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Eric Wagner, Randy Oplinger Fisheries Experiment Station, UDWR Don Bone JP Egan State Fish Hatchery.

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Presentation on theme: "Eric Wagner, Randy Oplinger Fisheries Experiment Station, UDWR Don Bone JP Egan State Fish Hatchery."— Presentation transcript:

1 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 10 secTest Chemical Sterile rinse Sterile rinse 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

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) LD 10 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 TreatmentPercent hatch (SD) Yellow CFU (% of green eggs positive) Yellow CFUs (% of eyed eggs positive) Penicillin 67.9 (6.0) 0.0 a12.5 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) Control Pen-Strep % (SD) Pen-Strep Control % (SD) Pen-Strep %(SD) Eyeup 2 wk98.5 (1.0) 90.0 (14.7) 96.5 (4.1) 95.2 (3.2) 4 wk93.1 (5.0) 88.9 (12.9) 86.1 (9.9) 96.6 (1.7) Eggs with No CFUs 2 wk78.7 (30.6) 68.7 (29.8) 88.7 (7.5) 86.2 (17.0) 4 wk23.8 (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 Control Pen-Strep 2 wk Prevalence (%) 4 wk Prevalence (%)6.316.311.3 N80

13 Brood Antibiotic Injection Ovarian fluid bacteriology: no significant difference in total bacteria abundance between treatments for either 2 or 4 wks. Female treatment % negative0 CFU1-100 CFU>100 CFU 2 wk Control 37.5 9141 Pen-strep.52.212110 4 wk Control33.3693 Pen-strep.0061

14 Brood Antibiotic Injection Ovarian fluid bacteriology: yellow bacteria Female treatment % yellow CFU N 2 wk Control41.724 Pen-strep.34.823 4 wk Control33.318 Pen-strep.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

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