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Efficacy of orally administered florfenicol and
oxolinic acid for the treatment of vibriosis in cod
(Gadus morhua)
Ole Bent Samuelsen*, Øivind Bergh
Department of Aquaculture, Institute of Marine Research, P.O. Box 1870 Nordnes, N-5817, Bergen, Norway
Received 1 March 2003; received in revised form 31 May 2003; accepted 15 June 2003
Abstract
This study was performed to determine the efficacy of orally administered oxolinic acid and
florfenicol in the treatment of experimentally induced vibriosis in cod Gadus morhua. The
Vibrio anguillarum strain HI-610 was used. This strain has minimum inhibitory concentration
(MIC) values of 0.016 mg/l against oxolinic acid and 0.5 mg/l against florfenicol. Ten groups of
40 fish each were challenged by bath, 8.5� 106 cells/ml for 1 h. Three days following
challenge, medication with oxolinic acid or florfenicol was introduced in eight of the groups.
The dosages used were 10 or 20 mg/kg day for both antibacterials and administered at days 1,
2, 4, 6, 8 and 10 following initiation of treatment for oxolinic acid and daily for 10 consecutive
days for florfenicol. Among challenged unmedicated fish, the mortality started at day 3 post-
challenge reaching a final cumulative mortality of 87.5% at day 22 when the experiment was
terminated. In the medicated groups, the majority of deaths occurred from days 3 to 5 post-
challenge reaching final cumulative mortalities of 34% and 28%, respectively, for the fish
treated with 10 and 20 mg/kg of oxolinic acid and 31% and 20%, respectively, for the fish
treated with 10 and 20 mg/kg of florfenicol. Survival of medicated fish in all groups were
significant ( p < 0.005) greater than survival of challenged unmedicated fish. No significant
difference ( p>0.1) in survival was however found between groups with parallel treatment or
between groups given different drugs, dosages or medication regimens. Twenty-four hours
following last medication, fish (n = 5) given a daily dosage of 10 mg/kg of florfenicol had mean
plasma and muscle concentrations of 5.0F 1.6 mg/l and 4.6F 0.9 mg/kg, respectively.
Corresponding values for fish given 20 mg/kg day of florfenicol were 6.5F 1.3 mg/l (plasma)
and 7.0F 2.7 mg/kg (muscle). The plasma and muscle concentrations for fish treated with
0044-8486/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0044-8486(03)00446-0
* Corresponding author. Present address: Department of Pharmacology, University of Bergen, Armauer
Hansens Hus, 5021 Bergen, Norway. Tel.: +47-55-974615; fax: +47-55-974605.
E-mail address: [email protected] (O.B. Samuelsen).
www.elsevier.com/locate/aqua-online
Aquaculture 235 (2004) 27–35
oxolinic acid were 0.8F 0.2 mg/l and 1.9F 0.4 mg/kg, respectively, when administered a
dosage of 10 mg/kg day and 1.4F 0.6 mg/l and 3.4F 1.1 mg/kg, respectively, for the fish given
a dosage of 20 mg/kg day.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Vibriosis; Cod; Antibacterial agents; Efficacy
1. Introduction
The cod is regarded as an interesting candidate for domestication in Norway, but it has
long been known that cod is susceptible to vibriosis (Colwell and Grimes, 1984; Egidius,
1987). Infections, especially due to Vibrio anguillarum serotype 02, cause severe losses in
the production of cod fry. Clinical infections associated with vibriosis in cod are usually
associated with low grade of mortality over a long period (Torrissen et al., 1993). Since
outbreaks usually occur prior to or in connection with vaccination, antimicrobial therapy is
necessary to treat the infection (Bergh, 2002). Medication of cod has until now been based
on dosages and dosage regimens originally intended for Atlantic salmon (Salmo salar) and
the drugs in use have been oxolinic acid and florfenicol. Oxolinic acid is at present the
most used antibacterial agent in Norwegian aquaculture (Grave et al., 1999). This drug
possesses excellent activity against fish pathogens and has been used to treat systemic
bacterial infections in fish for years (Austin et al., 1983; Rodgers and Austin, 1983;
Samuelsen et al., 1999). The second most used antimicrobial, florfenicol, is a synthetic
drug with chemical structure and spectrum of antibacterial activity similar to chloram-
phenicol. In vitro investigations with florfenicol have demonstrated potent activity against
several bacteria pathogenic to fish including cod (Gadus morhua) (Fukui et al., 1987;
Inglis and Richards, 1991; Samuelsen, unpublished data).
Observations made at farms showed that oxolinic acid (25 mg/kg for 6 days) was
highly effective in treating vibriosis in cod, whereas florfenicol (10 mg/kg daily for 10
days) showed low efficacy in spite of isolation of florfenicol sensitive pathogens (J.P.
Pedersen, Institute of Marine Research, Bergen, Norway and S. Nygaard, FoMAS,
Haugesund, Norway, personal communications). For florfenicol, this was somewhat
surprising since a recently performed single dose pharmacokinetic study administering a
dosage of 10 mg/kg showed this drug to possess excellent pharmacokinetic properties in
cod (Samuelsen, unpublished data). Therefore, in order to verify or invalidate the field
observations, efficacy studies performed under controlled laboratory conditions were
deemed necessary. The dosage and dosage regimen applied at the farms were used and
in addition, the effect of doubling the dosage was investigated.
The design of treatment regimens and the prediction of their possible clinical outcomes
represent a practical application of pharmacokinetic data. Traditionally, the clinical
significance of pharmacokinetic data has been related to an assumption that the in vivo
plasma concentration of the agent should exceed its minimum inhibitory concentration
(MIC) value for the relevant pathogen by a factor of 3–4 (Stamm, 1989). Recently,
Shojaee AliAbadi and Lees (2000) published a paper on dosage regimen optimisation for
O.B. Samuelsen, Ø. Bergh / Aquaculture 235 (2004) 27–3528
the treatment of animals with antibacterial agents. They suggested that an optimal dosage
regimen should maintain concentrations at the site of infection in excess of MIC90 for the
entire medication period for bacteriostatic drugs and bactericidal drugs acting primarily by
time-dependant mechanisms while an AUIC value (AUC/MIC ratio) of at least 100 and a
Cmax/MIC ratio of at least 8 should be provided for bactericidal agents acting mainly by
concentration-dependant mechanisms (AUC= area under curve). Oxolinic acid is bacte-
ricide and acts by a concentration-dependant mechanism, whereas florfenicol is bacterio-
static. In a single dose pharmacokinetic study of oxolinic acid in cod using an oral dosage
of 25 mg/kg, an AUC value of 140 h mg/l was found (Samuelsen et al., 2003). Combined
with a MIC value of 0.016 mg/l, an AUIC value of more than 8000 could be calculated.
Therefore, from a theoretical point of view, a reduction in dosage is a possibility that
should be investigated. A reduction in dosage is favourable since medicated feed is
expensive and the use of oral administered antibacterials is controversially from an
environmental point of view.
Hence, this study was initiated to evaluate the efficacy of orally administered oxolinic
acid and florfenicol, given in two different dosages, in controlling mortality in cod
experimentally infected with V. anguillarum.
2. Materials and methods
2.1. Experimental fish
Non-vaccinated cod with a mean weight of 65F 12 g obtained from Parisvannet
Research Station (Institute of Marine Research, Norway) were maintained in circular
(1� 2.5 m inside diameter) flow-through seawater storage tanks at the laboratory of the
Institute of Marine Research, Bergen, Norway. The seawater had a salinity of 33x, a flow
rate of 12 l/min and a temperature of 12F 0.5 jC. The fish were fed a non-medicated
ration of 1% body weight per day of dry pellets from Ewos Aqua (Skarer, Norway).
Prior to challenge, fish from the storage tanks were randomly assigned to 10 groups,
each of 40 fish and transferred to 10 circular (0.8� 0.8 m inside diameter) flow-through
seawater tanks maintained at 12F 0.5 jC and with a flow rate of 8 l/min. The fish were
acclimatised for 2 weeks and at the end of the acclimatisation period, the fish consumed
the apportioned amount of unmedicated feed of 1% body weight per day.
2.2. Cultivation of bacteria
The strain used, V. anguillarum HI-610, was originally isolated from cod from
Parisvannet suffering from vibriosis. Apparently identical strains, according to their API
20E and API 50CH (Bio Merieux, MI, USA), profiles have been frequently isolated from
moribund cod juveniles at this facility. The bacteria were grown in Tryptone Soya Broth
(Oxoid) with 1% (wt/vol) NaCl in shaking culture (100 rpm) at 20 jC. They were
precultivated for 24 h, whereafter 2% (vol/vol) of the culture was resuspended in the
medium and cultivated for 24 h. The optical density at 600 nm was measured by a Hitatchi
U-1100 Spectrophotometer. The numbers of colony-forming units (CFU) per ml were
O.B. Samuelsen, Ø. Bergh / Aquaculture 235 (2004) 27–35 29
assessed by a dilution series in sterile 25-ppt seawater. Bacteria were then plated out on
Petri dishes with Tryptone Soya Broth with 1.5% Agar (Bacto) and incubated at 20 jC.
The Petri dishes were counted after 4 days. The strain was passed through cods in a pre-
challenge using the same conditions as the final challenge experiment and subsequently
recultivated from kidney samples from moribund fish and cultivated on nutrient blood agar
(Oxoid) supplemented with 5% sheep blood and 1.5% NaCl. After confirming that the
strain’s biochemical profile, using an API 20E kit, was identical to the original strain, the
recultivated strain was used for the challenge experiment.
2.3. Minimum inhibitory concentration (MIC) determinations
Determination of MIC values for strain HI-610 against florfenicol and oxolinic acid
was performed using the agar dilution method with some modifications (Washington,
1985; Samuelsen and Lunestad, 1996). Two bacterial strains, LT25 and LT62, which have
known MIC values for florfenicol and oxolinic acid (Torkildsen et al., 2000) were used as
internal standards. Strains were maintained on Mueller Hinton agar (Difco) supplemented
with 2% NaCl. Inocula were transferred to Erlen-Meyer bottles with 10 ml Mueller Hinton
broth with 2% NaCl and incubated for 48 h in shaking culture at 18 jC giving a final cell
density of approximately 5� 108 cells/ml determined by optical density measurements.
Using a 10-Al inoculation loop, bacteria from this broth were distributed on the surface of
Mueller Hinton Agar supplemented with 2% NaCl and containing varying concentrations
of florfenicol or oxolinic acid. The two strains used as internal standards were inoculated
to different sectors of the same Petri dishes as strain HI-610. This testing was performed in
triplicate. The agar plates were prepared within 24 h prior to use. The antibacterial agents
were added to the agar in twofold dilutions from a newly prepared stock solution with an
initial concentration of 16 Ag/ml and a final concentration of 0.016 Ag/ml. The temperature
of the agar on addition of the agent was 50 jC. Following incubation at 18 jC for 72 h, the
plates were examined for bacterial growth. The lowest concentration of drug at which
complete inhibition occurred was recorded as the MIC value.
2.4. Challenge
A bath challenge model was developed in this study, based on previous studies with
halibut (Samuelsen, 1997). The fish were starved for 48 h before challenge. The water
supply to the tanks was turned off immediately before the bacterial suspension was added.
The concentration of bacteria in the tanks was measured by the above described plate
counts to 8.5� 106 cells/ml. During challenge, the tanks were oxygenated. The fish were
maintained in the bath for 1 h, after which the water supply was restored.
2.5. Medicated feeds
The medicated feed were made by mixing 2 or 4 g oxolinic acid (100% pure) or 4 or 8 g
florfenicol premix (50%) with 1 kg feed. This corresponds to a daily drug dosage of 10 or
20 mg/kg fish based on a feeding ration of 0.5% of the fish body weight per day. The drugs
were coated onto the surface of the pellets using capelin oil.
O.B. Samuelsen, Ø. Bergh / Aquaculture 235 (2004) 27–3530
2.6. Treatment
Medication started 3 days post-challenge in 8 of the 10 groups. The fish were fed once a
day and approximately 1 h after feeding, excess food was collected from each tank and the
percentage of medicated feed consumed each day estimated. The amount of feed
administered each day was readjusted to account for fish that had died in the tanks during
the last 24 h. The fish in groups 1 and 2 were treated with 10 mg/kg day of oxolinic acid
and groups 3 and 4 with 20 mg/kg day. The treatment regime for oxolinic acid was at days
1, 2, 4, 6, 8 and 10 following initiation of medication giving a total dose of 60 mg/kg for
groups 1 and 2 and 120 mg/kg for groups 3 and 4. At days 3, 5, 7 and 9, the fish were
given non-medicated feed. Groups 5 and 6 were treated with 10 mg/kg day of florfenicol
and groups 7 and 8 with 20 mg/kg day. The treatment regime for florfenicol was daily for
10 consecutive days following initiation of medication giving a total dose of 100 mg/kg
for groups 5 and 6 and 200 mg/kg for groups 7 and 8. Groups 9 and 10 were given non-
medicated feed at a ration of 0.5% fish body weight per day and served as controls.
In the post-medication period, the feed and feeding regime were as described for the
acclimatisation period. The fish were observed for 22-day post-challenge and mortality
was recorded daily. Dead fish were removed from the tanks once a day. Twenty-four hours
after last medication, five fish from each dosage regime were killed by a blow to the head
and samples of plasma and muscle collected for drug analysis. Kidney samples were
collected for microbiological analysis.
Treatments were compared on the basis of cumulative mortality at the end of the study
using the mean value of parallel treated groups and the chi-square test.
2.7. Microbiological analysis
Kidney samples from the dead fish and from five fish taken from each dosage regimen
24 h following terminated medication were inoculated on Petri dishes with nutrient blood
agar with 5% sheep blood and 1.5% NaCl. The isolated strains were tentatively identified
by an agglutination test (Mono-Va, Bio-Nor, Norway) followed by verification of the
biochemical profiles using the API 20E kit.
2.8. High-performance liquid chromatography (HPLC)
Plasma and muscle samples were prepared and analysed for florfenicol residues
following the HPLC methods of Horsberg et al. (1996) or oxolinic acid following the
methods described in Samuelsen and Ervik (1999). These methods have quantitation limits
of 20 and 75 Ag/kg (l), respectively, for florfenicol in muscle and plasma and 25 and 10
Ag/kg (l), respectively, for oxolinic acid in muscle and plasma.
3. Results
The first death of challenged fish occurred at day 3 following challenge. Among
challenged controls (groups 9 and 10), the mortality started at day 3 post-challenge and
O.B. Samuelsen, Ø. Bergh / Aquaculture 235 (2004) 27–35 31
rose rapidly to 85% after which a plateau was reached at day 8 post-challenge. A final
cumulative mortality of 87.5% was recorded at day 22 when the experiment was
terminated. In the medicated groups, mortality had plateaued at day 6 post-challenge,
corresponding to 3 days following initiation of treatment. The majority of deaths occurred
from days 3 to 5 post-challenge and final cumulative mortalities were 34% and 28%,
respectively, for the fish treated with 10 and 20 mg/kg day of oxolinic acid (Fig. 1).
Corresponding values for the fish treated with 10 and 20 mg/kg day of florfenicol were
31% and 20%, respectively (Fig. 1). The values are the mean of two parallel groups.
Survival of medicated fish in all groups was significant ( p < 0.005) greater than survival of
challenged unmedicated fish. No significant difference ( p>0.1) in survival was, however,
found between groups with parallel treatment or between groups given different drugs or
dosages although the fish medicated with 20 mg/kg day of florfenicol obtained the lowest
mortality rate (Fig. 1). The relative percent survival (RPS) (Inglis et al., 1991) was
calculated to be 61% and 68%, respectively, for the groups treated with 10 and 20 mg/kg
day of oxolinic acid and 64% and 77%, respectively, for the groups treated with 10 or 20
mg/kg day of florfenicol.
Bacteria with identical API 20E profile as HI-610 were isolated from the kidneys from
all dead fish from all groups and from all sampled non-treated fish, whereas the pathogen
was not isolated from any of the treated survivors sampled 24 h after terminated
medication.
In all tanks throughout the experiment, 100% of the ration of medicated feed was
consumed and the HPLC analysis showed high concentrations in the sampled fish with the
highest values in those administered the highest dosage. In fish given 10 mg/kg day of
florfenicol, the mean plasma and muscle concentrations were 5.0F 1.6 mg/l and 4.6F 0.9
Fig. 1. Cumulative mortality over time of four treatment groups and a non-medicated control group of cod
challenged with V. anguillarum. Each treatment is the mean value of two parallel groups.
O.B. Samuelsen, Ø. Bergh / Aquaculture 235 (2004) 27–3532
mg/kg, respectively. Corresponding values for the 20 mg/kg day dosage were 6.5F 1.3
mg/l (plasma) and 7.0F 2.7 mg/kg (muscle). For oxolinic acid, the plasma and muscle
concentrations were 0.8F 0.2 mg/l and 1.9F 0.4 mg/kg, respectively, when given 10 mg/
kg day and 1.4F 0.6 mg/l and 3.4F 1.1 mg/kg, respectively, when given 20 mg/kg day.
Statistical treatment of the data (t-test) showed that the mean concentration of oxolinic acid
in muscle of fish treated with 20 mg/kg day was significant higher ( p < 0.05) than in the
muscle of fish treated with 10 mg/kg day. Although increasing with increasing doses, no
significant difference ( p < 0.05) was found between the mean plasma and muscle
concentrations in the fish treated with florfenicol or between the mean plasma concen-
trations in the fish treated with oxolinic acid.
4. Discussion
The culture and presumptive identification of V. anguillarum from the dead and
unmedicated fish from all groups was expected and validates the pathogen as the probable
cause of death. None of the medicated fish sampled 24 h following termination of
medication cultured positive for the bacterium, indicating the pathogen to be effectively
removed from the fish by both antibacterials and dosages.
Statistical treatment of data obtained in this investigation suggests that no drug, dosage
or medication regimen was superior to the others, although a higher efficacy was observed
applying the higher dosage of florfenicol. The differences in mortality between the groups
were however not large enough to be statistically significant, at least at the size of the
experimental groups employed in this study. Therefore, this study failed to establish the
lower dosages of efficacy for both the drugs. The better efficacy of the higher florfenicol
dosage is indicated in the improved RPS value of this treatment, 77%, compared to RPS
values of 61%, 68% and 64%, respectively, for both dosages of oxolinic acid (10 and 20
mg/kg day) and florfenicol (10 mg/kg day). Similar RPS values were reported by other
investigators using florfenicol and oxolinic acid to treat bacterial infections in fish. From
data presented by Nordmo et al. (1998), a RPS value of 56.4% could be calculated for
oxolinic acid in treating furunculosis infected Atlantic salmon when applying a daily
dosage of 25 mg/kg day for 10 successive days. Corresponding value for florfenicol
applying a daily dosage of 10 mg/kg day for 10 successive days was 70% (Nordmo et al.,
1998). Initiating medication at days 10, 13 and 16 following a cohabitational challenge of
Atlantic salmon with Aeromonas salmonicida subsp. salmonicida, a daily dosage of 10 mg
florfenicol for 10 successive days gave RPS values of 96%, 84% and 76%, respectively
(Samuelsen et al., 1998). Furthermore, Samuelsen et al. (1999) found a RPS value of 61%
when treating furunculosis infected Atlantic salmon with oxolinic acid using a dosage of
25 mg/kg day administered at days 1, 2, 4, 6, 8 and 10 following initiation of medication.
The bacterial strain used in this experiment has MIC values of 0.5 mg/l for florfenicol
and 0.016 mg/l for oxolinic acid. For a bacteriostatic drug like florfenicol, the length of the
period for which the concentration exceeds the MIC value is an important determinant of
the outcome of therapy (Shojaee AliAbadi and Lees, 2000). It has been shown that
florfenicol is rapidly absorbed in cod with a plasma Tmax value of 7 h but is slowly
eliminated with a plasma half-life (t1/2b) of 39 h (Samuelsen unpublished results). These
O.B. Samuelsen, Ø. Bergh / Aquaculture 235 (2004) 27–35 33
results and the concentrations of florfenicol found in plasma and muscle 24 h after
terminated medication indicate that for both dosages the concentrations in plasma and
muscle exceed the MIC value of 0.5 mg/l with good margin for the entire medication
period, hence confirming the high efficacy obtained for both dosages (Shojaee AliAbadi
and Lees, 2000).
The high AUIC value calculated for oxolinic acid when administered at a dosage of 25
mg/kg indicated the possibility of obtaining satisfactory effect using a lower dosage. This
is confirmed by the results from this investigation. According to Shojaee AliAbadi and
Lees (2000), prevention of the development of bacterial resistance is correlated with a
preferred Cmax/MIC ratio of at least 8. If the plasma and muscle concentrations measured
24 h after last medication is accepted as Cmax values, then Cmax/MIC ratios of 50–212
could be calculated and hence is well above the recommended value of 8.
In conclusion, this investigation shows that no statistical significance in efficacy was
found between the two drugs or dosages and no verification of the field observations
proclaiming low efficacy of florfenicol in treating vibriosis in cod could be made.
However, due to this discrepancy between the field observations and the laboratory
studies, we feel that additional field studies using florfenicol should be performed before a
final recommendation can be made. Furthermore, our findings show that acceptable
efficacy was achieved using a dosage of 10 mg/kg day of oxolinic acid which is less than
half the dosage normally used for Atlantic salmon (25 mg/kg day).
5. Uncited references
Martinsen and Horsberg, 1995
Poher and Blanc, 1998
Rogstad et al., 1993
Samuelsen et al., 2000
Acknowledgements
The technical assistance of Mrs. Kari Andersen and Mr. Hari Rudra is highly
appreciated.
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