ASSESSMENT OF Flavobacterium columnare FROM GOLDEN SHINERS Notemingus crysolucas SUBJECT TO CROWDING STRESS  

Intensive aquaculture practices and exposure to environmental stressors can trigger outbreaks of Flavobacterium columnare, a bacterial pathogen that causes columnaris disease in commercially important fish including Golden Shiners. A rapid assessment of the bacterial load is essential to prevent outbreaks and subsequent economic losses. Classical methods using bacterial plates and Polymerase Chain Reaction (PCR) is time consuming and requires sample preparation.

In this study we present a technique to enumerate F. columnare using flow-cytometry. Two 5-d trials were conducted at 22 and 28° C to determine the effect of temperature and crowding stress on the survival of Golden Shiners infected with F. columnare. Both trials consisted of four treatments: (1) low density (600 fish/m³) and no bacterial challenge (control), (2) low density and bacterial challenge, (3) high density (2400 fish/m³) and no bacterial challenge, and (4) high density and no bacterial challenge. Golden Shiners, weighing 2.62 (±0.78) g each, were stocked in 40, Ultra Low Flow (ULF, after Mitchell and Farmer 2010) tanks containing 10 L of water. The tanks were inoculated with a strain of F. columnare, MS94-081. The study was considered complete 48 h post bacterial challenge. Mortality was recorded. Bacterial pellets were formed by centrifuging 50 mL of tank water and the resulting pellets were resuspended in 2 mL of the supernatant. An antibody labelled with fluorescein dye (2µL) was mixed with 100 µL of the concentrated tank water to be analyzed by flow cytometry. The bacterial counts were validated by qPCR. Results from the study showed significantly high mortality in fish exposed to bacterial challenge and fish held at 28°C. No significant differences were found between the high density and low density treatments. Bacterial counts from water samples obtained by flow cytometry and qPCR showed significantly higher numbers of F. columnare in tanks exposed to bacterial challenge. The counts did not vary in the density or temperature treatments. Bacterial counts using flow cytometry is a rapid and accurate for assessing bacterial load in a water body.