Use of Penicillin and Streptomycin to Reduce Spread of Bacterial Coldwater Disease II: Efficacy of Using Antibiotics in Diluents and During Water Hardening.

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, has lead to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid and can enter the egg during fertilization. The addition of antibiotics to diluents and water-hardening solutions could prevent the spread of the disease. In separate trials, a mixture of 0.197 mg/mL penicillin plus 0.313 mg/mL streptomycin was added to both a 0.5% sodium chloride fertilization diluent and hatchery well water during hardening. Tests showed that the addition of the antibiotics to the diluent and during up to 60 min of water hardening had no effect on the eye-up, hatch and deformity rates of Rainbow Trout Oncorhynchus mykiss eggs compared with the nonantibiotic-treated controls. Also, significant reductions in the prevalence of F. psychrophilum on the surface and inside eggs were observed when compared with controls. These results indicate that the addition of penicillin and streptomycin to diluents and during water hardening can prevent the vertical transmission of bacterial coldwater disease.

Use of penicillin and streptomycin to reduce spread of bacterial coldwater disease I: antibiotics in sperm extenders.

Bacterial coldwater disease caused by Flavobacterium psychrophilum has led to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid. Methods for disinfecting ovarian fluid and unfertilized eggs are available, but methods for disinfecting sperm have not been described. In this study we determined whether sperm extenders containing a mixture of penicillin and streptomycin can be used to eliminate F. psychrophilum. In vitro trials demonstrated that when Rainbow Trout Oncorhynchus mykiss sperm is mixed with an extender, a 15-min exposure to 0.197 mg penicillin plus 0.313 mg/mL streptomycin is effective at killing the bacteria and has no effect on sperm motility. Small-scale trials showed that egg fertilization rates were not reduced when sperm held in an extender solution containing the same antibiotic mixture for 15 min was used to fertilize eggs. Production-scale trials, however, showed a roughly 18% decrease in egg fertilization rate when sperm stored in an antibiotic containing extender was used. To determine why a reduction in fertilization capacity was observed, a small-scale experiment testing the fertilization of eggs with larger quantities of sperm was performed and showed that increasing the volume of sperm used did not increase fertilization rates. Our results demonstrate that extenders containing penicillin and streptomycin can be used to disinfect sperm, especially when small quantities of eggs are fertilized, but factors negatively affecting egg fertilization and survival on a production scale still need further investigation.

Comparison of the susceptibility of four rainbow trout strains to cold-water disease.

Abstract Susceptibility to cold-water disease was compared among four strains of Rainbow Trout Oncorhynchus mykiss: Arlee strain from Ennis National Fish Hatchery, Montana (AL-EN), the Arlee strain from Jocko River Hatchery, Montana (AL-JR), a cold-water disease-resistant strain (WV), and the Harrison-Hofer strain (HH). Bacterial challenges were either by bath or intraperitoneal injection (50 µL of 0.65 optical density). Each strain was exposed at 75 d after hatch to either the CSF 259-93 (Idaho) or 09-104 isolate (Utah) of Flavobacterium psychrophilum. Injection controls received a phosphate-buffered saline (PBS) solution and bath controls were exposed to uninoculated sterile broth (tryptone yeast extract salts) mixed 1:1 with hatchery well water. For injected fish, the WV had significantly lower mortality (20.0-36.7%) than HH and AL-EN (76.7-96.7%) but did not significantly differ from AL-JR (46.7-56.7%). Injected fish had significantly higher mortality than bath-exposed fish. For bath-exposed fish, the WV had significantly lower mortality (0%) than the HH (10.0-26.7%), but both Arlee strains had intermediate mortality values (0-13.3%) that did not significantly differ from either the HH or WV strain. There were no significant differences between the two bacterial isolates, indicating similar virulence and similar resistance response of WV to another novel isolate of F. psychrophilum. Received November 5, 2013; accepted April 15, 2014.

Toxicity of copper sulfate to Flavobacterium psychrophilum and Rainbow Trout eggs.

Tests were conducted to determine the concentrations of copper sulfate needed to kill Flavobacterium psychrophilum, the cause of bacterial coldwater disease, either in vitro or on Rainbow Trout Oncorhynchus mykiss eggs. For the in vitro test, a plastic strip dipped in a solution of F. psychrophilum was exposed for 15 min to copper sulfate solutions of 0, 1, 5, 10, 20, 35, 50, 75, or 100 mg/L. Bacteria were "too numerous to count" at concentrations ≤10 mg/L CuSO4; significant reductions in prevalence relative to untreated controls were noted for concentrations ≥35 mg/L. However, CFUs were still observed at 50 and 75 mg/L (20% of plates with tryptone yeast extract salts media). No yellow-pigmented CFUs typical of F. psychrophilum were observed at 100 mg/L CuSO4. For the in vivo test, eggs were exposed for 15 min to 100, 300, 500, and 700 mg/L CuSO4 or 100 mg/L iodine (control). Survival to hatch was significantly lower at 500 (44.3 ± 15.2%, mean ± SD) or 700 mg/L CuSO4 (1.7 ± 0.8%) than for controls treated with 100 mg/L iodine (93.6 ± 0.9%) or at copper sulfate concentrations ≤300 mg/L. The 15-min LD50 and LD10 for copper sulfate were 461 mg/L (95% confidence interval: 457-466 mg/L) and 259 mg/L (251-266 mg/L). The prevalence of yellow CFUs at 100 mg/L CuSO4 (40.0%) was significantly higher than in untreated controls. Significant reductions in yellow CFUs were achieved using 300, 500, or 700 mg/L CuSO4 (7.5, 2.5, or 0.0% of plates with CFUs, respectively) or 100 mg/L iodine (2.5%), relative to untreated control eggs. Overall, since the concentrations of copper sulfate required to eliminate F. psychrophilum were toxic to the eggs, copper sulfate is not recommended for coldwater disease control in Rainbow Trout eggs based on conditions and parameters in this study.

Control of Flavobacterium psychrophilum: tests of erythromycin, streptomycin, osmotic and thermal shocks, and rapid pH change.

Flavobacterium psychrophilum, the etiological agent that causes bacterial coldwater disease, has been implicated in significant reductions in the numbers of salmonids reared at fish hatcheries. In this study, we performed a series of in vitro experiments to test the effectiveness of rapid temperature, pH, and osmotic pressure changes in killing three strains of the bacterium. We also evaluated the effectiveness of antibiotics (erythromycin, streptomycin, and a penicillin-streptomycin mixture) against F. psychrophilum. The bacterium tolerated temperatures of 40-50°C for up to 30 min (when acclimated to 15°C). The bacterium can survive lower temperatures for >60 min. Although temperatures ≥55°C appeared to kill F. psychrophilum on contact, we found that eyed eggs of Rainbow Trout Oncorhynchus mykiss were not able to survive short (<60-s) exposures at these temperatures. We found that rapid changes in pH (15-min exposures to pH 3.0, 4.0, 5.0, 7.0, 9.0, 10.0, and 11.0) and osmotic pressure (15-min exposures to 0, 6, 8, 10, 12% sodium chloride) were not effective at killing the bacterium. Erythromycin concentrations up to 2,000 mg/L for 15 min were also ineffective. However, streptomycin concentrations ≥5,000 mg/L killed the bacterium during a 15-min exposure. The combination of penicillin and streptomycin was also effective, killing the bacterium at doses as low as 2.5 × 10(6) IU penicillin + 2,500 mg/L streptomycin. Our trials demonstrate that elevated temperatures and the combination of penicillin and streptomycin can kill F. psychrophilum under in vitro conditions. Erythromycin and rapid changes in pH and osmotic pressure are not effective at killing the bacterium.

Comparison of hatchery and field performance between a whirling-disease-resistant strain and the Ten Sleep strain of rainbow trout.

A whirling-disease-resistant strain of rainbow trout Oncorhynchus mykiss (GRHL strain) derived from a backcross of an F1 hybrid of two strains (German strain x Harrison Lake strain) with German strain females, was compared with the Ten Sleep (TS) strain of rainbow trout. The GRHL strain had consistently superior growth and feed conversion in two consecutive hatchery trials. Hatching and mortality rates were similar between strains. Both strains were stocked into two Utah reservoirs (Hyrum, Porcupine), and a third, Causey Reservoir, was monitored as a control for seasonal variation in prevalence of Myxobolus cerebralis. A total of 1,323 salmonids captured by gill net in spring and fall sampling between 2006 and 2008 were tested for M. cerebralis via pepsin-trypsin digest methods. Only eight of these (< 1% per species) had clinical signs consistent with whirling disease. In both reservoirs, GRHL survived better than the TS and had higher growth rates. The prevalence of M. cerebralis was significantly lower for GRHL (18.1%) than TS (50.0%) in Porcupine Reservoir. In Hyrum Reservoir the trend was similar, but prevalence was lower and did not significantly differ between GRHL (9.6%) and TS (23.1%). For infected fish, no significant differences were observed between strains in myxospore counts in either Hyrum (GRHL = 911-28,244 spores/fish [spf], TS = 1,822-155,800 spf) or Porcupine (GRHL = 333-426,667spf, TS = 333-230,511 spf) reservoirs. Unmarked rainbow trout in both reservoirs had significantly higher myxospore counts than stocked fish of either strain. There were significant differences in M. cerebralis prevalence and myxospore loads among other naturally reproducing salmonids in the reservoirs. The trend in susceptibility was cutthroat trout Oncorhynchus clarkii > kokanee Oncorhynchus nerka > brown trout Salmo trutta. The GRHL performed well in both hatchery and field settings and is recommended for stocking programs.

Effects of media ingredient substitution and comparison of growth of Flavobacterium psychrophilum among four media.

The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.

Disinfection of contaminated equipment: evaluation of benzalkonium chloride exposure time and solution age and the ability of air-drying to eliminate Flavobacterium psychrophilum.

Disinfection of equipment that comes in contact with fish can help to minimize the spread of Flavobacterium psychrophilum (the etiological agent of bacterial coldwater disease) within and among fish culture facilities. We present the results of three studies that evaluated the potential use of benzalkonium chloride and air-drying to kill surface-attached F. psychrophilum. In the first study, we established a vat with a 600-mg/L benzalkonium chloride solution and sampled this solution 0, 14, 35, 56, 70, and 84 d after creation. The solution was kept outdoors and subjected to typical hatchery use. Plastic test strips were dipped in a solution containing F. psychrophilum and were then immersed in benzalkonium chloride for 0, 1, 10, 30, or 60 min. The strips were then rinsed with sterile water and streaked across a plate containing tryptone yeast extract salts (TYES) medium. No culturable bacteria were detected from any strips immersed for 10, 30, or 60 min. Bacteria were detected on 17% of the strips that were immersed for 1 min. The age of the benzalkonium chloride solution had no effect on disinfection ability. In the second study, plastic strips were immersed in a solution containing F. psychrophilum and then were dipped in a 600-mg/L benzalkonium chloride solution for 10 s. The strips were then air-dried for 1 h and were streaked onto TYES medium. No bacterial growth was observed from any strips in the second experiment. The third study determined whether air-drying alone was sufficient to kill F. psychrophilum. Plastic strips were dipped in a solution containing F. psychrophilum; were allowed to dry at room temperature for 0, 24, 48, or 96 h; and were then streaked across TYES medium. Bacteria were cultured from strips representing each drying interval, indicating that air-drying times of 96 h or less are insufficient to kill F. psychrophilum.