A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.
Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture. INTRODUCTIONFlavobacterium psychrophilum is a yellow-pigmented, Gram-negative, gliding bacterium that predominantly affects salmonid fish (Borg, 1960), such as coho salmon (Oncorhynchus kisutch) or rainbow trout (Oncorhynchus mykiss), and occasionally other fish species, such as ayu (Plecoglossus altivelis) (Iida & Mizokami, 1996). This bacterium is therefore responsible for considerable economic losses in fish aquaculture. Infections with F. psychrophilum have several clinical manifestations, the most significant of which include mortality associated with haemorrhagic septicaemia and spleen hypertrophy in juvenile fish, referred to as rainbow trout fry syndrome, and in adults, septicaemia preceded by extensive necrotic lesions, called cold-water disease (CWD) (Bernardet & Bowman, 2006). However, the actual mechanism of pathogenesis is not well understood, although virulence has been suspected to be related to the ability of F. psychrophilum to produce exotoxins (Dalsgaard, 1993), extracellular metalloproteases (Fpp1-2; Secades et al., 2001Secades et al., , 2003 or enzymes involved in the degradation of products such as chondroitin sulfate, collagen and fibrinogen (Bertolini et al., 1994). Clearly, the flavobacterial outer membrane (OM) is important when we consider interactions of bacteria with host cells and tissues in the context of pathogenesis and immunity to infection. Several surface components of F. psychrophilum have been implicated in flavobacterial pathogenesis and identified as possible vaccine and diagnostic candidate macromolecules; they include lipopolysaccharide O antigen (MacLean et al., 2001) and surface-exposed antigens [e.g. 20 kDa antigen and OmpA (Merle et al., 2003;Dumetz et al., 2007)], some of which may be good candidates for an F. psychrophil...
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