eric wagner, randy oplinger fisheries experiment station, udwr don bone jp egan state fish hatchery

Antibiotic Effects on Flavobacterium psychrophilum

the cause of cold-water diseaseEric Wagner, Randy OplingerFisheries Experiment Station, UDWR

Don Bone JP Egan State Fish Hatchery

OverviewIn vitro tests with 4 antibioticsPenicillin toxicity testsMilt treatment with penicillin-streptomycinBrood InjectionOther CWD tests

In-Vitro Test Process

10 sec Test Chemical

Sterilerinse

Sterilerinse

• Methods:• Dip plastic strip into bacteria solution (n

= 5)• Transfer strip to antibiotic solution• Let sit for 15 min• Dip strip twice in rinse water• Streak across agar plate• Incubate and look for growth

Penicillin In-Vitro Test ResultsFor 28 h exposure, ≥333 IU/mL was needed to kill

F. psychrophilumFor 15 min exposure,

≥ 10,000 IU/mL needed

Antibiotic Effects on F. psychrophilumAntibiotic Effects on F. psychrophilum

• Antibiotics: • Streptomycin (0, 100, 500, 1,000, 5,000 and

10,000 mg/L) • Erythromycin (0, 10, 100, 500, 1,000, and 2,000

mg/L)• Penicillin-Streptomycin (0 to 100% of stock:

10,000 IU penicillin + 10 mg/L streptomycin)

Antibiotic Effects on F. psychrophilum• Streptomycin:

• No F.p. growth on 1,000 and 10,000 mg/L strips• Growth on 1 strip treated at 5,000 mg/L• Growth on all strips at ≤ 500 mg/L

• Erythromycin:• F.p. growth on all strips at all concentrations

• Penicillin-streptomycin:• Generally no growth at >25% of stock (1 strip+ at

50%); stock = 10,000 IU penicillin + 10 mg/L streptomycin

Milt Treatment with Penicillin-Streptomycin

• Added milt to sperm extender with commercial penicillin/streptomycin for 15 min

• >2,500 IU penicillin and 2.5 µg/mL streptomycin effective at killing coldwater bacterium

• No effects of up to 2X full antibiotic strength on motility

• Progeny from treated sperm and injected females had similar hatch percentage as controls

Penicillin 96 h Toxicity TestsTests doses up to 100,000 IU/mL on RT eggs and

these doses were safe (65-75% hatch)LD10 for fingerling rainbow trout was 52,868 IU/mLBrood RT injected with up to 800,000 IU/mL; no

significant effect on mortality (0-7%)

Penicillin Egg TestUsed 10,000 IU/ml during water hardening (for 1 hr, after post-

fertilization rinse) on RT eggsNo significant impact on hatching (P = 0.6)No reduction in yellow CFU abundance relative to iodine

Treatment Percent hatch (SD)

Yellow CFU (% of green eggs positive)

Yellow CFUs (% of eyed eggs positive)

Penicillin 67.9 (6.0) 0.0 a 12.5 a

UntreatedControl

10.0 b

Iodine control

70.4 (2.9) 3.3 ab 2.1 b

Brood Antibiotic InjectionEach female (N = 50 RTGH) injected with antibiotic

(50,000 IU penicillin and 50 mg streptomycin); controls got saline

at 2 wk after injection, eggs were taken; green females at 2 wk injected again with same concentration

Eggs taken again at 4 wksEggs fertilized with control or antibiotic treated miltEgg bacteriology: ovarian fluid, homogenized eggs

Brood Antibiotic Injectionno significant difference in egg survival or prevalence of eggs with no bacteria

Female:Male:

ControlControl% (SD)

ControlPen-Strep% (SD)

Pen-StrepControl% (SD)

Pen-StrepPen -Strep%(SD)

Eyeup 2 wk 98.5 (1.0) 90.0 (14.7) 96.5 (4.1) 95.2 (3.2)

4 wk 93.1 (5.0) 88.9 (12.9) 86.1 (9.9) 96.6 (1.7)

Eggs withNo CFUs 2 wk 78.7 (30.6) 68.7 (29.8) 88.7 (7.5) 86.2 (17.0)

4 wk 23.8 (3.4) 24.0 (3.8) 25.5 (3.2) 26.6 (3.1)

Brood Antibiotic Injection

Yellow bacteria in homogenized eggs: no significant difference

Female:Male:

ControlControl

ControlPen-Strep

Pen-StrepControl

Pen-StrepPen-Strep

2 wkPrevalence (%) 11.2 22.5 7.5 10.0

4 wkPrevalence (%) 6.3 16.3 11.3 11.3

N 80 80 80 80

Brood Antibiotic InjectionOvarian fluid bacteriology:

no significant difference in total bacteria abundance between treatments for either 2 or 4 wks.

Female treatment

% negative 0 CFU 1-100 CFU >100 CFU

2 wk Control

37.5 9 14 1

Pen-strep. 52.2 12 11 0

4 wk Control 33.3 6 9 3

Pen-strep. 0 0 6 1

Brood Antibiotic Injection

Ovarian fluid bacteriology: yellow bacteria

Female treatment

% yellow CFU

N

2 wk Control 41.7 24

Pen-strep. 34.8 23

4 wk Control 33.3 18

Pen-strep. 85.7* 7

*P= 0.015

Other cold-water study results:Things that worked

Drip treatment with formalin or hydrogen peroxide recommended

1000 mg/l iodine safe and effectivePre-fertilization iodine or PBS rinses reduced F.p.,

though 10-15% lower survival on average is a concern

SummaryStreptomycin at ≥ 10,000 mg/L has potential to kill F.

psychrophilumPenicillin-streptomycin very effective together; >50%

of stock solution worked (5,000 IU+5 mg/L)Brood Injections led to higher mortality with >1

injectionNo reduction in bacteria noted for antibiotic

treatments in broodMilt treatment with penicillin-streptomycin effective

(in-vitro) and safe

Questions?

Chirolophis japonicus Photo by A. Semenov