Complementary approaches to diagnosing marine diseases: a union of the modern and the classic.

Linking marine epizootics to a specific aetiology is notoriously difficult. Recent diagnostic successes show that marine disease diagnosis requires both modern, cutting-edge technology (e.g. metagenomics, quantitative real-time PCR) and more classic methods (e.g. transect surveys, histopathology and cell culture). Here, we discuss how this combination of traditional and modern approaches is necessary for rapid and accurate identification of marine diseases, and emphasize how sole reliance on any one technology or technique may lead disease investigations astray. We present diagnostic approaches at different scales, from the macro (environment, community, population and organismal scales) to the micro (tissue, organ, cell and genomic scales). We use disease case studies from a broad range of taxa to illustrate diagnostic successes from combining traditional and modern diagnostic methods. Finally, we recognize the need for increased capacity of centralized databases, networks, data repositories and contingency plans for diagnosis and management of marine disease.

Sphaerospora elwhaiensis sp.n. (Myxosporea: Sphaerosporidae) from landlocked sockeye salmon Oncorhynchus nerka (Salmoniformes: Salmonidae) in Washington State, USA.

A new species of sphaerosporid myxosporean, Sphaerospora elwhaiensis sp. n., is described from kidney of non-anadromous sockeye salmon (kokanee) Oncorhynchus nerka (Walbaum) from Lake Sutherland in the northern Olympic Peninsula, Washington, USA. Infection with the parasite was detected in 45% of 177 kokanee examined over 5 years. While conforming to the morphological criteria by which members of the genus are defined, the parasite is distinguished from congeners in salmonids of western North America by a unique combination of valvular sculpting of the myxospore, the relatively large size of the myxospore and monosporous development within the pseudoplasmodium. In addition, nucleotide sequences of the parasite's small and large subunit ribosomal RNA gene are unique. Phylogenetic analyses of these sequences suggested that the parasite is most closely related to freshwater Myxidium spp. and Zschokkella spp. The molecular data have provided further evidence for a polyphyletic association previously recognized among members of the genus and emphasize the need for a taxonomic revision of Sphaerospora Thélohan, 1892 and related genera.

A quantitative enzyme-linked immunosorbent assay and filtration-based fluorescent antibody test as potential tools to screen broodstock for infection with Flavobacterium psychrophilum.

There is strong evidence that Flavobacterium psychrophilum, the etiologic agent of coldwater disease, is transmitted vertically; it has been hypothesized that disease management at hatchery facilities can be improved through broodstock screening and implementation of culling programs. This paper describes the development of two assays used to screen broodstock tissues (kidney and ovarian fluid) for the presence of F. psychrophilum. Four monoclonal antibodies (MAbs) were generated against outer membrane preparations of F. psychrophilum strain CSF (Clear Springs Foods) 259-93. Of these, MAb FL43 was selected for assay development; this MAb reacted with 67 isolates of F. psychrophilum but exhibited no reaction with two strains of F. columnare or single strains of F. pectinovorum, F. aquatile, F. branchiophilum, and F. saccharophilum. An enzyme-linked immunosorbent assay (ELISA) was developed using MAb FL43 as the capture antibody and MAb FL43 conjugated to horseradish peroxidase (enzyme number 1.11.1.7; IUBMB 1992) as the secondary detection antibody. The ELISA had a lower F. psychrophilum detection boundary of approximately 1.6 X 10(3) colony-forming units (CFU)/mL in kidney tissue homogenates spiked with known bacterial concentrations. Asymptomatic broodstock of coho salmon Oncorhynchus kisutch (n = 50 fish) were sampled, and 100% tested positive for infection by ELISA analysis of kidney tissue; bacterial load was estimated at 2.0 x 10(3) to 9.4 x 10(3) CFU/mL. Ovarian fluid was also collected from these same coho salmon as well as from broodstock of rainbow trout O. mykiss; however, the ELISA proved to be unsuitable for use with ovarian fluid. A filtration-based fluorescent antibody test (FAT) was subsequently developed by conjugating MAb FL43 to Alexa Fluor 488. This FAT was able to detect F. psychrophilum in 74% of ovarian fluid samples collected from coho salmon and 42% of ovarian fluid samples from rainbow trout. Interestingly, yellow-pigmented bacteria were isolated on culture plates from 100% of kidney and ovarian fluid samples. All yellow-pigmented colonies were tested by polymerase chain reaction, and 100% of the coho salmon and rainbow trout were confirmed positive for infection with F. psychrophilum.