Phylogeny of the coral pathogen Vibrio coralliilyticus and the development of a qPCR-based diagnostic assay for its detection

Coral disease has emerged over recent decades as a significant threat to coral reef ecosystems, with declines in coral cover and diversity of Caribbean reefs providing an example of the impacts of disease at regional scales. If similar trends are to be mitigated or avoided on reefs worldwide, a deeper understanding of the factors underlying the origin and spread of coral diseases, as well as the steps that can be taken to prevent, control, or reduce their impacts is required. In recent years, an increased focus on coral microbiology has revealed several coral pathogens that could serve as targets for a new generation of coral disease diagnostic tools. The development and implementation of novel diagnostic techniques, which specifically target these pathogens, will allow accurate coral disease detection and diagnosis, pathogen load monitoring, and identification of pathogen sources, vectors, and reservoirs. These capabilities will allow reef managers to discern the threats that may affect the occurrence, prevalence, or severity of diseases so their sources can be identified, and possibly reduced through better management practices. In the first chapter, I establish the need for sensitive and specific molecular-based coral pathogen detection, outline the emerging technologies that could serve as the basis of a new generation of coral disease diagnostic assays, and address the unique challenges inherent to the application of these techniques to environmentally-derived coral samples.\ud \ud The coral pathogen Vibrio coralliilyticus represents a good model system to study coral disease. It has been implicated as the etiological agent responsible for bleaching and tissue lysis in a number of scleractinian coral species throughout the Indo-Pacific and has been the focus of research efforts characterizing the organism's genome, proteome, and metabolome. Little is known, however, about the population genetics of V. coralliilyticus, its evolutionary history, or the population dynamics of this widely distributed species. In order to determine whether this bacterium exists as a single cosmopolitan clonal population, which might indicate rapid spread of a pandemic strain, or is grouped into endemic and genotypically distinct strains, a phenotypic and phylogenetic comparison of geographically disparate isolates was conducted. Five phylogenetic marker genes (16S, rpoA, recA, pyrH, and dnaJ) frequently used for discriminating closely related Vibrio species and a zincmetalloprotease gene (vcpA) linked to pathogenicity were sequenced in thirteen isolates collected from the Red and Caribbean Seas, and Indian, Pacific, and Atlantic Oceans. No evidence of clonality or consistent lineage structure was observed, suggesting that V. coralliilyticus represents an endemic components of coral reef ecosystems that varies genetically among the globally distributed geographic locations sampled.\ud \ud To gain a more complete understanding of the epidemiology of V. coralliilyticus, including information on its distribution, incidence of infection, and rates of transmission through populations, a real-time quantitative PCR (qPCR)-based detection assay for V. coralliilyticus was developed. The assay, which targets the dnaJ gene, a housekeeping gene encoding for heat shock protein 40, was highly sensitive, detecting as little as 0.1 pg of purified V. coralliilyticus DNA and 104 colony forming units (CFU) per reaction (20 μL) for pure bacterial cultures. Inhibition of the assay by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. These findings support the utility of this assay to target the pathogen within the complex coral holobiont. This assay represents a novel approach to coral disease diagnosis and provides a useful tool for coral pathogen detection and accurate diagnosis, which will play a vital role in advancing the field of coral disease research