Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae

Authors


Correspondence: Jose R. López, IFAPA Centro Agua del Pino, Junta de Andalucia, Carretera El Portil-El Rompido s/n, 21450 Cartaya, Huelva, Spain. Tel.: +34 959 024900; fax: +34 959 024929; e-mail: lasacias@yahoo.es

Abstract

The aims of this work were to characterize the 16S–23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNAIle and tRNAAla genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 105. The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.

Introduction

Tenacibaculosis caused by bacteria belonging to the genus Tenacibaculum is one of the more devastating infectious diseases of farmed marine finfish worldwide (Hansen et al., 1992; Toranzo et al., 2005). Tenacibaculum soleae is a recently described species which has been reported causing high mortalities in commercially valuable species as Senegalese sole (Solea senegalensis), Wedge sole (Dicologoglossa cuneata) and Brill (Scophthalmus rhombus), being virulent also for Turbot (Scophthalmus maximus; Piñeiro-Vidal et al., 2008; López et al., 2010). At present, T. soleae is detected from fish by cultivation and subsequent identification using biochemical and serological techniques, which are frequently inconclusive and time-consuming. Moreover, isolation from diseased fish is problematic because of the slow growth of the pathogen and the overgrowth and/or inhibition by other bacteria present within the lesions.

PCR has proved to be useful for identification and detection of bacterial pathogens from samples without any need of cultivation (Cepeda et al., 2003; Gonzalez et al., 2003). The gene for the 16S rRNA is widely used in bacterial taxonomy as it contains variable stretches that have been used successfully for specific PCR primer design (Wiklund et al., 2000; Del Cerro et al., 2002; Oakey et al., 2003). However, it has been widely shown that the internal spacer region (ISR) between the 16S and 23S rRNA genes is more variable between bacterial species than ribosomal genes themselves in both sequence and length (Barry et al., 1991; Hassan et al., 2003; Osorio et al., 2005). Species-specific primers derived from these sequences have also been reported (Kong et al., 1999; Lee et al., 2002; Hassan et al., 2008). In this study, we sequenced the ISR from T. soleae and designed species-specific primers, targeting both the 16S rRNA gene and ISR region, for its identification and detection by PCR.

Materials and methods

Bacterial strains

The strains used in this study are listed in Table 1. Together with 32 reference strains, 57 isolates obtained in our laboratory from diseased flatfish were also used. These isolates were identified based on 16S gene sequencing and biochemical tests. All strains were cultured aerobically at 20 °C on tryptic soya agar (TSA) made with seawater, with the exception of those belonging to Tenacibaculum maritimum, which were grown on Flexibacter medium (FMM; Pazos et al., 1996).

Table 1. Bacterial species and strains used in this work
SpeciesStrainsSourceGeographic origin
Tenacibaculum soleaeNCIMB 14368TSenegalese sole (Solea senegalensis)Spain
a11Brill (Scophthalmus rhombus)Spain
a47, a50, a216, a462, a467Wedge sole (Dicologoglossa cuneata)Spain
a410, a469Senegalese sole (S. senegalensis)Spain
Tenacibaculum ovolyticumLMG 13025Atlantic halibut (Hippoglossus hippoglossus) eggs 
Tenacibaculum maritimumCECT 4276Fishblack sea bream (Acanthopagrus schlegeli)Japan
Lg326Senegalese sole (S. senegalensis)Spain
a274, a388, a442, a443, a444, a461, a523Wedge sole (Dcuneata)Spain
Tenacibaculum gallaicumDSM 18841TSeawater 
Tenacibaculum discolorDSM 18842TSenegalese sole (S. senegalensis)Spain
Tenacibaculum litoreumJCM 13039TTidal flat sedimentKorea
Tenacibaculum sp.a3Wedge sole (D. cuneata)Spain
Polaribacter sp.a500, a502Wedge sole (D. cuneata)Spain
Flavobacterium marinotypicumCECT 578T  
Flavobacterium johnsoniaeCECT 5015GrassUK
UW 101T (ATCC 17061T)SoilUK
Flavobacterium psychrophilumNCIMB 1947TCoho salmon (Oncorhynchus kisutch)USA
OSU THCO2-90Coho salmon (O. kisutch)USA
Aeromonas salmonicida ssp. salmonicidaCECT894TSalmon (Salmo salar) 
Aliivibrio fischeriCECT 524T  
Vibrio harveyiCECT 525TAmphipod (Talorchestia sp.)USA
CECT 5156Sea bass 
Lg 123, a10Senegalese sole (S. senegalensis)Spain
a9, a20, a26, a82, a87, a91, a102, a106, a417Wedge sole (D. cuneata)Spain
Vibrio alginolyticusCECT 521THorse mackerel (Trachurus trachurus)Japan
CECT 436Food 
a134Brill (S. rhombus)Spain
a241Wedge sole (D. cuneata)Spain
Vibrio parahaemolyticusCECT 511THuman patient suffering food poisoningJapan
Vibrio campbelliiCECT 523TSeawaterHawaii
Vibrio natriegensCECT 526TSalt marsh mudUSA
Vibrio tubiashiiCECT 4196THard clam (Mercenaria mercenaria), USAUSA
Vibrio tapetisCECT 4600TClam (Tapes philippinarum)France
a255Wedge sole (D. cuneata)Spain
Vibrio splendidusCECT 528TMarine fish 
Vibrio spp.a7, a29, a35, a54, a107, a256Wedge sole (D. cuneata)Spain
a6Senegalese sole (S. senegalensis)Spain
Photobacterium damsela ssp. piscicidaCECT 5895Sea bass (Dicentrarchus labrax) 
Lg 122, a316, a319, a321, a335, a356Senegalese sole (S. senegalensis)Spain
Photobacterium damsela ssp. damselaeCECT 626TDamsel fish (Chromis punctipinnis)USA
Photobacterium leiognathiCECT 4191TTeleostean fish (Leiognathus equula)Thailand
Photobacterium angustumCECT 5690TSeawater 
Photobacterium phosphoreumCECT 4192T  
Photobacterium spp.a185, a197Wedge sole (D. cuneata)Spain
Pseudoalteromonas spp.a220, a250Wedge sole (D. cuneata)Spain
Psychrobacter sp.a328Wedge sole (D. cuneata)Spain
Pseudomonas fluorescensCECT 378TWater tankUK
Pseudomonas putidaCECT 385Soil 
Pseudomonas anguillisepticaCECT 899TEel (Anguilla japonica)Japan
Pseudomonas aeruginosaATCC 27853  
Pseudomonas baeticaa390T, a391, a393, a398, a399, a600Wedge sole (D. cuneata)Spain
Escherichia coliATCC 25922  

DNA extraction

Template DNA from pure cultures was prepared by boiling bacterial colonies for 10 min in distilled water followed by centrifugation at 12 400 g for 1 min to sediment the cell debris. DNA from tissue samples was extracted as follows: after homogenizing 100 mg of fish tissue in TE buffer (Sigma), SDS (1%) and proteinase K (100 μg mL−1) were added and the solution was incubated for 3 h or overnight at 56 °C. Thereafter, pancreatic RNAse (20 μg mL−1) was added and incubation was performed for 1 h at 37 °C. The solution was transferred to a phase-lock gel (Eppendorf) and the DNA was purified using the common phenol/chloroform/isoamyl alcohol procedure and finally precipitated with ethanol and dissolved in distilled water. The concentration and purity of genomic DNA were calculated from measurements of absorbance at 260 and 280 nm, recorded using a NanoDrop 1000 spectrophotometer.

16S rRNA gene and ISR region characterization

Partial 16S rRNA gene sequences were obtained using primers 20F and 1500R (Weisburg et al., 1991), and ISR sequences were obtained using primers 16/23S-F and 16/23S-R (Lee et al., 2002). PCR products were electrophoresed in a 1% agarose gel and purified with the kit GenElute PCR Clean-up (Sigma) following the manufacturer's instructions. The purified products were cloned in pGEM-T Easy Vector System II kit (Promega) or directly used for sequencing. Sequencing was accomplished using the kit BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems) and an ABI Prism 3130 DNA Sequencer (Applied Biosystems). The sequences were analyzed using chromaslite v2.01 and seqmanii (DNASTAR) programs and subjected to blast searches to retrieve the most closely related sequences. The presence of tRNA genes was determined using tRNAscan-SE 1.21 software (Lowe & Eddy, 1997).

Primer design

Previously reported 16S rRNA gene and ISR sequences from T. soleae and related species, retrieved from GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) and those obtained in this study, were aligned by using the program clustalw (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and examined for areas of similarity and variability between different species and strains. On the basis of the alignments, two variable regions were chosen and a pair of primers was designed by using the primer3 program (http://frodo.wi.mit.edu/; Rozen & Skaletsky, 2000). Primers were synthesized by Thermo Scientific (Ulm, Germany).

PCR amplification

The PCR amplifications were carried out using the commercial kit RedTaq ReadyMix (Sigma), which included all necessary reagents except the primers and DNA template. The PCR mixture consisted of reaction buffer (10 mmol L−1 Tris–HCl pH 8.3, 50 mmol L−1 KCl, 1.5 mmol L−1 MgCl2), 200 μmol L−1 of each dNTP, 200 nmol L−1 of each primer, 3 U of Taq DNA polymerase, template DNA, and double-distilled water up to a final volume of 50 μL. The amplification was performed in a Mastercycler gradient (Eppendorf) as follows: an initial denaturation at 94 °C for 5 min followed by 45 amplification cycles (denaturation at 94 °C for 1 min, annealing at 57 °C for 45 s, and extension at 72 °C for 1 min), and a final elongation at 72 °C for 5 min. DNA from strain T. soleae a47 was included as a positive control and distilled water as a negative control. PCR products were electrophoresed on a 1% agarose TBE gel stained with SYBR Safe DNA Gel Stain (Invitrogen); a 1-kb DNA ladder (Biotools) was included as a molecular weight marker.

Specificity and sensitivity of the PCR

To test the specificity of the primers in the PCR procedure, nine T. soleae strains, isolated from three different hosts and including the type strain, and 81 strains of other species, most of them taxonomically and/or ecologically related, were used as positive and negative controls, respectively (Table 1). For PCR amplification, 100 ng DNA template was used for each strain. The detection limit was evaluated using 10-fold serially diluted DNA, isolated from strain T. soleae a47, over the range 100 ng to 100 fg. Large amounts of DNA (0.5–3 μg) were also assayed. To test the sensitivity in the presence of tissue debris or other bacterial species, the same 10-fold DNA dilutions were used mixed with 1 μg of DNA from healthy Wedge sole liver or from a mixture of pure cultures of a number of marine bacteria (T. maritimum, Vibrio harveyi, Photobacterium damsela, Psychrobacter sp., and Pseudomonas baetica). Each assay was performed at least in duplicate.

Detection of T. soleae in naturally infected fish tissues

Ulcer samples from six Wedge sole with suspected tenacibaculosis caused by T. soleae on the basis of medical history and the presence of filamentous bacteria in wet-mount preparations, together with samples (ulcers, liver or kidney) from four fish (one Brill, one Senegalese sole and two Wedge sole) diagnosed by culture as positive for T. soleae, were analyzed for the presence of the pathogen using PCR. DNA from samples was extracted as outlined above and 1 μg used for each PCR reaction.

Results

ISR region analysis

Twenty-one 16S and ISR nucleotide sequences were determined from T. soleae or related organism strains (accession numbers FR734188, FN433006, FN646547FN646565). The ISR PCR products from T. soleae strains a11, a47, a50, a216, a410, a462, a467 and a469 were analyzed by agarose gel electrophoresis; each strain seemed to contain only one type of operon, as a single band of about 1200 bp, including partial 16S and 23S rRNA genes, was found (data not shown). Direct sequencing of ISR from some strains (a11, a47, a50, a410, a462) seemed to support this possibility; an unambiguous reading of nucleotide sequences was possible, and the sequences obtained by cloning and by direct sequencing were similar. Sequence analysis showed that T. soleae 16S–23S spacers were basically similar in length (586–596 bp) and belonged to a unique ISR class (ISRIA), carrying tRNA genes for isoleucine (Ile) and alanine (Ala). Similarity between T. soleae strains ISR sequences was of 90.6–100%. The main differences between strains were due to the presence of a variable region, of approximately 90 bp and located near the 3′ end, which contained different short sequence blocks. On the basis of variation in this region, T. soleae ISR sequences could be grouped into two basic types, the first including those obtained from strains a11, a47, a216, and a410 (96.3–100% similarity), and the second comprising strains a50, a462, a467 and a469 (97.5–99.2%). Similarity values with other related species were clearly lower, the closest strains being Tenacibaculum ovolyticum LMG 13025 (85.2% similarity) and T. maritimum a523 (71.9%). The tRNAIle and tRNAAla genes were similar both in length (74 bp) and in nucleotide composition for all the T. soleae strains tested, and were also similar to those found in other species of the genus as T. maritimum and T. ovolyticum, differing only at one or two positions, or at none at all.

Primer design and specificity

A pair of primers to identify T. soleae, forward G47F (5′-ATGCTAATATGTGGCATCAC-3′), and reverse G47R (5′-CGTAATTCGTAATTAACTTTGT-3′), were designed at the 5′ region of the 16S gene and of the ISR, respectively (Fig. 1), flanking a 1555-bp fragment.

Figure 1.

Diagrammatic representation of a T. soleaerRNA operon, showing the relative locations of primers G47F and G47R, the tRNA genes and the ISR intraspecific variable region. Primers are represented as black arrows. Primer G47F was designed at the beginning of the 16S gene and corresponds to nucleotides 137–156 in the sequence with GenBank accession no. AM989478. Primer G47R was designed within the 16S–23S intergenic spacer, between the two tRNA genes present, and corresponds to nucleotides 193–214 in the sequence with GenBank accession no. FN646550.

The specificity was tested experimentally as indicated using pure cultures from target and non-target strains (Table 1). All T. soleae strains produced a clear PCR band of the expected size (1555 bp). A phantom band of about 750 bp was sometimes also visible. Conversely, no PCR product was detected from non-target species (Fig. 2).

Figure 2.

Profile of PCR using primers G47F and G47R and DNA templates from T. soleae and some non-target related species. MW: 1-kb ladder molecular weight marker. Lanes 1–8: T. soleae strains NCIMB 14368T, a11, a47, a50, a216, a410, a462 and a467, respectively. Lanes 9–19: Tenacibaculum sp. a3, T. ovolyticumLMG 13025, T. maritimum strains CECT 4276, Lg326 and a443, Polaribacter sp. a502, Flavobacterium psychrophilum strains NCIMB 1947 and OSU THCO2-90, Flavobacterium johnsoniae strains CECT 5015 and UW101, and Flavobacterium marinotypicumCECT 578, respectively. Numbers on the left indicate the position of molecular size marker in bp. Only T. soleae strains showed the expected 1555-bp band.

Sensitivity

The detection limit of the PCR assay, when purified DNA of T. soleae was used as template, was as little as 1 pg in a 50-μL reaction volume. A 100-fg template could sometimes be detected, although this product was extremely weak and not always reproducible. Conversely, large DNA amounts gave positive results, showing that the optimum template concentration was from 2 μg to 100 ng (Fig. 3). When DNA extracted from fish tissues was seeded with different concentrations of T. soleae DNA and used as template, the detection limit was of 10 pg of T. soleae DNA in 1 μg of fish DNA. Thus, the assay was capable of detecting one T. soleae genomic copy among 105 copies from fish tissues. Similar results were found when this assay was made with DNA from mixed cultures of marine bacteria instead of from fish tissues.

Figure 3.

Detection limit of the PCR protocol with primers G47F and G47R, determined with different dilutions of purified DNA of T. soleae. MW: 1-kb ladder molecular weight marker. Lanes 1–11: 3, 2, 1 μg, 500, 100, 10, 1 ng, 100, 10, 1 pg and 100 fg of DNA, respectively; lane 12, negative control. Numbers on the left indicate the position of molecular size marker in bp.

Detection of T. soleae in naturally infected fish tissues

Results obtained with naturally infected fish samples indicated that the proposed protocol was more sensitive than agar cultivation for detecting T. soleae. When the samples used were from fish suspected of suffering tenacibaculosis by T. soleae, three of the six fish tested proved positive by PCR. Although filamentous bacteria had been observed in these samples by microscopy, none grew in culture medium, presumably because of inhibition or overgrowth by environmental bacteria. On the other hand, when fish diagnosed by culturing as positive for T. soleae were used, all four samples gave positive results.

Discussion

Because of their specificity, sensitivity and rapid performance, PCR-based methods constitute one of the strongest tools for bacteria diagnosis, and specific protocols have been developed for many major bacterial pathogens in aquaculture (Toyama et al., 1996; Wiklund et al., 2000; Pang et al., 2006; Beaz-Hidalgo et al., 2008). PCR constitutes a useful tool not only for detecting pathogens in diseased fish, but also in asymptomatic carriers, in the environment, or for selecting pathogen-free egg stocks.

In this study, we developed a PCR protocol against T. soleae, an emerging pathogen in marine aquaculture whose identification is tedious and time-consuming, requiring prior isolation of the bacteria and the utilization of phenotypic tests, which require days or weeks to perform. The PCR assay described here is specific and sensitive, enabling quicker and easier identification of the pathogen.

The 16S rRNA gene and the ISR region were selected as primer targets to take the greatest advantage of these two DNA regions. Although 16S rRNA gene is highly conserved in eubacteria and contains only small regions of variation, the vast database of sequences available makes finding and comparison with close relatives feasible. The ISR region, in comparison, is generally more variable than rRNA genes, allowing better discrimination of closely related species, even among strains of the same species (Lee et al., 2002; Osorio et al., 2005). However, few sequences are available from the Flavobacteriaceae species. This is the first report of ISR sequences from Tenacibaculum species, namely T. soleae, T. maritimum and T. ovolyticum, which will facilitate the identification of other specific primers for Flavobacteriaceae species.

Tenacibaculum soleae strains ISR showed only minor size variations in length and belonged to a single ISR class, containing tRNAIle and tRNAAla genes. The presence of a single ISR class is frequent in bacteria. For example, the analysis of the ISR region of 155 bacterial strains belonging to a variety of taxa, carried out by Stewart & Cavanaugh (2007), revealed that only 41% of the strains had two or more ISR classes. In the same study, the presence of tRNAIle and tRNAAla genes was also common, being detected in 48% of the ISR sequences obtained by these authors; nevertheless, its frequency varied depending on the bacterial taxa, being absent, for example, in Actinobacteria. However, in Flavobacteriaceae, the tRNAIle-tRNAAla combination appears to be dominant, being present in different genera of the family, such as Flavobacterium or Cellulophaga (Figueiredo et al., 2005; Welker et al., 2005; Holmfeldt et al., 2007; Ford, 2008), as well as in all the Tenacibaculum and Polaribacter strains tested by our group. ISR intraspecific variation in T. soleae was of 0–9.4%, a lower value than that reported by Stewart & Cavanaugh (2007) when comparing sequences from the same species and ISR class (0–12.1%). Differences between T. soleae ISR sequences were due mainly to the absence/presence of distinct sequence blocks, as reported by other authors for a variety of bacterial species, including fish pathogens such as Photobacterium damselae (Gürtler & Barrie, 1995; Chun et al., 1999; Osorio et al., 2005; Stewart & Cavanaugh, 2007). On the other hand, ISR sequences proved useful for differentiating T. soleae from related species, displaying lower interspecific similarity values than obtained with 16S rRNA gene. For example, the similarity of T. soleae a47 and T. ovolyticum LMG 13025 was 97.7% when 16S rRNA gene sequences were compared, but only 85.2% with ISR sequences. In this sense, it is important to note that although the ISR region generally displays greater nucleotide divergence than 16S rRNA gene, this is not always the case. In fact, Stewart & Cavanaugh (2007) noted that the ISR region was less discriminating than 16S rRNA gene for 24% of the strains tested.

The specificity of the proposed PCR protocol was validated in nine T. soleae strains and 81 strains belonging to other species, most of these from marine environments, including several common fish pathogens. No cross-reactions with any of the non-target organisms were observed. The sensitivity experiments showed a detection limit with DNA extracted from pure cultures of 1 pg DNA/PCR tube, equivalent to 30 or fewer bacterial cells, a result that agrees with those described with other PCR methods designed for bacterial fish pathogens (Del Cerro et al., 2002; Mata et al., 2004; Romalde et al., 2004; Hong et al., 2007). The isolation of T. soleae from diseased fish is in many cases unsuitable due to the slow growth of the pathogen and overgrowth or inhibition by other faster growing bacteria present within the lesions. Thus, the usefulness of the proposed PCR protocol to detect the bacteria from mixed cultures and fish tissue samples was also tested. The results from seeding DNA extracted from fish tissues or from a mixture of bacterial cultures confirmed the sensitivity of the method (10 pg of T. soleae DNA was detected at a target/background ratio of 1: 105), although as expected the detection level was lower than that with pure cultures, probably due to the presence of some PCR inhibitor. It has been reported that high levels of non-target DNA, constituents of bacterial cells, and different compounds found in animal tissues can have an adverse effect on PCR (Wilson, 1997; Becker et al., 2000). When naturally infected fish were subjected to the PCR assay, positive results were recorded for all the confirmed cases, and in half of the suspected cases in which cultures failed to detect the bacteria. The PCR-assay was therefore more sensitive than agar culturing for detecting T. soleae from tissue samples, offering a useful tool for rapid diagnosis and examination of the epidemiology of this pathogen.

In summary, the present study reports the first PCR protocol suitable for identifying this pathogen from pure or mixed cultures, as well as for detection from fish tissue samples.

Acknowledgements

This work was supported by INIA project 2005-00215-C03 (Spanish Ministerio de Educación y Ciencia), the European Union FEDER program and a PhD grant from IFAPA (Junta de Andalucía, Spain). We thank Dr Y. Santos, Dr J. A. Guijarro and Dr S. Arijo for sending us different strains.

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