APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2010, p. 5282–5286 0099-2240/10/$12.00 doi:10.1128/AEM.00330-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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Detection and Quantification of the Coral Pathogen Vibrio coralliilyticus by Real-Time PCR with TaqMan Fluorescent Probes䌤† F. Joseph Pollock,1,2,3,4 Pamela J. Morris,2,3,5 Bette L. Willis,4 and David G. Bourne1* Australian Institute of Marine Science, PMB 3, Townsville 4810, Australia1; College of Charleston, Charleston, South Carolina 294122; Hollings Marine Laboratory, Charleston, South Carolina 294123; ARC Centre of Excellence for Coral Reef Studies, School of Marine and Tropical Biology, James Cook University, Townsville 4811, Australia4; and Baruch Institute for Marine and Coastal Sciences, University of South Carolina, Charleston, South Carolina 294125 Received 7 February 2010/Accepted 25 May 2010

A real-time quantitative PCR-based detection assay targeting the dnaJ gene (encoding heat shock protein 40) of the coral pathogen Vibrio coralliilyticus was developed. The assay is sensitive, detecting as little as 1 CFU per ml in seawater and 104 CFU per cm2 of coral tissue. Moreover, inhibition by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. This assay represents a novel approach to coral disease diagnosis that will advance the field of coral disease research. 23694) were obtained through extraction of total DNA using a Promega Wizard Prep DNA Purification Kit (Promega, Sydney, Australia), PCR amplification, and sequencing using primers and thermal cycling parameters described by Nhung et al. (8). A 128-bp region (nucleotides 363 to 490) containing high concentrations of single nucleotide polymorphisms (SNPs), which were conserved within V. coralliilyticus strains but differed from non-V. coralliilyticus strains, was identified, and oligonucleotide primers Vc_dnaJ_F1 (5⬘-CGG TTC GYG GTG TTT CAA AA-3⬘) and Vc_dnaJ_R1 (5⬘-AAC CTG ACC ATG ACC GTG ACA-3⬘) and a TaqMan probe, Vc_dnaJ_TMP (5⬘-6-FAM-CAG TGG CGC GAA G-MGBNFQ-3⬘; 6-FAM is 6-carboxyfluorescein and MGBNFQ is molecular groove binding nonfluorescent quencher), were designed to target this region. The qPCR assay was optimized and validated using DNA extracted from V. coralliilyticus isolates, nontarget Vibrio species, and other bacterial species grown in marine broth (MB) (Table 1), under the following optimal conditions: 1⫻ TaqMan buffer A, 0.5 U of AmpliTaq Gold DNA polymerase, 200 ␮M deoxynucleotide triphosphates (with 400 ␮M dUTP replacing deoxythymidine triphosphate), 0.2 U of AmpErase uracil N-glycosylase (UNG), 3 mM MgCl2, 0.6 ␮M each primer, 0.2 ␮M fluorophore-labeled TaqMan, 1 ␮l of template, and sterile MilliQ water for a total reaction volume to 20 ␮l. All assays were conducted on a RotoGene 300 (Corbett Research, Sydney, Australia) real-time analyzer with the following cycling parameters: 50°C for 120 s (UNG activation) and 95°C for 10 min (AmpliTaq Gold DNA polymerase activation), followed by 40 cycles of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). During the annealing/extension phase of each thermal cycle, fluorescence was measured in the FAM channel (470-nm excitation and 510-nm detection). The qPCR assay specifically detected 12 out of 13 isolated V. coralliilyticus strains tested in this study (Table 1). The exception was one Caribbean strain (C2), which failed to give specific amplification despite repeated attempts. Positive detection of the target gene segment was determined by the increase in fluorescent signal beyond the fluorescence threshold value (normalized fluorescence, 0.010) at a specific cycle, referred to

Vibrio coralliilyticus has recently emerged as a coral pathogen of concern on reefs throughout the Indo-Pacific. It was first implicated as the etiological agent responsible for bleaching and tissue lysis of the coral Pocillopora damicornis on Zanzibar reefs (2). More recently, V. coralliilyticus has been identified as the causative agent of white syndrome (WS) outbreaks on several Pacific reefs (14). WS is a collective term describing coral diseases characterized by a spreading band of tissue loss exposing white skeleton on Indo-Pacific scleractinian corals (16). V. coralliilyticus is an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease (13) and therefore provides an ideal model for the development of diagnostic assays to detect coral disease. Current coral disease diagnostic methods, which are based primarily upon field-based observations of macroscopic disease signs, often detect disease only at the latest stages of infection, when control measures are least effective. The development of diagnostic tools targeting pathogens underlying coral disease pathologies may provide early indications of infection, aid the identification of disease vectors and reservoirs, and assist managers in developing strategies to prevent the spread of coral disease outbreaks. In this paper, we describe the development and validation of a TaqMan-based real-time quantitative PCR (qPCR) assay that targets a segment of the V. coralliilyticus heat shock protein 40-encoding gene (dnaJ). Nucleotide sequences of the dnaJ gene were retrieved from relevant Vibrio species, including V. coralliilyticus (LMG 20984), using the National Center for Biotechnology Information’s (NCBI) Entrez Nucleotide Database search tool (http: //www.ncbi.nlm.nih.gov/). Gene sequences of strains not available in public databases (V. coralliilyticus strains LMG 21348, LMG 21349, LMG 21350, LMG 10953, LMG 20538, LMG 23696, LMG 23691, LMG 23693, LMG 23692, and LMG

* Corresponding author. Mailing address: Australian Institute of Marine Science, PMB3, Townsville 4810, Australia. Phone: 61 7 47534139. Fax: 61 7 47725852. E-mail: [email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 4 June 2010. 5282

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TABLE 1. Species, strain, and threshold cycle for all bacterial strains testeda Strainb

Species

Vibrio coralliilyticus

LMG 23696

LMG 21348

Nelly Bay, Magnetic Island, Australia Majuro Atoll, Republic of Marshall Islands Nikko Bay, Palau Nikko Bay, Palau Nikko Bay, Palau Indian Ocean, Zanzibar, Tanzania Red Sea, Eilat, Israel

LMG 21349

Red Sea, Eilat,

LMG 21350

Red Sea, Eilat,

LMG 10953

Kent, United Kingdom

LMG 20538

Atlantic Ocean, Floriano ´polis, Brazil Caribbean Sea, La Parguera, Puerto Rico Caribbean Sea, La Parguera, Puerto Rico

LMG 23691 LMG LMG LMG LMG

23693 23692 23694 20984T

C1 C2 Vibrio alginolyticus Vibio brasiliensis Vibrio calviensis Vibrio campbellii Enterovibrio campbellii Alliivibrio fischeri Vibrio fortis Vibrio furnissii Vibrio harveyi Vibrio natriegens Vibrio neptunius Vibrio ordalii Vibrio parahaemolyticus Vibrio proteolyticus Vibrio rotiferianus Vibrio splendidus Vibrio tubiashii Vibrio xuii Escherichia coli Psychrobacter sp. Shewanella sp.

Origin

ATCC 17749 DSM 17184 DSM 14347 ATCC 25920T LMG 21363 DSM 507 DSM 19133 DSM 19622 DSM 19623 ATCC 14048 LMG 20536 ATCC 33509 ATCC 17802 ATCC 15338 LMG 21460 ATCC 33125 ATCC 19109 LMG 21346 ATCC 25922 AIMS 1618 AIMS C041

CT ⫾ SEMc

dnaJ gene sequence accession no.

Reference

Montipora aequituberculata Acropora cytherea

12.43 ⫾ 0.20

HM215570

14

14.07 ⫾ 1.33

HM215571

14

Pachyseris speciosa Pachyseris speciosa Pachyseris speciosad Pocillopora damicornis Pocillopora damicornis Pocillopora damicornis Pocillopora damicornis Crassostrea gigas (oyster) larvae Nodipecten nodosus (bivalve) larvae Pseudopterogorgia americana Pseudopterogorgia americana

10.83 ⫾ 2.76 9.40 ⫾ 0.36 12.54 ⫾ 0.24 12.80 ⫾ 0.71

HM215572 HM215573 HM215574 HM215575

14 14 14 2

13.81 ⫾ 0.49

HM215576

3

12.98 ⫾ 0.94

HM215577

3

11.49 ⫾ 0.19

HM215578

3

10.53 ⫾ 0.40

HM215579

3

12.13 ⫾ 0.50

HM215580

3

14.53 ⫾ 0.28

HM215568

15

HM215569

15

Host organism

NA 33.74 ⫾ 0.33 37.84† 27.06 ⫾ 0.52 39.10† 37.33 ⫾ 2.41 31.36 ⫾ 1.42 NA NA NA 28.56 ⫾ 0.60 NA 25.56 ⫾ 0.41 NA 30.00 ⫾ 0.89†† NA 32.31 ⫾ 0.82 NA NA NA NA 25.34 ⫾ 0.45

a

Origin, host organism, and dnaJ gene sequence accession numbers are shown for V. coralliilyticus strains. Strain designations beginning with LMG were derived from the Belgian Coordinated Collections of Microorganisms, ATCC strains are from the American Type Culture Collection, DSM strains are from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH culture collection, AIMS strains are from the Australian Institute of Marine Science culture collection, and C1 and C2 were provided by Pamela Morris. c †, amplification in one of three reactions; ††, amplification in two of three reactions; NA, no amplification. d Isolated from seawater above coral. b

as the threshold cycle (CT). Specific detection was further confirmed by gel electrophoresis, which revealed a PCR product of the correct theoretical size (128 bp) (data not shown), and DNA sequencing, which confirmed the target amplified product to be a segment of the dnaJ gene. No amplification with the assay was detected for 13 other closely related Vibrio strains, including the closely related Vibrio neptunius and two non-Vibrio species (Table 1). A total of five other Vibrio strains and one non-Vibrio strain (Shewanella sp.) exhibited CT values less than the cutoff of 32 cycles. However, CT values for these strains (mean ⫾ standard error of the mean [SEM], 27.96 ⫾

2.40) were all much higher than those for V. coralliilyticus strains (12.30 ⫾ 1.52), and no amplicons were evident in postqPCR gel electrophoresis (data not shown). The detection limit for purified V. coralliilyticus genomic DNA was 0.1 pg of DNA, determined by performing 10-fold serial dilutions (100 ng to 0.01 pg per reaction), followed by qPCR amplification. Similarly, qPCR assays of serial dilutions of V. coralliilyticus (LMG 23696) cells cultured overnight in MB (108 CFU ml⫺1 to extinction) were able to detect as few as 104 CFU (Fig. 1). Standard curves revealed a strong linear negative correlation between CT values and both DNA and cell

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FIG. 1. Standard curves delineating threshold (CT) values of fluorescence for indicators of pathogen presence: (A) concentration of V. coralliilyticus DNA and (B) number of V. coralliilyticus cells in pure culture. Error bars indicate standard error of the mean for three replicate qPCRs.

concentrations of V. coralliilyticus over several orders of magnitude, with r2 values of 0.998 and 0.953 for DNA and cells, respectively (Fig. 1). Little interference of the qPCR assay was observed when purified V. coralliilyticus (LMG 23696) DNA (10 ng) was combined with 10-fold serial dilutions (0.01 to 100 ng per reaction) of non-V. coralliilyticus DNA (i.e., Vibrio campbellii [ATCC 25920T]). Over the entire range of nontarget DNA concentrations tested, the resulting CT values (mean ⫾ SEM, 17.76 ⫾ 0.53) were not significantly different from those of a control treatment containing 10 ng of V. coralliilyticus DNA and no nonspecific DNA (16.75 ⫾ 0.18; analysis of variance [ANOVA], P ⫽ 0.51) (Table 2). Detection of V. coralliilyticus (LMG 23696) bacterial cells (104, 105, 106, 107, or 108 CFU per ml) in a background of non-V. coralliilyticus cells (i.e., V. campbellii [ATCC 25920T] at 0, 10, 104, or 107 CFU per ml) showed little reduction in assay sensitivity (see Fig. S1 in the supplemental material). For example, when V. coralliilyticus was seeded at 107 cells with similarly high concentrations of nontarget cells, little inhibition of the assay was observed. The assay’s detection limit in seawater was tested by inoculating 10-fold serial dilutions of V. coralliilyticus (LMG 23696) cultures (grown overnight in MB medium, pelleted at 14,000 rpm for 10 min, and washed twice with sterile phosphate-

APPL. ENVIRON. MICROBIOL.

buffered saline [PBS]) into 1 liter of seawater (equivalent final concentrations were 106 to 1 CFU ml⫺1). The entire volume of V. coralliilyticus-seeded seawater was filtered through a Sterivex-GP filter (Millipore), and DNA was extracted using the method described by Schauer et al. (11). The lowest detection limit for V. coralliilyticus cells seeded into seawater was 1 CFU ml⫺1 (Fig. 2), with no detection in a 1-liter volume of an unseeded seawater negative control. Standard curves revealed a strong correlation between CT values and the concentrations of V. coralliilyticus bacteria seeded into the seawater over several orders of magnitude (r2 of 0.968) (Fig. 2). The detection limit in seeded coral tissue homogenate was determined by seeding 10-fold dilutions (1010 to 103 CFU ml⫺1) of pelleted, PBS-washed and resuspended (in 10 ml of sterile PBS) V. coralliilyticus cells onto healthy fragments (⬃10 cm2) of the coral Montipora aequituberculata collected from Nelly Bay (Magnetic Island, Australia). Corals were collected in March 2009 and maintained in holding tanks supplied with flowthrough ambient seawater. Resuspended cells were inoculated onto M. aequituberculata fragments, each contained in an individual 3.8-liter plastic bag, allowed to sit at room temperature for 30 min, and then air brushed with compressed air until only white skeleton remained. One-milliliter aliquots of the resulting slurry (PBS, bacteria, and coral tissue) was vortexed for 10 min at 14,000 rpm, and DNA was extracted using a PowerPlant DNA Isolation Kit (Mo Bio, Carlsbad, CA). The lowest detection limits for V. coralliilyticus cells seeded onto coral fragments was 104 CFU per cm2 of coral tissue (Fig. 2). Again, standard curves revealed a strong correlation between CT values and the concentrations of seeded bacteria over several orders of magnitude (r2 of 0.981) (Fig. 2). When a 1-ml aliquot of the slurry was also inoculated into 25 ml of MB and enriched for 6 h at 28°C (with shaking at 170 rpm), the detection limit increased by 1 order of magnitude, to 103 CFU of V. coralliilyticus per cm2 of coral tissue (Fig. 2). The slope of the standard curve reveals some inhibition, particularly at the highest V. coralliilyticus concentrations, which could result from lower replication rates in the cultures with the highest bacterial densities (i.e., 109 CFU). However, since this effect is most pronounced only at the highest bacterial concentrations, the detection limit is still valid. In all trials, unseeded coral fragments and enrichment cultures derived from uninoculated coral fragments served as negative controls. The current study describes the first assay developed to

TABLE 2. Effect of nontarget bacterial DNA on the detection of 10 ng of purified V. coralliilyticus DNA Amt of nontarget DNA (ng)

CT (mean ⫾ SEM)

100 ................................................................................. 16.97 ⫾ 0.33 10 ..................................................................................... 16.9 ⫾ 0.08 1 .......................................................................................16.74 ⫾ 0.10 0.1 .................................................................................... 17 ⫾ 0.09 0.01 ..................................................................................16.37 ⫾ 0.43 0a .....................................................................................16.75 ⫾ 0.18 NTCb ...............................................................................35.04 ⫾ 0.02 a V. coralliilyticus (LMG 23696) DNA (10 ng) free of nontarget DNA and cells served as positive controls. b A qPCR mixture containing no bacterial DNA served as a no-template, or negative, control (NTC).

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FIG. 2. Standard curves showing CT values of the fluorescent signal versus the number of V. coralliilyticus cells per ml seawater (ƒ), and cells per cm2 of M. aequituberculata tissue, with (E) or without ( 䡠 ) enrichment. Each dot represents an independent experiment. Error bars indicate standard error of the mean for three replicate qPCR runs.

detect and quantify a coral pathogen using a real-time quantitative PCR (qPCR) approach. While previous studies have utilized antibodies or fluorescent in situ hybridization (FISH) to detect coral pathogens (1, 6), the combination of high sensitivity and specificity, low contamination risk, and ease and speed of performance (5) make qPCR technology an ideal choice for rapid pathogen detection in complex hosts, such as corals. The assay developed is highly sensitive for V. coralliilyticus, detecting as few as 1 CFU ml⫺1 of seawater and 104 CFU cm⫺2 of coral tissue (103 CFU cm⫺2 of coral tissue with a 6-h enrichment). These detection limits are likely to be within biologically relevant pathogen concentrations. For example, antibodies for specific detection of the coral bleaching pathogen Vibrio shiloi showed that bacterial densities reached 8.4 ⫻ 108 cells cm⫺3 1 month prior to maximum visual bleaching signs on the coral Oculina patagonica (6). Each seeded seawater and coral (enriched and nonenriched) dilution assay was performed in triplicate. The linearity of the resulting standard curves indicates consistent extraction efficiencies over V. coralliilyticus concentrations spanning 6 orders of magnitude (Fig. 2) and provides strong support for the robustness of the assay. In addition, the presence of competing, non-V. coralliilyticus bacterial cells and DNA had a minimal impact on the detection of V. coralliilyticus. This is an important consideration for accurate detection within the complex coral holobiont, where the target organism is present within a matrix of other microbial and host cells. V. coralliilyticus, like V. shiloi (10), is becoming a model pathogen for the study of coral disease. Recent research efforts have characterized the organism’s genome (W. R. Johnson et al., submitted for publication), proteome (N. E. Kimes et al., submitted for publication), resistome (15), and metabolome (4) and enhanced our understanding of the genetic (7, 9) and physiological (7, 13) basis of its virulence. Before effective management response plans can be formulated, however, continuing research on the genetic and cellular aspects of V. coralliilyticus must be complemented with knowledge of the epide-

miology of this pathogen, including information on its distribution, incidence of infection, and rates of transmission throughout populations. The V. coralliilyticus-specific qPCR assay developed in this study will provide important insights into the dynamics of pathogen invasion and spread within populations (6) while also aiding in the identification of disease vectors and reservoirs (12). These capabilities will play an important role in advancing the field of coral disease research and effective management of coral reefs worldwide. This work was supported in part by an Australian Research Council DP to B. L. Willis, a National Science Foundation Biodiversity Surveys and Inventories Grant (DEB0516347) to P. J. Morris, and a Fulbright Postgraduate Fellowship to F. J. Pollock. We thank Rose Cobb, Vivian Cumbo, Rochelle Soo, Kimberley Lema, and Jean-Baptiste Raina for their assistance in the laboratory and for many lively and interesting discussions. Also, we thank Lone Hoj for supplying many of the bacterial strains used in this study. Finally, we thank Megan Kent for her assistance in the preparation of the manuscript. REFERENCES 1. Ainsworth, T. D., M. Fine, L. L. Blackall, and O. Hoegh-Guldberg. 2006. Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities. Appl. Environ. Microbiol. 72:3016–3020. 2. Ben-Haim, Y., and E. Rosenberg. 2002. A novel Vibrio sp. pathogen of the coral Pocillopora damicornis. Mar. Biol. 141:47–55. 3. Ben-Haim, Y., F. L. Thompson, C. C. Thompson, M. C. Cnockaert, B. Hoste, J. Swings, and E. Rosenberg. 2003. Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis. Int. J. Syst. Evol. Microbiol. 53:309–315. 4. Boroujerdi, A. F., M. I. Vizcaino, A. Meyers, E. C. Pollock, S. L. Huynh, T. B. Schock, P. J. Morris, and D. W. Bearden. 2009. NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus. Environ. Sci. Technol. 43:7658–7664. 5. Espy, M. J., J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter, J. D. Yao, N. L. Wengenack, J. E. Rosenblatt, F. R. Cockerill III, and T. F. Smith. 2006. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin. Microbiol. Rev. 19:165–256. 6. Israely, T., E. Banin, and E. Rosenberg. 2001. Growth, differentiation and death of Vibrio shiloi in coral tissue as a function of seawater temperature. Aquat. Microb. Ecol. 24:1–8. 7. Meron, D., R. Efrony, W. R. Johnson, A. L. Schaefer, P. J. Morris, E. Rosenberg, E. P. Greenberg, and E. Banin. 2009. Role of flagella in virulence

5286

8.

9.

10. 11.

12.

POLLOCK ET AL.

of the coral pathogen Vibrio coralliilyticus. Appl. Environ. Microbiol. 75: 5704–5707. Nhung, P. H., M. M. Shah, K. Ohkusu, M. Noda, H. Hata, X. S. Sun, H. Iihara, K. Goto, T. Masaki, J. Miyasaka, and T. Ezaki. 2007. The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species. Syst. Appl. Microbiol. 30:309–315. Pollock, F. J., B. Wilson, W. R. Johnson, P. J. Morris, B. L. Willis, and D. G. Bourne. 8 February 2010. Phylogeny of the cosmopolitan coral pathogen Vibrio coralliilyticus. Environ. Microbiol. Rep. doi:10.1111/j.17582229.2009.00131.x. Rosenberg, E., C. A. Kelloff, and F. Rohwer. 2007. Coral microbiology. Oceanography 20:146–154. Schauer, M., R. Massana, and C. Pedros-Alio. 2000. Spatial differences in bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting. FEMS Microbiol. Ecol. 33:51–59. Sussman, M., Y. Loya, M. Fine, and E. Rosenberg. 2003. The marine fireworm Hermodice carunculata is a winter reservoir and spring-summer vector

APPL. ENVIRON. MICROBIOL.

13.

14.

15.

16.

for the coral-bleaching pathogen Vibrio shiloi. Environ. Microbiol. 5:250– 255. Sussman, M., J. C. Mieog, J. Doyle, S. Victor, B. L. Willis, and D. G. Bourne. 2009. Vibrio zinc-metalloprotease causes photoinactivation of coral endosymbionts and coral tissue lesions. PLoS One 4:e4511. Sussman, M., B. L. Willis, S. Victor, and D. G. Bourne. 2008. Coral pathogens identified for White Syndrome (WS) epizootics in the Indo-Pacific. PLoS One 3:e2393. Vizcaino, M. I., W. R. Johnson, N. E. Kimes, K. Williams, M. Torralba, K. E. Nelson, G. W. Smith, E. Weil, P. D. Moeller, and P. J. Morris. 2010. Antimicrobial resistance of the coral pathogen Vibrio coralliilyticus and Caribbean sister phylotypes isolated from a diseased octocoral. Microb. Ecol. 59:646–657. Willis, B. E., C. A. Page, and E. A. Dinsdale. 2004. Coral disease on the Great Barrier Reef, p. 69–104. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag Publishing, Berlin, Germany.