pRS26 had 1 large insert (7.4 kb) and 1 small insert (1.2 kb), while pRS47 and pRS49 had only 1 insert each of 2.4 kb and 2.6 kb size, respectively.

Two fish in another tank received only peptone saline with-out bacteria, and these served as unchallenged controls. Fish were euthanized 51 d after challenge. The kidneys were removed aseptically and homogenized in a stomacher blender (Lab Blend 400 Stomacher, Seward Medical, London, U.K.) for one minute with 20 vol. peptone saline. Ten microliters of samples were plated onto KDM-2 agar plates and incubated for up to 6 wk at 15°C. The remaining RESTRICTION MAPPING OF THE homogenate was used for FAT and for R. salmoninarum SPECIFIC GENOMIC DNA extraction for dot blot hybridiza- CLONES In order to understand the relationtion. For FAT, Bac Trace fluorescein labelled affinity purified antibody to ships between these R. salmoninarum R. salmoninarum (Kirkegaard & Perry specific plasmids, a brief restriction

analysis was done using BamHI, EcoRI, and PstI. A preliminary restriction map of plasmids, pRS47, pRS49, and pRS26 was deduced from the data of the restriction analysis (Fig. 1), which indicated that there were no overlaps between these plasmids. SPECIFICITY AND SENSITIVITY OF

CLONED R. salmoninarum DNA PROBES AND THE DETECTION OF THE BKD BACTERIUM IN FISH KIDNEY SAMPLES

infections by culture has the disadvantage that it can take up to 6 wk to obtain a result. Further, overgrowth with fast growing contaminants can lead to negative results (17). Storage of samples and freezing also reduce the sensitivity of culture for lightly infected samples (18). Rapid tests such as FAT are widely used alternatives because of the simplicity and general reliability (17), but some workers have reported concerns with FAT (7,19). Austin and Austin (17) have observed that gene probes for BKD may enter the armory of the diagnostician in the near future. Etchegaray et al. (20) described a DNA fragment from R. salmoninarum, isolated from plasmid pS84, after digesting with EcoRI and HindIII. Although this fragment gave negative reactions with several Gramnegative bacteria, it hybridized with Corynebacterium striatum in addition to the test strain of R. salmoninarum. Coryneform groups of bacteria and Arthrobacter are closely related to R. salmoninarum (1,21). An oligonucleotide probe developed by Mattsson et al. (9) did not hybridize with Corynebacterium species, but did cross react with Arthrobacter. A DNA probe which did not cross hybridize with several fish bacteria was reported recently (1 1). Our probe pRS47/BamHI was specific to R. salmoninarum, and did not hybridize with 9 other bacterial species which included both Gramnegative and Gram-positive bacteria associated with fish, as well as bacteria genetically related to R. salmoninarum. The dot blot assay results also agreed with culture results in the case of kidney samples from experimentally infected fish. It is interesting that FAT, a rapid procedure, gave results generally similar to those obtained with culture and dot blot hybridization assay. It has been suggested that some of the problems associated with FAT can be overcome using purified antibodies (22). The commercial FAT reagent used in our test contained

Two cloned R. salmoninarum DNA probes, pRS26/EcoRI insert (7.4 kb), and pRS47/BamHI insert (1.7 kb), were purified from low temperaturemelting-agarose gel and labelled with photobiotic acetate. Southern blot and dot blot hybridization of the bacterial genomic DNAs, and plasmids pRS26, and pRS47, with these 2 probes proved their specificity to R. salmoninarum and cross reactivity between pRS26 and pRS47, and showed that the sensitivity of the dot blot method with these probes to be 5 ng DNA of R. salmoninarum strain FH106 (unpublished results). The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA (1 p,g, 0.5 jig, 0.1 jIg) from 3 strains of R. salmoninarum and 9 other strains of bacteria, and gave positive results (hybridization) only with R. salmoninarum strains (Fig. 3). No hybridization occurred with the DNA of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. The aforementioned probe was used on DNA extracts from 12 fish kidney samples, and the results were compared with those obtained with culture and FAT (Table I). The culture and dot blot hybridization results agreed' 100%. Two samples (Fish no. 2 and 7) which showed 1 fluorescent cell/50 fields were negative in both culture and the dot blot assay. Samples positive in culture and dot blot hybridiza- affinity purified antibody. tion showed high numbers of fluoAlthough our assay requires that rescing cells. the specimen DNA be extracted and applied to nitrocellulose membranes, unlike radio-labelled probes, it has the DISCUSSION advantage that the results can be read Confirmatory diagnosis of subclini- by visual inspection of the memcal Renibacterium salmoninarum branes. Biotinylated probes provide

reagents with long shelf lives, and require minimal facilities for storage and handling. The assay using these probes is easy to perform, rapid, and without irradiation hazard. Compared to the PCR, our DNA probe method has the advantage that it does not need expensive equipments. On the other hand, the PCR can be made more sensitive and specific if the PCR products are detected by a DNA probe in Southern blot hybridization. The probe can therefore complement the PCR method. Diagnostic procedures with higher sensitivity may be more useful in detecting R. salmoninarum within fish eggs. In summary, nonradioactive nucleic acid hybridization such as reported here is a useful tool and lends itself to applications in diagnostic and research laboratories. DNA probes have been found to be of value in difficult or doubtful identifications obtained with conventional methods. (23)

ACKNOWLEDGMENTS This study was supported in part by a General Research Grant to the Faculty of Veterinary Medicine, University of Prince Edward Island, from the National Sciences and Engineering Research Council of Canada. We thank Leslie Holt for assistance in the processing of fish kidney samples, and James Bryenton and Mada Coles for assistance with the fluorescence antibody tests.

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