1. Antibiotic Effects on Flavobacterium psychrophilum the cause of cold-water disease
Eric Wagner, Randy Oplinger
Fisheries Experiment Station, UDWR
Don Bone
JP Egan State Fish Hatchery

2. Overview In vitro tests with 4 antibiotics Penicillin toxicity tests
Milt treatment with penicillin-streptomycin
Brood Injection
Other CWD tests

3. In-Vitro Test Process Methods: 10 sec Test Chemical Sterile rinse
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

4. Penicillin In-Vitro Test Results
For 28 h exposure, ≥333 IU/mL was needed to kill F. psychrophilum
For 15 min exposure, ≥ 10,000 IU/mL needed

5. Antibiotic 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)

6. 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

7. 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

8. Penicillin 96 h Toxicity Tests
Tests 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/mL
Brood RT injected with up to 800,000 IU/mL; no significant effect on mortality (0-7%)

9. Penicillin Egg Test
Used 10,000 IU/ml during water hardening (for 1 hr, after post-fertilization rinse) on RT eggs
No 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
Untreated
Control
10.0 b
Iodine
control
70.4 (2.9)
3.3 ab
2.1 b

10. Brood Antibiotic Injection
Each 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 wks
Eggs fertilized with control or antibiotic treated milt
Egg bacteriology: ovarian fluid, homogenized eggs

11. Brood Antibiotic Injection no significant difference in egg survival or prevalence of eggs with no bacteria
Female:
Male:
Control
% (SD)
Pen-Strep
Pen -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 with
No CFUs
78.7 (30.6)
68.7 (29.8)
88.7 (7.5)
86.2 (17.0)
23.8 (3.4)
24.0 (3.8)
25.5 (3.2)
26.6 (3.1)

12. Brood Antibiotic Injection Yellow bacteria in homogenized eggs: no significant difference
Female:
Male:
Control
Pen-Strep
2 wk
Prevalence (%)
11.2
22.5
7.5
10.0
4 wk
6.3
16.3
11.3 N 80

13. Brood Antibiotic Injection Ovarian 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
4 wk
33.3 6 3

14. 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
33.3 18
85.7* 7
*P= 0.015

15. Other cold-water study results: Things that worked
Drip treatment with formalin or hydrogen peroxide recommended
1000 mg/l iodine safe and effective
Pre-fertilization iodine or PBS rinses reduced F.p., though 10-15% lower survival on average is a concern

16. Summary
Streptomycin at ≥ 10,000 mg/L has potential to kill F. psychrophilum
Penicillin-streptomycin very effective together; >50% of stock solution worked (5,000 IU+5 mg/L)
Brood Injections led to higher mortality with >1 injection
No reduction in bacteria noted for antibiotic treatments in brood
Milt treatment with penicillin-streptomycin effective (in-vitro) and safe

17. Questions?
Chirolophis japonicus
Photo by A. Semenov