The Coral Health and Disease Consortium:
New Information on Coral Disease

During January 22-25, 2002, I was an invited participant in the first official workshop, "Coral Health and Disease: Developing a National Research Plan," of the Coral Disease and Health Consortium (CDHC). The CDHC was created in response to the Coral Reef Task Force's National Action Plan with the expressed purpose of organizing and coordinating scientific resources to focus specifically on coral health issues (Woodley 2002).

The workshop participants were experts in varied disciplines, and included those involved in coral disease research, as well as people from veterinary medicine, the health profession, and biotechnology. This cross section of disciplines was an intentional effort to enlist techniques and ideas from various fields to help accelerate the slow progression of advances in the study of coral disease.

Before delving into the proceedings and results of the workshop, I will briefly make a few comments regarding this event - several findings that came as somewhat of a surprise to me. Since the mid-to late-1990's, I have been suggesting that coral disease is likely to have a strong correlation to stress, and that coral immunity should be looked at in terms of its relation to coral disease (Borneman and Lowrie 1998a, 1998b). My initial thoughts were met with some controversy, yet I felt - and feel - strongly enough about it that I have made it the focus of my doctoral dissertation work. It came as a quite a pleasant surprise to see that a focus of this workshop was towards beginning to look at coral immunity and the relation of stress to coral disease. In fact, our working group (one of only four) was designated with the title "Environmental Factors Affecting Susceptibility and Infectivity." To say I was thrilled to be part of this group would be an understatement.

Paradoxically, this same workshop gave me some considerable measure of pause regarding my thoughts on the roles of pathogens in coral disease. For an equally long period of time, I have been adamantly saying that the number of people suggesting that "mystery pathogens and bacteria" seem to be the root of all coral disease were probably mistaken. I feel more strongly than ever, and based largely on the amount of supporting research in this area (Herndl and Velimirov 1986, Koh 1997, Paul et al. 1986, Rowher et al. 2001, Santavy 1995, Shasar 1994), that bacteria and corals are associatively linked and in most cases have benign or even beneficial relationships. However, I am also more convinced that bacterial pathogens may be more prevalent or have a greater role in disease than I had previously wanted to admit. I hope that this view will be clarified over the following article.

The White Papers

Prior to the workshop, and included in the materials given to us, were a number of papers by coral disease researchers that summarized current status in the field or that presented new information that was either currently in press or being submitted for publication. Thus, we got a "sneak preview" of some exciting work. Unfortunately, I cannot relate the exact nature of some of this material until it has been published. There is plenty, though, that I can relate.

Coral Histology

Esther Peters, one of the pre-eminent coral pathologists, presented information summarizing the use and progress of histological techniques used to study coral disease. Histology is the study of tissues and their structure function, usually through microscopy. She is a leader in this area, and in addition to showing some of the tools currently available to study histology in corals, she made several important comments. The study of the histopathology of corals is still in its infancy and those contributions in the etiology (factors that cause or play a role in the expression) of diseases will be helped by interdisciplinary studies, "incorporating information from analytical chemistry, biochemistry, microbiology, molecular biology, and physiology, as well as oceanography and ecology, and new developments in histotechniques." She pointed out how important histopathology is in understanding coral disease, and that so much work is need to determine how disease "might be the result of infections with different pathogens and/or physical or chemical stressors." (Peters 2002).

The Scope and Management of Coral Disease

Ernesto Weil presented material based on his current and upcoming publications discussing primarily the scope and range of coral diseases throughout the Caribbean (Weil 2002a, 2002b). He mentioned that coral disease is found in the Pacific, as well. Much of the study of coral disease is in the Caribbean, and Ernesto showed photos of disease events throughout the region. He also showed photos of new putative coral diseases, many of which I had also brought to show the group from my own diving in the region. A real question is, to what extent the signs of these diseases represent separate phenomena or are simply different appearances of the same phenomena? Answering this question is hampered greatly by the difficulty and slow progress in determining real causes of coral disease (and of course, one of the very reasons for this workshop).

This Colpophyllia natans, photographed at the Flower Garden Banks, had a highly aberrant mottled pattern on its surface that consisted of both bleached areas and necrotic areas. This is an undescribed condition that has been seen elsewhere (Weil pers comm), but the coral apparently recovered within several months (Wiseman pers. comm).

The darkened rings on this Montastraea faveolata in Puerto Rico is not normal. The tissue itself looked healthy, and the cause of this banding is not known or described.

The Diplora strigosa in these photos from the Flower Gardens were not unusual. Numerous colonies have this unusual patttern that seems to be a version of the common hypertrophic growths (tumors) that are evident on many corals in this area. The cause of this condition is unknown and undescribed.

This ring-type pattern is being seen more commonly in Sidearastrea siderea, a species well known to display the condition called Dark Spots Disease. There is no known cause for either condition, and the rings may be a variation of the more spotty Dark Spots. However, the consistency of variation in the signs of the disease, albeit with some overlap, is remarkable. Photographed at Mona Island.

These pale ring shapes were seen on several species of Diploria and Colpophyllia around Mona Island. The tissue appeared healthy, but the coloration was aberrant enough that we tagged the colonies for future observations. Apparently, the colonies had recovered their normal pigmentation several months later (Bruckner pers comm.).

This is a disease known as Ridge Mortality Disease and is only reported on Diploria sp. Some researchers feel that it is not a disease, but results from the nipping actions of damselfish. There are indeed cases where this is likely to be the case, but damselfish tend to work in patches and non-linearly along a ridge. Furthermore, no damsels were seen nesting around this colony. Photographed at the Flower Garden Banks.

A band of unknown origin on Siderastrea siderea. Photogrpahed at Southwater Caye, Belize.

This type of pattern would be called Patchy Necrosis by some definitions, but Patchy Necrosis is supposed to only occur on Acropora palmata. Photographed in Tavernier Key, Florida.

A new outbreak of Patchy Necrosis on A. palmata has recently been reported following a warm, still period in Parguera, Puerto Rico. I photographed this specimen many months earlier than the reported "outbreak" at the same location. Thse signs resemble "RTN" in aquarium corals.

This would ordinarily be called White Plague on Montastraea faveolata. However, Plague usually forms a solid line across the tissue, and across corallites. Here, the outline follows each polyp exactly. It is unusual in this regard. Photographed in Cozumel, Mexico.

Doctor Andrew Bruckner gave a presentation on his comprehensive (49 pages!) white paper entitled "Priorities for Effective Management of Coral Diseases." Andy suggested the following points as being important in the management of disease in his summary:

1)	an early warning system to predict and identify disease outbreaks;
2)	documentation of spatial distribution and temporal variations of coral diseases and other syndromes at local to global scales;
3)	elucidation of relationships of environmental stressors, localized anthropogenic impacts, and widespread phenomena such as global warming and El Niño on coral health, disease, degradation and recovery;
4)	development of standardized terminology for diseases and other syndromes through a characterization of the visual appearance, pathology and etiology, and the development of molecular probes and other tools to identify and verify diseases in the field;
5)	identification of factors that facilitate the introduction, spread and transmission of pathogens;
6)	research on the effects of disease on coral species and populations, associated species, and ecosystem structure and function; and
7)	implementation of measures to mitigate disease impacts, including strategies that reduce anthropogenic stressors responsible for the proliferation or spread of diseases and the development of novel techniques to treat affected corals and improve habitat quality.
	(Bruckner, 2002)

Laurie Richardson and Richard Aronson also had a paper outlining the various techniques being currently used in coral disease, the need for interdisciplinary action, and the relatively poor state of knowledge that exists, given the impact and extent of current coral diseases (Richardson and Aronson 2002). Garriet Smith summarized his research and progress in isolating coral pathogens and in determining the extent to which they represent normal or abnormal microbial flora to coral surfaces in a presentation titled, "Disease Identification: Technologies." There was also an excellent presentation by Dr. Jonas Almeida, titled "Bioinformatics and Coral Disease," on the use of bioinformatics and neural networks to begin working with databases to increase relative performance of existing data and to help detect trends not easily available with current non-centralized efforts.

Despite the quality and importance of these papers and presentations, there were several others that, for me, "stole the show." I will limit the remainder of this article to discussing aspects of those talks, and perhaps delve into results of the workshop in the next article.

Standards for Disease in Other Fields

Pam Parnell of the Clemson Veterinary Diagnostic Center presented ideas that were to become a focal point of the entire workshop in her presentation titled, "Disease Investigation: The Process." In particular, her words have great importance to aquarists, as well. Coral disease research has been somewhat of a reactive science in that once a disease is noticed, frantic work is begun to try and uncover the etiology of the disease. The goals and methods used by the community have little in common with established practice in other health areas, and she used her expertise in veterinary medicine to state the major points. First, in all medicine there is a very strict and complete language and terminology used to describe disease. This encompasses sometimes exceedingly intricate detail in describing lesions and other aspects of the disease. The language is developed such that everyone involved in the field can communicate and know exactly what is being described. No such language exists in the coral disease field. This stems, in part, from our relative ignorance about exactly what these things are, but another part is a lack of cohesiveness among the researching body. Without being able to describe and articulate what various observers are seeing, the understanding of epizootics and disease will remain stifled. This problem is apparent in the terminology of the diseases where terms such as White Plague, White Plague Type II, and now White Plague type III exist. Similarly, Yellow Blotch Disease, Yellow Band Disease, and Yellow Line Disease may be used to describe the same condition. Arnfried Antonius, despite having been integral in the description of recognized "white" diseases (Antonius 1995), now recognizes this limitation of the nomenclature and lumps all diseases with unknown etiologies that are characterized by a white line delineating healthy tissue from denuded skeleton, as "White Syndromes."

Parnell also discussed the need for a centralized "center of operations" or communication center to facilitate the dissemination of reports, research, findings, databases, reference libraries, and discussions. Furthermore, she emphasized the need for a coral "guinea pig," a need also recognized almost unanimously by the attendees. In other words, a model coral needs to be established that can be used for both baseline information (genetics, physiology, etc.) and for disease studies.

For aquarists, these suggestions are also very important. In The Coral Forum on Reef Central, countless requests are made that describe a coral in poor health. It is very difficult to assess the actual nature of the problem, syndrome, or disease without being able to adequately communicate the event in question. In that regard, I will pass along those core standards to the hobby in a future publication, perhaps with practical revisions once they are established, so that we can effectively communicate within the hobby, as well. Furthermore, our observations, if properly documented and communicated, can be valuable additions to the coral disease body of knowledge, in general.

The Use of Biotechnology

Craig Downs is one of the founders of EnVirtue Biotechnologies. While not expressly involved with corals, Craig has various assays and technologies either available or potentially available to make coral disease work progress much more quickly. Many of the technologies he discussed in his talk, titled "Abiotic Factors Infecting Infectivity and Suceptibility," were very similar to ones being used in other fields for years. Michael Gerdes and Frank Marini, both Reef Central members, have discussed this many times with me. Michael works at NIH and astounds me with the technologies available in human medicine, as does Frank who works at the MD Anderson Cancer Research Center. They know the powerful nature of the available tools and how little they are being used in marine science. Fortunately, this may be changing. Among the many topics discussed was the use of conserved areas of the genome to probe for various stresses and for the identification of putative pathogens. Of particular interest were assays available for stress proteins that are nearly, if not totally, conserved among animals and even bacteria, such as ubiquitin, NADPH oxidase, heat shock proteins (particularly hsp60), and others. Also of great interest was the investigation of iron in coral tissue and coral surfaces. Iron is normally sequestered by organisms because bacteria need it for growth. It is an immunoresponsive action. While corals are not vertebrates, many basic immunological processes are similar, analogous, or are likely to be found but are as yet undetermined. Iron sequestration by molecules such as lactoferrin and transferrin, or analogues, would be easily investigated using existing methods and would likely be productive. Finally, Craig introduced another buzzword that many of us were already thinking about - apoptosis.

Cells die in one of two ways - necrosis and apoptosis. Necrosis is a passive degenerative process that entails that destructive enzymatic processes are involved.

So, what is the significance of apoptosis? Apoptosis is programmed and induced cellular suicide. The last few years have witnessed an explosion of interest and knowledge about apoptosis, the process by which a cell actively commits suicide. It is now well recognized that apoptosis is essential in many aspects of normal development and is required for maintaining tissue homeostasis, such as the limited lifespan and renewal of white blood cells. Failure to properly regulate apoptosis can have catastrophic consequences. Cancer and many diseases (AIDS, Alzheimer's disease, Parkinson's disease, heart attack, stroke, etc.) are thought to arise from deregulation of apoptosis. Apoptosis has emerged as a key biological regulatory mechanism (www.apopnet.com). In senescent organisms, apoptosis is hard wired into the genetic code, and numerous apoptosis genes have already been discovered (bcl-2, bax, bcl-Long, bcl-Short, caspase (several), and fas). However, apoptosis can also be triggered by stress, and bacteria are known that produce toxins and other molecules that can trigger apoptosis.

Several years ago, I argued that RTN had the appearance of autolysis (Borneman and Lowrie 1998a, 1998b). Autolysis has also been estimated as occurring with members of the family Xeniidae (Fabricius and Aldeslade 2001), Stylophora pistillata (Muller et al. 1986), and other corals as described in Borneman and Lowrie (1998a, 1998b). Over a year ago, I looked at an Acropora sp. from my tank that had contracted "RTN," as part of a coral histopathology workshop. Esther Peters described the tissue at the time as a coagulative necrosis. Under the microscope, the tissue was sloughing in blobs, with many zooxanthellae still intact within the blobs. There were no unusual bacteria noticed within the tissue, or external to the tissue. However, this was but a single sample and the fixation process could have removed external pathogens. Similarly, staining and light microscopy may not have been able to easily resolve intracellular bacteria. I am currently embedding more specimens with RTN for examination, but this is not the focus of this finding. The description of apoptosis is that apoptotic bodies are produced by a process called blebbing. What this means is that blobs of cellular material are produced as the cell kills itself. Needless to say, this will be an area of great interest and work for me in the coming years.

An Acropora sp. with RTN, also known as Shut Down Reaction, currently under study from Houston, Texas.

Trachyphyllia geoffroyi with RTN in Houston, Texas. Is this apoptosis?

A typical Shut Down Reaction, also known as RTN, in Galaxea fascicularis, photographed in Jakarta, Indonesia.

The Amazing Case of Vibrio shiloi.

Several years ago, an article appeared that described the bacteria, Vibrio shiloi, as causing bleaching in the Mediterranean coral, Oculina patagonica (Kushmaro 1996). At the time, most people were of the opinion that the conditions of this were unusual. It seemed to occur in a single species in a non-coral reef area. Most researchers were relatively unconcerned. Julian Sprung spoke vocally about this event in a discussion on NOAA's coral-list, and it was similarly met with some skepticism that it could be much of an issue for corals, in general. To be sure, I was one of them.

However, one could have heard a pin drop during the elegant and outstanding presentation by Dr. Eugene Rosenberg of Tel Aviv University (Rosenberg 2002). This man single handedly threw the proverbial monkey wrench into the coral world that morning. In the years since the original articles have been published, Rosenberg's team has not only fulfilled Koch's postulates for this pathogen in a textbook-like fashion, but has proceeded to describe the etiology in an extremely impressive manner. I would urge those interested to read the articles listed below that relate to this disease.

A wild Acropora cervicornis with classic Shut Down Reaction, or RTN, in my aquarium hours after being collected and shipped from Puerto Rico.

This Trachyphyllia geoffroyi is showing signs of a progressive and spreading-type bleaching. Is this bacterial bleaching? The coral is currently under study.

In short, Vibrio shiloi is a newly described species of bacteria, related to V. mediterranei, with an as-yet undetermined reservoir; that is, it is not known where or if the presence of this bacteria is normal to the environment, or if it is somehow just recently showing up to affect the area. It follows the temperature cycles of the area precisely, and causes bleaching in warm months followed by recovery as the water temperature declines.

To infect Oculina patagonica, V. shiloi forms an adhesion with a ß -galactoside receptor on the coral surface and is specific for the coral and the bacteria. One pathogen, one host (although V. shiloi has infected other corals in laboratory studies). The temperature at which adhesion occurs is critical, with adhesion only occurring if the bacteria (and not necessarily the coral) is grown at the elevated temperature required to induce the virulence factor. Even more recently, the receptor was found to exist in the coral mucus, and that photosynthesis by zooxanthellae is required for the synthesis and secretion of the receptor. Interestingly, it only takes 120 bacteria to cause an infection, and the bacteria can reproduce to 10⁹ bacteria/cm³ in five days!! With water cooling below the virulence temperature, the bacteria die rapidly.

Another fascinating aspect is that the virulence factor is, in fact, a housekeeping gene (a normal metabolic gene). Superoxide dismutase (SOD) is produced both by corals and V. shiloi at high temperatures. Mutants of V. shiloi which lack the gene to make SOD, adhere to the coral, penetrate it, and die from the oxygen radicals produced by the photosynthesizing zooxanthellae. It may be that the zooxanthellae have a function in protecting corals from infection.

Another virulence factor of V. shiloi is a type of extracellular proline rich toxin that can inhibit photosynthesis of zooxanthellae by forming a membrane channel that allows NH3 to pass, changing the pH gradient that exists across the algal membrane, and blocking photosynthesis. It, and other as yet uncharacterized high molecular weight toxins, then bleach and lyse the algal cells. The levels of toxin production are also correlated with the high temperature.

The reader may ask the same question that has occurred before, and was described above. So what? It's a Vibrio that is found not on coral reefs, but is specific to one coral species that we don't keep and will likely never see. The implications are certainly interesting, but what does it mean to tropical corals? Rosenberg had an answer to this, too. Knowing the skepticism that existed in the community, he has recently gone into the Indian Ocean and the Red Sea and looked at bleached Pocillopora damicornis. Is everyone ready?

A new species of bacteria, Vibrio corallyticus, was consistently found in the tissues of the bleached Pocillopora at a level that already fulfills the first of Koch's postulates. The virulence is even more amazing. At 23° C, there are no visible signs of disease. At 25° C, bleaching occurs. At 27° C, there is rapid tissue lysis. A virulence factor is being produced by this bacteria that correlates extremely well with the temperatures commonly cited as causing coral bleaching. Furthermore, Rosenberg describes the bleaching as spreading; a characteristic seen all too often by both field observers and aquarists.

The implications of Rosenberg's work are almost indescribable. He is of the opinion that probably all bleaching is caused by bacteria. Unfortunately, there are many studies where bleaching has been caused by low temperature, UV radiation, darkness, chemicals, etc. (see Borneman 2002). However, the importance of looking at bleaching in an entirely new light is now at hand. It has often been questioned why corals in the wild would bleach with only a 1-2° C temperature change when other areas (including tanks) routinely experience far greater vacillations without any bleaching incidence. The fact that virulence can be expressed with this small temperature increase makes such accounts explainable. Furthermore, with temperatures in the oceans having warmed over the past fifty years, and with bleaching events being more common in recent years, the existence of bacterial bleaching under such temperature increases may explain not only the increased incidence of bleaching, but also explain why mortality is so common in some bleaching events while recovery happens in others.

As an example, if corals have been growing in water averaging 26° C, more or less, for the past several thousand years, and over the past fifty years the temperatures in the water are now 27° C. Virulence of a microbe is expressed at 28° C to cause bleaching. Now, it only takes a 1° C change to cause virulence genes to be turned on and cause bleaching, and this occurs much more frequently than the 2° C change that it took previously. Furthermore, if the water temperature gets to 29° C, it may not be that the corals have exceeded their upper thermal limit, but that virulence genes that cause tissue lysis have been expressed.

Several points regarding this work should be made, however. First, Oculina patagonica is a facultatively zooxanthellate temperate to sub-tropical coral. It is not from coral reefs, and as far as we know, neither is the Vibrio that causes a problem. The results with a bleaching Vibrio may still be relatively unique to this coral and bacterium. Second, the results involving P. damicornis and V. corallyticus are in their infancy. The degree to which bacteria play a role in any other events is a long way away, and no conclusions should be drawn at this point regarding other similar events. The water temperatures were low compared to most reef areas and other factors (both biotic and abiotic) have not yet been fully considered in this finding. The potential implications are what are notable.

The major questions remaining to be answered are many. First, to what extent are bacteria involved in bleaching events? What is the variation in virulent species or strains? Are these normal microbial flora that are heat-activated opportunists? Are they new to the environment? What is their reservoir and why are they now being expressed? Are there immune mechanisms that can deal with these pathogens, and to what degree do various stressors act in the expression of infectivity and susceptibility?

I have recently prepared samples of a Trachyphyllia geoffroyi with spreading signs of bleaching, and of a foliose Montipora sp. with similar signs from another aquarium. This latter case was especially intriguing as another colony of a different species of Montipora (M. digitata) had "caught" the spread of bleaching. No other corals in the tank were bleached. Rest assured I will report what I find at a future date.

A Stylophora pistillata from a store in Houston, Texas displaying a classic white band-type disease. The coral is currently under study. Interestingly, the band line stopped immediately upon the coral’s placement in my aquarium (after sampling the tissue) and is recovering.

A Hydnophora sp. from a store in Houston Texas, also with a white syndrome. Here, the tissue was slowly receding, but the tissue was actually free at the disease line margin and appeared to be released from its skeleton.

As a final note to this incredible tale, and as if the reader has not had enough already, Rosenberg also found that Oculina in shallow water, even in high temperature and exposed to V. shiloi, rarely bleached. They found that UV radiation acted as an effective sterilizer for V. shiloi on the coral surface!

To be continued......