Bruce L. Nicholson

Fish Cell Culture: An Update

Publisher Summary This chapter provides an overview of the techniques for the preparation and maintenance of fish cell cultures and also presents a more comprehensive view of the broad range of current and potential uses of such cell cultures. The methodologies and growth media for the preparation and maintenance of fish cell cultures generally do not differ from those used for the culture of cells from homeotherm vertebrates. The selection of fish species and appropriate tissues for the initiation of primary cell cultures is usually dictated by the cell type or function to be studied and/or the ultimate use of the cell culture. In many ways, the initiation of cell cultures from fish is actually easier than from homeotherm vertebrates. Most new fish cell cultures can be subcultured for varying periods of time before reaching senescence. The primary impetus for the initiation of fish cell cultures, and until recently the most widespread use of these cultures, has been for the isolation and characterization of fish viruses that are the causative agents of epizootics of commercially important aquaculture or fish farm species. The potential usefulness of fish cell and tissue cultures as teaching tools should not be overlooked. Most fish cell cultures are relatively easy to initiate and/or maintain. They grow over a wide temperature range and most can be propagated at ambient room temperature.

Case Report: Viral Infectious Pancreatic Necrosis in Farmed Rainbow Trout from Mexico.

This case report provides pathologic and confirmatory diagnostic documentation of the first reported clinical epizootic of infectious pancreatic necrosis (IPN) in farmed rainbow trout Oncorhynchus mykiss from central Mexico. Both the gross and microscopic pathology were consistent with IPN. A virus was isolated in cell culture with the cytopathic effect typical of the IPN virus (IPNV). Positive identification as IPNV was achieved by means of an IPNV-specific indirect fluorescent antibody test and reverse transcription-polymerase chain reaction. Further genotyping identified this isolate as the Buhl strain of IPNV, which is a member of the West Buxton (A1) serotype of aquatic birnavirus serogroup A.

Use of monoclonal antibodies in identification and characterization of fish viruses

Abstract Monoclonal antibodies (MAbs) for fish viruses have been used increasingly in fish health management programs and as tools in research. The initial and most widespread use has been in the development of improved diagnostic and virus identification assays. MAbs with broad reactivity that provide standardized reagents for use in general screening assays for particular groups of fish viruses have been developed, whereas MAbs with more restricted specificities permit the rapid serotyping of fish viruses and, in some cases, the identification of specific virus strains. The large number and variety of MAbs now available for some fish viruses, such as the aquatic birnaviruses and rhabdoviruses, present an ideal opportunity to select appropriate MAbs to construct standardized virus identification assays for use by all fish pathologists worldwide. Furthermore, these MAbs have been used to identify specific epitopes that stimulate a protective immune response to many fish viruses, and to investigate the mechanisms of antibody-mediated neutralization of fish viruses. They also have been used to study the epidemiology of specific viruses as well as antigenic variation and the mechanisms by which new strains arise. Such information is important for the development of reliable diagnostic assays for short-term control methods as well as the development of effective vaccines for long-term control of virus diseases in fish. This article presents a brief overview of MAb production and a review of some current applications of MAbs in the identification and characterization of viruses of fish.

Expression of Capsid Proteins from Infectious Pancreatic Necrosis Virus (IPNV) in the Marine Bacterium Vibrio Anguillarum

V. anguillarum is a marine fish pathogen with serotype O1 strains comprising the major disease-causing serotype (Crosa et al., 1980). Two components of a high-affinity iron uptake system encoded by the virulence plasmid pJM1 are the secreted siderophore, anguibactin, and the outer membrane protein receptor for iron-siderophore complexes, OM2. Because formalin-inactivated serotype O1 strains are used commercially as vaccines for the prevention of vibriosis in salmonids, we explored the use of V. anguillarum as an expression host for protective epitopes of the major capsid protein VP2 from infectious pancreatic necrosis virus (IPNV). IPNV causes primarily a disease of salmonid fry, but survivors become life-long carriers and serve as reservoirs of future infection (Dobos & Roberts, 1983). Segment A of the bisegmented dsRNA IPNV genome is 2.9 kb and is translated from a single open reading frame into a polyprotein which is subsequently cleaved into three proteins, VP2, NS and VP3 (Huang et al., 1986). The major capsid protein of the virus, VP2 (59 kDa), contains most of the neutralization epitopes (Caswell-Reno et al., 1986). NS is the non-structural polyprotein protease and VP3 is a minor structural protein. We show here that IPNV cDNA in a translational fusion with V. anguillarum DNA results in production of IPNV and OM2 hybrid proteins expressed in V. anguillarum.