Antibiotic Effects on Flavobacterium psychrophilum the cause of cold-water disease

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

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

In-Vitro Test Process Sterile rinse Sterile rinse 10 sec Test Chemical • 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 Results • For 28 h exposure, ≥333 IU/mL was needed to kill F. psychrophilum • For 15 min exposure, ≥ 10,000 IU/mL needed

Antibiotic Effects on F. psychrophilum 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)

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 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%)

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

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

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

Brood Antibiotic InjectionYellow bacteria in homogenized eggs: no significant difference

Brood Antibiotic InjectionOvarian fluid bacteriology: no significant difference in total bacteria abundance between treatments for either 2 or 4 wks.

Brood Antibiotic InjectionOvarian fluid bacteriology: yellow bacteria *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 effective • Pre-fertilization iodine or PBS rinses reduced F.p., though 10-15% lower survival on average is a concern

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

Questions? Chirolophis japonicus Photo by A. Semenov

Antibiotic Effects on Flavobacterium psychrophilum the cause of cold-water disease