Vibrio coralliilyticus Strain OCN008 Is an Etiological Agent of Acute Montipora White Syndrome Blake Ushijima,a,b Patrick Videau,a Andrew H. Burger,b,c Amanda Shore-Maggio,a,b Christina M. Runyon,a,b Mareike Sudek,b,d Greta S. Aeby,b Sean M. Callahana,b,c Department of Microbiology, University of Hawai‘i, Honolulu, Hawai‘i, USAa; Hawai‘i Institute of Marine Biology, Ka៮ne‘ohe, Hawai‘i, USAb; Department of Molecular Biosciences and Bioengineering, University of Hawai‘i, Honolulu, Hawai‘i, USAc; Victoria University, Wellington, New Zealandd Identification of a pathogen is a critical first step in the epidemiology and subsequent management of a disease. A limited num- ber of pathogens have been identified for diseases contributing to the global decline of coral populations. Here we describe Vibrio coralliilyticus strain OCN008, which induces acute Montipora white syndrome (aMWS), a tissue loss disease responsible for substantial mortality of the coral Montipora capitata in Ka៮ne‘ohe Bay, Hawai‘i. OCN008 was grown in pure culture, recreated signs of disease in experimentally infected corals, and could be recovered after infection. In addition, strains similar to OCN008 were isolated from diseased coral from the field but not from healthy M. capitata. OCN008 repeatedly induced the loss of healthy M. capitata tissue from fragments under laboratory conditions with a minimum infectious dose of between 107 and 108 CFU/ml of water. In contrast, Porites compressa was not infected by OCN008, indicating the host specificity of the pathogen. A decrease in water temperature from 27 to 23°C affected the time to disease onset, but the risk of infection was not significantly reduced. Temperature-dependent bleaching, which has been observed with the V. coralliilyticus type strain BAA-450, was not observed during infection with OCN008. A comparison of the OCN008 genome to the genomes of pathogenic V. coralliilyticus strains BAA-450 and P1 revealed similar virulence-associated genes and quorum-sensing systems. Despite this genetic similarity, infections of M. capitata by OCN008 do not follow the paradigm for V. coralliilyticus infections established by the type strain. acteria have been implicated as etiological agents in many that include the mussel (Perna canaliculus) larva (22) and the rain- Bcoral diseases; however, a causal relationship between a bow trout (Oncorhynchus mykiss)(23). pathogen and disease has been established in only a limited num- Although various strains of V. coralliilyticus infect a broad ber (1–11). Disease presentation is frequently characterized by range of hosts, pathogenesis has been studied predominantly in one or both of two physical processes. Bleaching is the loss of the strains BAA-450 and P1, which infect the corals Pocillopora photosynthetic symbiotic zooxanthellae from coral cells (12), and damicornis and Montipora aequituberculata, respectively (4, 8, 20). tissue loss is necrosis and eventual sloughing of infected tissue that Water temperature plays a prominent role in the infection of P. exposes the calcium carbonate skeleton (13). Both of these re- damicornis by BAA-450 (24). Infections at temperatures from 24 sponses can lead to the death of entire coral colonies. Most corals to 25°C cause bleaching of the coral host, whereas infections at 27 appear to have a limited number of gross morphological responses to 29°C cause tissue loss, and BAA-450 is avirulent at temperatures to disease; tissue loss or bleaching can also result from environ- below 22°C. Increased concentrations of proteins that contribute mental stressors, so pathogen identification and precise disease to the virulence of other bacterial pathogens are present in or description are critical first steps toward understanding disease secreted by V. coralliilyticus strains BAA-450 and P1 at the higher processes (14). temperature range, perhaps explaining the temperature-depen- Koch’s postulates of disease causation have been utilized in the dent change in disease presentation (25, 26). The level of one field of coral disease research as guidelines for identifying a micro- protein, FlhA, increased 10-fold when the temperature was shifted organism, rather than an environmental stressor, as the cause of a from 24 to 27°C (26), and an intact flhA gene was required for specific disease (15). In general, a causal relationship between dis- chemotaxis, attachment, and virulence with the coral P. damicor- ease and pathogen is inferred after isolation of the pathogen from nis (27). diseased but not healthy hosts, isolation of the microorganism in In Ka៮ne‘ohe Bay, Hawai‘i, Montipora capitata, one of the major pure culture, experimental induction of disease in healthy hosts by reef-building corals, is affected by a tissue loss disease called Mon- the microorganism, and reisolation of the identical microorgan- tipora white syndrome (MWS) (28). MWS has two disease presen- ism from experimentally infected hosts. Five coral pathogens have fulfilled Koch’s postulates: Aurantimonas coralicida (16, 17), Ser- ratia marcescens (3, 18), Vibrio shiloi (19), Vibrio owensii (11), and Received 22 October 2013 Accepted 13 January 2014 Vibrio coralliilyticus (8, 20). Published ahead of print 24 January 2014 V. coralliilyticus is a pathogen of particular concern because it Editor: K. E. Wommack has broad host and geographic ranges. Strains of V. coralliilyticus Address correspondence to Sean M. Callahan, [email protected]. have been isolated from diseased corals in the Indian Ocean and Supplemental material for this article may be found at http://dx.doi.org/10.1128 Red Sea (4), the Caribbean (21), and the Great Barrier Reef and /AEM.03463-13. Micronesia (8), and strains have been shown to infect coral of the Copyright © 2014, American Society for Microbiology. All Rights Reserved. genera Pocillopora (20) and Pachyseris, Acropora, and Montipora doi:10.1128/AEM.03463-13 (8). V. coralliilyticus has also been shown to infect noncoral hosts 2102 aem.asm.org Applied and Environmental Microbiology p. 2102–2109 April 2014 Volume 80 Number 7 Vibrio coralliilyticus Strain OCN008 and Coral Disease tations: a progressive infection designated chronic MWS (cMWS) increments and ranging from 1% to 10% in 0.5% increments. Growth rate that is caused by Vibrio owensii strain OCN002 and is character- was determined with aerated liquid GASW cultures inoculated 1:10,000 ized by subacute tissue loss and a comparatively faster acute infec- from an overnight culture at 23 or 27°C in triplicate. Every hour for 36 h, tion designated acute MWS (aMWS) (11, 28). The infectious the optical density at 600 nm (OD600) was recorded, and dilutions were potential of aMWS was observed during outbreaks of rapid, wide- plated on solid GASW to calculate the numbers of CFU/ml. spread tissue loss in 2010 and 2011, with transmission observed Laboratory infection trials. Coral fragments were acclimatized to in- fection trial temperatures over the course of a day prior to inoculation. between neighboring coral colonies in contact with one another Seawater passed through a 0.2-␮m-pore-size filter (filtered seawater (G. S. Aeby, unpublished data). Tissue loss from corals with [FSW]) was used for all laboratory trials. Infection trials utilized a block aMWS was hypothesized to be caused by an infectious agent like design with fragments from the same colony exposed to FSW, control that which causes cMWS and not an environmental stressor. bacterium OCN004, or strain OCN008, as previously described (11), with This study focused on the pathogenesis of V. coralliilyticus the following minor modifications. Each replicate in each infection trial strain OCN008, which contrasts with the temperature-dependent was collected from a unique coral colony. Control bacterium OCN004 is infections of BAA-450. Koch’s postulates were fulfilled for strain a strain of Alteromonas previously isolated from healthy M. capitata (11). OCN008, establishing it as an etiological agent of acute Montipora Each fragment was tested separately in its own primary 4-liter container white syndrome. OCN008 induced tissue loss on M. capitata frag- filled with 3 liters of FSW. Each set of primary containers was maintained ments but not fragments of Porites compressa, suggesting host in a larger, freshwater-filled secondary container that was fitted with water pumps and heaters for temperature control. Water was heated to 23 or specificity for OCN008 infections. Comparative genomics showed ៮ that OCN008 possesses many of the putative virulence factors 27°C, the average low and high sea surface temperatures of Kane‘ohe Bay, respectively (32). For infection trials testing the effect of temperature, upregulated in strains BAA-450 and P1 in response to increases in fragments at 27°C had corresponding fragments from the same colony at temperature, despite the reduced influence of temperature on vir- 23°C to account for genotypic differences between coral colonies. ulence. OCN008 is the first pathogenic V. coralliilyticus strain iso- To prepare inocula for the infection trials, an overnight culture of the lated from Hawai‘i. desired strain was diluted to an OD600 of approximately 0.03 in GASW and incubated at either 23 or 27°C, depending on the infection tempera- MATERIALS AND METHODS ture. Cultures grown to an OD600 of 1.6 were washed and diluted with FSW to an OD of 0.8 for inoculation. Each tank was inoculated to a Coral collection, bacterial strains, and growth conditions. Samples of 600 final concentration of 108 CFU/ml of water unless indicated otherwise. Montipora capitata and Porites compressa for infection trials measured For determination of the minimal infectious dose, serial 10-fold dilutions approximately 3 by 3 by 1 cm and were collected from a fringing reef of the standard inoculum were used to generate final concentrations of surrounding the island Moku o Lo‘e in Ka៮ne‘ohe Bay, Hawai‘i, which is 107,106, and 105 CFU/ml of seawater. Cultures were diluted in FSW dominated by these two coral species (28).