Development of an improved medium for primary isolation of Flavobacterium psychrophilum,
cause of bacterial coldwater disease.
Cliff Starliper
USGS National Fish Health
Research Laboratory
11649 Leetown Road, Kearneysville
WV. 25430
Ph: 304-724-4433
cliff_starliper@usgs.gov
Sue Marcquenski
Wisconsin
Department of Natural Resources
101 S. Webster St.
Madison, WI. 53707
Ph:
608-266-2871
Andrew Noyes
New York Department of
Environmental Conservation
8314 Fish Hatchery Road
Rome,
NY. 13440
Ph: 315-337-0910
adnoyes@gw.dec.state.ny.us
August 2007
ABSTRACT:
Cold Water Disease (CWD) is a
serious disease and cause of mortality to salmonid
fish species in the northern United
States. The causative bacterium of CWD is Flavobacterium psychrophilum.
Primary recovery of the pathogen and diagnosis of CWD is problematic because of
the difficulty in isolation of F. psychrophilum
using cytophaga medium (AO), a medium currently used
by many fish diagnostic laboratories. On
AO, 3-4 days are required to produce F. psychrophilum colonies 2-4 mm in diameter. This incubation time can allow contaminants
to overgrow the primary plates, thus impeding diagnosis of the disease. Early identification of F. psychrophilum can be greatly enhanced through the use
of a bacteriological medium that will improve the bacterium’s growth and retard
or inhibit overgrowth by common environmental contaminants. An improved medium would serve as a useful
tool for early treatment of infected fish and reducing overall mortality. This study evaluated a new developmental
medium, #2, in comparison to two other media, AO and EAO+FBS, the latter being
the best medium for F. psychrophilum reported in the literature. The
ingredients in #2 are 0.5% tryptone, 0.05% yeast extract,
0.05% beef extract, 0.02% sodium acetate, 0.02% calcium chloride, 0.05% magnesium
sulfate, and 5% fetal bovine serum. Results from comparative growth studies in
broth showed a significant increase (p £ 0.043) in cfu/mL with #2,
when compared to AO and EAO+FBS. On agar format, there was no
quantitative differences in the cfu/mL yield
of the three media; however, colonies on #2 grew more luxuriant, and more
importantly, could typically be enumerated one day sooner, compared to AO. The
individual medium components of #2 were varied and evaluated to optimize the
recipe; significant responses were noted with various serum supplements. Optimum
performance was with fetal bovine serum. Isolates of F. psychrophilum
and representative contaminants collected from primary isolation plates that
were cultured from coldwater diseased fish were screened against antimicrobials
to identify potential selective agents to incorporate into #2. Although fetal
bovine serum does add cost to the medium, the other ingredients are all very
inexpensive, particularly considering the small amounts used of each component.
We feel the cost of serum is justified given the excellent colony growth of our
large number of F. psychrophilum
isolates, and the reliability that the #2 medium demonstrated; we did not have
one culture that did not grow.