FLOW CYTOMETRIC ENUMERATION OF Flavobacterium columnare FROM GOLDEN SHINERS SUBJECTED TO SPAWNING STRESS

High energy costs associated with the response to stressors compromise the immune and metabolic responses of the fish. Stressed fish are more vulnerable to subsequent challenges and especially compromised in their ability to defend against pathogens such as Flavobacterium columnare that causes columnaris disease in golden shiners. Using flow cytometery to as a rapid assessment of the bacterial load in ponds as well as from the skin and gills of the fish could be a useful tool to prevent outbreaks. The objective of this study was to determine the correlation between detectable levels of columnaris in the water and the incidence of columnaris disease in golden shiners Notemigonus crysoleucas subjected to spawning stress.       

Reproductively mature and juvenile golden shiners were held in 8, 35-L tanks. Ten pairs of reproductively mature males and females were held in each of the 4 tanks. A set of 20 juvenile fish were placed in each of the remaining 4 tanks to serve as a control. Artificial spawning mats were placed in all the tanks to provide a substrate for spawning. When the reproductively mature fish started to spawn, an isolate of a virulent strain of F. columnare (ATCC, Manassas, VA) was added to the water in each experimental tank at a concentration of 1.5 x 10 ^-7 CFU per mL. After the reproductively mature fish stopped spawning, the fish in both the treatment groups were harvested and euthanized immediately in an overdose of MS-222.  Whole body cortisol analysis was conducted on a pooled sample of 10 fish from each tank. Fish were examined for external infections and lesions. Bacterial swab samples were prepared for enumeration by flow cytometry (Figure 1) and by the plate method from the skin and gills from a set of 10 individuals. Data are currently being analyzed and results will be presented.