CONTENTS
Introduction
Bacterial diseases represent some of the most significant challenges
facing
fish farming worldwide. According to the 2000 United States Department
of Agriculture National Animal Health Monitoring System (USDA-NAHMS)
report, bacterial diseases account for approximately 70% of all diseases
affecting catfish (Ictalurus punctatus) in the southeastern
US.
The most important bacterial disease in commercial catfish aquaculture
is enteric septicemia of catfish (ESC), a highly fatal systemic disease
caused by infection with Edwardsiella ictaluri. Signs
of E. ictaluri infection include inflammation through the sutra
fontanel of the skull (“hole in the head”); hemorrhages on
the skin, fins, and/or gills; white spots or nodules on the skin and/or
fins; oral hemorrhages; abdominal distention (ascites); and exophthalmia.
Mortality rates in susceptible catfish can reach 100%; and in
surviving fish, growth rates and weight gains may be significantly affected.
 Outbreaks
of ESC typically occur in the spring and fall months, when water temperatures
are 20° – 28°C and other conditions are optimal for growth
of E. ictaluri. Husbandry and environmental stress play significant
roles in determining the clinical and economic impact of E. ictaluri infections.
Nevertheless, this organism is considered a primary pathogen and is capable
of causing substantial losses even on well-managed farms.
For successful treatment during an outbreak of ESC, the diagnosis must
be made and treatment with an effective antibiotic initiated while the
majority of fish are still feeding. E.
ictaluri is susceptible to a variety of antibiotics, but
until recently only 2 antibacterial agents were approved for use in food
fish: sulfadimethoxine-ormetoprim (Romet 30) and
oxytetracycline (Terramycin).
Resistance to both of these antibiotics has been reported in fish. Inadequate
intake of medicated feed, whether from poor palatability (Romet) or
inappropriate pellet type (oxytetracycline), has
also contributed to the limited efficacy of these products. Clearly,
a more palatable and efficacious oral antibiotic is needed for the treatment
of ESC in catfish.
Aquaflor 50% Type A Medicated Article
is a feed premix containing florfenicol,
a broad-spectrum antibiotic. Florfenicol (FFC) has activity against a
wide range of fish pathogens in vitro and in vivo,
including E. ictaluri. Treatment with Aquaflor, incorporated
into floating catfish feed prior to pelleting and administered at a dose
rate of 10 mg FFC/kg body weight daily for 10 consecutive days, results
in a rapid decline in mortality rates due to enteric septicemia of catfish
associated with Edwardsiella ictaluri.
Key Characteristics of Aquaflor:
- Highly palatable
- Administered in floating feed
- Well tolerated by catfish
- Highly effective against E.
ictaluri infection
- Effective in a range of water
temperatures favored by E.
ictaluri
- Effective against multiple antibiotic-resistant
organisms
- Minimal environmental effect
- Antibiotic developed exclusively
for veterinary medicine
Florfenicol is a synthetically produced antibacterial agent that has
been specifically developed for veterinary use. It is a fluorinated
analogue of thiamphenicol, a chloramphenicol analogue, and these structural
modifications confer advantages in activity, particularly against bacteria
resistant to thiamphenicol and chloramphenicol. Florfenicol
is chemically different from chloramphenicol and lacks the functional
group responsible for chloramphenicol’s human toxicity concerns
(bone marrow suppression and aplastic anemia).
Studies with florfenicol indicated potent activity against a number of
bacterial fish pathogens in vitro and in a variety of fish species in
vivo. Experimental efficacy has
been demonstrated against E. ictaluri in channel catfish, Photobacterium
damselae subsp. piscicida (formerly Pasteurella piscicida)
in yellowtail, Edwardsiella tarda in
eels, Vibrio anguillarum in
goldfish, Aeromonas salmonicida, Vibrio
salmonicida in Atlantic salmon, and Yersinia
ruckeri and Flavobacterium psychrophilum in trout (data
on file).
There is a limited number of products approved for treatment of bacterial
disease in fish, and antibacterial resistance to some of these products
has already become widespread. Oral
administration of antimicrobials is the preferred route of chemotherapy
in finfish aquaculture due to the ease of use and lack of any additional
stress to the fish during treatment. Reduction in appetite due to clinical
disease can be addressed by higher feed inclusion rates of the medication.
Florfenicol was first approved for aquaculture use in Japan in 1990 for
treatment of susceptible bacterial diseases, including pasteurellosis
and streptococcosis in yellowtail, red sea bream, coho salmon, horse
mackerel, rainbow trout, sweetfish, tilapia and eel. Since then it has
been approved for use in Atlantic salmon in Norway, Chile, Canada and
in the U.K. Most recently Aquaflor has
been approved in the U.S. for control of mortality in catfish due to
enteric septicemia of catfish associated with Edwardsiella ictaluri.
Aquaflor was developed by the Research
Division of Schering-Plough Animal
Health Corporation specifically to provide fish producers with a product
that combines highly effective control of susceptible bacterial diseases
with safety for fish, palatability ease of administration and a different
mode of action from other antibiotics. Back
to top
Chemistry
The following data describe the active ingredient.
Florfenicol is the active ingredient in Aquaflor.
It is a monofluorinated derivative of thiamphenicol, a chloramphenicol
analogue in which the p-nitro group on the aromatic ring is
substituted with a sulphonylmethyl group.
Chemical Structure:
Scientific Name: ([R-(R*,S*)]-2,2-dichloro-N-[(-(fluoromethyl)-(-hydroxy-(-[4-(methylsulfonyl)
phenyl]ethyl]-acetamide
Generic Name: florfenicol
Molecular Formula: C12H14Cl2FNO4S
Molecular Weight: 358.21
Mode of Action
Florfenicol is a synthetic,
broad-spectrum antibiotic active
against many Gram-negative and
Gram-positive bacteria. Florfenicol
acts by binding to the 50S ribosomal
subunit, thereby preventing bacterial
protein synthesis.
In vitro activity has
been demonstrated against commonly
isolated bacterial fish pathogens
including Edwardsiella ictaluri, Aeromonas
salmonicida, Edwardsiella
tarda, Flavobacterium
psychrophilum, Photobacterium
damselae subsp. piscicida, Vibrio
anguillarum, V. salmonicida,
other Vibrio spp., and Yersinia
ruckeri (see Table 1-1).
Bacteria resistant to chloramphenicol, through chloramphenicol acetyltransferase
production, are sensitive to florfenicol. In
vitro tests with fish pathogens and a range of chemotherapeutants,
including chloramphenicol, streptomycin, tetracycline, ampicillin, trimethoprim,
furazolidone, kanamycin, naladixic acid, amoxycillin, oxolinic acid and
florfenicol, demonstrated that florfenicol had greater antibacterial
activity than the other compounds tested. Back
to top
Dosage Form
Premix: Aquaflor is
a 50% (w/w) Type A Medicated Article for inclusion into fish feed.
The composition is 50% florfenicol and 50% inert carriers. Aquafloris:
- Supplied in 2-kg (4.4 lb) foil
packages, with eight 2-kg packages
in a fiber drum
- Has a shelf-life of 3 years
Medicated Feed Production (see
label for explanation):
- The product should be mixed in
unmedicated fish feed prior to
pelleting
- It should be administered in
feed to deliver 10 mg florfenicol
per kg body weight daily (See Table
1-2 for recommended Aquaflorfeed
inclusion rate).
Dose Rate:
- Recommended dose rate is 10 mg
of florfenicol/kg body weight/day
for 10 consecutive days
Feeding Directions: Aquaflor Type
C medicated feed should only be
administered once the disease associated
with Edwardsiella ictaluri has
been appropriately diagnosed. Feeding
fish at a percent of biomass and
corresponding florfenicol concentration
included in the table above will
deliver 10 mg florfenicol per kg
of fish. Back
to top
Veterinary Feed Directive Drug
AquaflorType
A Medicated Article is a Veterinary
Feed Directive (VFD) drug.
Please note the following cautionary
information for sale and use of
Aquaflor Type A Medicated
Article: Federal law limits
this drug to use under the professional
supervision of a licensed veterinarian.
Animal feed bearing or containing
this veterinary feed directive
drug shall be fed to animals only
by or upon a lawful veterinary
feed directive issued by a licensed
veterinarian in the course of the
veterinarian’s professional
practice.
Extra-label use (i.e., use of this VFD feed in a manner other than as
provided for the VFD drug approval) is strictly prohibited.
Feed containing Aquaflor (florfenicol)
should not be fed to catfish for
more than 10 days. Following administration,
fish should be re-evaluated by a
licensed veterinarian before initiating
further therapy. The expiration date
for a VFD for Aquaflor (florfenicol)
must not exceed 15 days from the
date of issuance. VFDs for Aquaflor(florfenicol)
should not be refilled. Back
to top
Pharmacokinetics
While the pharmacokinetics of florfenicol have not been
studied in catfish, the absorption, distribution, metabolism
and excretion of florfenicol have been studied in numerous other
species, including cattle, pigs, Muscovy ducks, broiler chickens,
horses, rainbow trout and Atlantic salmon (in freshwater and
seawater). Conclusions from
these studies were consistent for all species: florfenicol is
well absorbed, and excreted in bile, feces and urine.
Studies
in Fish
Atlantic salmon (Salmo salar) were used in a number of studies
to determine the fate of florfenicol in fish; one study was a whole body
autoradiography study with salmon in seawater at 8°–11°C;
other studies were radiolabeled residue depletion studies conducted with
salmon in seawater at 5°C and 10°C. Results from the whole body
autoradiography study indicated that florfenicol had the following properties:
- Rapidly absorbed from the intestinal
tract and transferred to other
tissues
- Widely distributed to tissues,
with maximum levels found at 12
hours after the end of treatment
with similar concentrations in
blood and muscle, but lower levels
in fat and brain
- Maximum levels of florfenicol
achieved in the muscle exceeded
the Minimum Inhibitory Concentration
(MIC) values reported for most
fish pathogenic bacteria
In radiolabeled residue depletion
studies, salmon received either a
single dose of feed containing radiolabeled
florfenicol or 9 days of florfenicol-medicated
feed and 1 day of feed containing
radiolabeled florfenicol at 10 mg/kg
body weight. Results showed that:
- Maximum radioactivity concentrations
in all tissues occurred within
6–24 hours after final dose
delivery, and the highest levels
were observed in the kidneys and
liver
- Tissue radioactivity declined
faster at 10°C than at 5°C,
indicating that florfenicol is
cleared from tissues faster at
higher temperatures
- Residue concentrations were
lower in muscle than in skin and
depleted somewhat faster from muscle
than from skin Back
to top
Toxicology
A complete toxicological evaluation
has been conducted with florfenicol.
This includes extensive published
and unpublished studies in the mouse,
rat, cattle, dog, swine, poultry,
rainbow trout, salmon and bluegill
sunfish. From
studies with florfenicol on reproduction
in rats, a No Observable Effect Level
(NOEL) of 1.0 mg/kg has been established.
Food Safety and Residues
The metabolism of florfenicol in fish is qualitatively similar to the
metabolism in the rat. With a safety factor of 100, an acceptable daily
intake (ADI) has been calculated at 10 µg/kg/day. Thus, based on
toxicological assessment, an ADI of 10 µg/kg/day has been set for
florfenicol and its metabolites in fish tissues.
Maximum
Residue Limit or Safe Concentration
An HPLC assay based on the quantitative
conversion of florfenicol and its
metabolites to florfenicol amine
has been validated in fish tissues
(muscle, skin, and muscle with
attached skin). The
Maximum Residue Limit (MRL) or
equivalent Safe Concentration is
based on the marker residue, florfenicol
amine. The US has established a
Safe Concentration for florfenicol
amine in catfish edible tissue
(muscle) of 1.0 mg/kg. Back
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Residue
Depletion Study in Catfish
The accepted Safe Concentration
of florfenicol amine is 1 ppm in
catfish muscle. As the following
data show, florfenicol amine residues
in catfish muscle are well below
this level within 7 days after
concluding treatment with florfenicol
(FFC) at the recommended dose rate
of 10 mg FFC/kg body weight/day
for 10 consecutive days.
Purpose: A study was
conducted in market-weight catfish,
raised under field conditions,
in order to determine marker residue
concentrations in edible catfish
tissue (muscle) following treatment
and, thus, to establish a withdrawal
period for FFC in catfish.
Study Design: Market-weight
catfish (average weight 2 lb at
the start of the study period)
were kept in a 0.1-acre test pond
under normal aquaculture conditions
at a stocking density of 7,000
fish/acre. Medicated, pelleted
fish feed was fed for 12 consecutive
days* at a nominal dose rate of
10 mg/kg (actual measured dose
rate = 9.3 mg/kg body weight/day).
The fish were then fed unmedicated
feed for the remainder of the study
period. The water temperature remained
below 25°C throughout the study,
with average daily high and low
water temperatures during treatment
being 21.9°C and 19.4°C,
respectively.
*Cold
weather reduced initial feed
intake for 2 days, so the feeding
period was extended to 12 days.
Following the
cessation of FFC treatment, 20
fish were sampled for florfenicol
residues on each of the following
days post-treatment: 1, 2, 4, 7,
14 and 21 days. Muscle samples
(fillets) were collected from each
fish and analyzed using a validated
assay for florfenicol amine (HPLC,
using UV detection). Means were
calculated for each group; individual
values below the limit of quantitation
(0.075 ppm) were not used to calculate
the means.
Results: Average muscle concentrations of florfenicol
amine are shown in Table 3-1. Levels of the marker declined
rapidly during the first 7 days following cessation of treatment,
from an average of 5.38 ± 7.01 ppm on day 1 to an
average of 0.23 ± 0.11 ppm on day 7. Average tissue
levels of florfenicol amine were below the accepted Safe
Concentration (1 ppm) by 4 days after the end of treatment,
and levels were below this threshold in all individual fish
by 7 days post-treatment.
Residue levels plateaued by 2 weeks post-treatment at an average of
0.16 ppm. Levels were 2–4X higher in males than in females on
days 2 and 4 post-treatment, but average residue levels for males and
females were comparable at all other time points (data not shown). Back
to top
*Catfish were fed florfenicol at
a dose rate of 9.3 mg/kg/day for 12 days. Fish were harvested and
muscle samples analyzed for florfenicol amine at various times
after the end of the treatment period.35, 36
Conclusions: Tissue levels of florfenicol amine
declined rapidly after the 12-day treatment period had ended. Using
an upper limit of 1 ppm in edible tissue and calculations based
on a 99% tolerance limit and a 95% confidence interval, the withdrawal
period for Aquaflor50% Type
A Medicated Article, administered orally in feed for 10 consecutive
days, is 12 days for channel catfish.
Withdrawal Period:
A withdrawal period of 12 days
has been established by the
United States Food and Drug
Administration for Aquaflor-treated
channel catfish.
Safety
of Florfenicol in Catfish—Study
1
A study was conducted to determine
the safety and palatability of orally
administered FFC in channel catfish
at 1X, 2X, 4X, and 10X the recommended
dose rate.
Study Design: Four hundred
(400) laboratory-reared, 5-month-old
channel catfish fingerlings with
no known history of exposure to E.
ictaluri were divided into 5
groups of 80 fish each:
- Group 1—fed unmedicated
feed
- Group 2—fed FFC at 10 mg/kg/day
for 10 days
- Group 3—fed FFC at 20 mg/kg/day
for 10 days
- Group 4—fed FFC at 40 mg/kg/day
for 10 days
- Group 5—fed FFC at 100
mg/kg/day for 10 days
Fish were weighed in groups at the
start of the study period. In Groups
2–5, treatment with FFC-medicated
feed began the next day and continued
for 10 consecutive days. All groups
were observed daily for feeding behavior,
signs of toxicity and mortality.
On day 11, all surviving fish were
counted, weighed in groups, euthanized
and submitted for gross and histopathologic
examination.
Throughout the study, feeding activity
was subjectively graded each day,
based on the amount of feed consumed.
A score of 2 was assigned if 50–100%
of the food was consumed. A score
of 1 was given if <50% of the
food was consumed. A score of 0 was
given if little or no food was consumed.
A palatability score was calculated
for each group at the end of the
study as the sum of the daily feeding
scores over the 10-day treatment
period. As the maximum feeding activity
score was 2, the maximum palatability
score was 20 (2 x 10 days).
Results: Palatability scores
and body weight gains for each group
are shown in Table 3-2. Weight gains
were not significantly different
between groups. Palatability
scores were not analyzed statistically,
as they were nearly identical for
all groups. Back
to top
*Average
daily feeding activity score (0–2)
x 10 days; maximum score = 20.
**Fed medicated feed containing FFC at the listed dosage for 10 consecutive
days.
No fish died and no signs of morbidity
were noted during the study period.
Only 6/400 fish (1.5%) examined at
necropsy had gross pathologic lesions.
Lesions were mild in all 6 fish,
consisting only of mild mottling
of the liver (3 fish in the 10 mg/kg
FFC group and 1 control fish) or
spleen (2 fish in the 20 mg/kg FFC
group and 1 fish in the 10 mg/kg
FFC group). There were no differences
in the histologic appearance of the
organs in treated versus untreated
fish.
Conclusions: Florfenicol, administered at up to 10X
the recommended daily dose rate of 10 mg/kg body weight for 10
consecutive days, was palatable to channel catfish fingerlings
and did not cause any treatment-related pathology.
Safety of Florfenicol in
Catfish—Study 2
The following study was conducted
to determine the safety and palatability
of orally administered FFC in channel
catfish at 1X, 3X, and 5X the recommended
dose rate for 2X the recommended
treatment duration.
Study Design: A total of
240 laboratory-reared channel catfish
fingerlings (mean weight 14.7 ± 3.8
g) were divided into 4 groups:
- Group 1—fed unmedicated
feed
- Group 2—fed FFC at 10 mg/kg/day
for 20 days
- Group 3—fed FFC at 30 mg/kg/day
for 20 days
- Group 4—fed FFC at 50 mg/kg/day
for 20 days
Fish were fed either unmedicated
feed (Group 1) or medicated feed
(Groups 2 – 4) for 20 consecutive
days. During that time, all groups
were monitored for feeding activity,
morbidity and mortality. At the end
of the treatment period, all surviving
fish were weighed, euthanized, necropsied,
and their tissues were examined histopathologically.
Results: No fish died and
no signs of morbidity were noted
during the study period. No changes
in behavior were detected in treated
fish relative to the controls. Although
feed consumption declined in the
latter part of the dosing period
at the 30 mg/kg and 50 mg/kg dose
rates, there were no significant
differences in body weight at the
terminal sampling.
No differences between groups were observed at gross necropsy. A very
mild, dose-dependent decrease in hematopoietic/lymphopoietic tissue was
observed microscopically in the kidneys and spleens of treated fish.
No other histopathologic changes were noted.
Conclusions: No significant changes attributable to FFC treatment
were observed, even at 5X the recommended dose rate for 2X the recommended
duration of treatment. Thus, FFC is considered safe for administration
to channel catfish at the recommended dose rate of 10 mg FFC/kg/day for
10 consecutive days. Back to top
Efficacy
Overview: Aquaflor 50%
Type A Medicated Article is recommended
for the control of mortality in catfish
due to enteric septicemia of catfish
associated with Edwardsiella
ictaluri. Aquafloris
administered in feed at an oral dose
rate of 10 mg FFC/kg body weight/day
for 10 consecutive days. The efficacy
of Aquaflor and
the validity of this dose regimen
is supported by several studies,
including in vitro sensitivity
data on E. ictaluri and in
vivo challenge studies. These
studies show the following:
- Aquaflor-medicated
feed is safe in channel catfish
at the recommended dose rate
- Aquaflor-medicated
feed is palatable to channel catfish
- Aquafloris
highly effective in the control
of mortality in channel catfish
caused by pathogenic strains of E.
ictaluri
In
Vitro Susceptility of E.
ictaluri to Florfenicol
The following study demonstrates
the in vitro susceptibility
of E. ictaluri to florfenicol.
Study Design: Twelve (12) field isolates of E. ictaluri,
obtained during natural outbreaks of ESC in commercial catfish
ponds in Mississippi, were tested. An additional 767 cultures
of E. ictaluri, obtained from channel catfish experimentally
infected with E. ictaluri, were also tested. Agar disk
diffusion susceptibilty testing was performed as outlined in
the National Committee for Clinical Laboratory Standards (NCCLS)
performance standards guidelines.
Results: Kirby-Bauer zones of inhibition and MIC values
for natural and experimental infections are shown in Table
4-1.
Conclusions: All tested
strains of E. ictaluri were
highly susceptible to FFC in
vitro. Parameters for the
zone of inhibition for FFC against E.
ictaluri are
not formally established. Using preliminary
interpretive standards for FFC in
cattle, a zone of inhibition >19
mm indicates that the organism is
sensitive. The
zones of inhibition for all isolates
of E. ictaluri in this study
were >30 mm. Thus, it can be concluded
that E. ictaluri is highly
susceptible to FFC. The low MIC value
for all tested isolates (0.25 µg/mL)
supports this conclusion. Back
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In
vivo EFFICACY
The following 4 studies confirm
the in vivo efficacy of
Aquaflor-medicated
feed at the recommended dose rate
in channel catfish exposed to pathogenic
strains of E. ictaluri.
Dose
Titration—Study 1
The following dose titration study
was conducted to confirm the safety,
palatability and efficacy of FFC-medicated
feed and to determine the optimal
dose rate in channel catfish exposed
to a pathogenic strain of E.
ictaluri.
Study Design: Four hundred
(400) laboratory-reared, 5-month-old
channel catfish fingerlings with
no known history of exposure to E.
ictaluri were divided into 5
groups of 80 fish:
- Group 1—not challenged
with E. ictaluri and fed
unmedicated feed
- Group 2—challenged with E.
ictaluri and fed unmedicated
feed
- Group 3—challenged and
fed FFC at 10 mg/kg/day for 5 days
- Group 4—challenged and
fed FFC at 20 mg/kg/day for 5 days
- Group 5—challenged and
fed FFC at 40 mg/kg/day for 5 days
Fish were weighed in groups and
then exposed for 2 hours to approximately
4.3 x 10CFU/mL
of a confirmed pathogenic isolate
of E. ictaluri, obtained
from a natural outbreak of ESC. Treatment
with FFC-medicated feed began in
Groups 3–5 the day after exposure
and continued for 5 consecutive days.
All surviving fish were then observed
for an additional 17 days, during
which time all groups were fed an
unmedicated ration.
Throughout the study, feeding activity
was subjectively graded each day,
based on the amount of feed consumed.
A score of 2 was assigned if 50–100%
of the food was consumed. A score
of 1 was given if <50% of the
food was consumed. A score of 0 was
given if little or no food was consumed.
The fish were also monitored daily
for abnormal behavior indicative
of morbidity (e.g., lethargy, abnormal
swimming patterns) and for adverse
events, such as piping at the water
surface or unexpected deaths.
Any fish that died during the study
were necropsied and bacterial culture
of E. ictaluri was attempted.
At the end of the study period (day
23), all surviving fish were counted,
weighed in groups, euthanized and
submitted for microbiologic and histopathologic
examination.
Results: Cumulative mortality rates over the 23-day
study period are shown in Figure 4-1. Following exposure, the
first mortalities occurred on day 7, in the challenged, untreated
fish (Group 2). Mortalities were seen in this group from day
7 to day 22, with the majority occurring between days 8 and 13.
The cumulative mortality rate in this group was 57.5%, with individual
tank mortality rates ranging from 35–100%.
Only 2/80 fish (2.5%) in the unchallenged, untreated group died. No mortalities
occurred in the 10 mg/kg FFC-treated fish (Group 3). One fish each in
the other 2 FFC-treated groups died (on day 22 or 23), for a cumulative
mortality rate of 1.25% for each group.
Mortality rates in the FFC-treated
fish (Groups 3–5) and in the
unchallenged, untreated fish (Group
1) were significantly lower than
the mortality rate in the challenged,
untreated fish (Group 2; p<0.0001
for each contrast). Pairwise contrasts
among FFC-treated groups and between
FFC-treated and unchallenged fish
showed no statistically significant
differences. Back
to top
A. Total
number of deaths. Includes fish
that died naturally and fish that
were euthanized at the end of the
study.
B. Challenged with E. ictaluri then treated with FFC at the
listed dose rate for 5 consecutive days.
No adverse events were noted during
the study. Feeding activity in the
unchallenged, unmedicated fish (Group
1) was scored at 2 throughout the
study. The average feeding activity
score for the challenged, unmedicated
fish (Group 2) was between 0 and
1 from days 4–17, and was consistently <1.5
until day 21.
Feeding activity in all FFC-medicated
groups was scored at 2 for all days
throughout the study, except day
5. On that one day, 2 tanks of fish
in Group 3 (10 mg FFC/kg) and one
tank of fish in Group 5 (40 mg FFC/kg)
had feeding scores of 1, dropping
the group average for that day to
1.5 and 1.75, respectively. At the
end of the study period, the average
body weight of surviving challenged,
untreated fish (Group 2) was lower
than that of surviving FFC-treated
fish (p<0.05).
Bacteria characteristic of E.
ictaluri were cultured from
94% of non-euthanized fish. Infection
rates for all fish, including surviving
fish euthanized at the end of the
study period, are shown in Figure
4-2.
A.
Total number of infected fish.
Includes fish that died naturally
and fish that were euthanized at
the end of the study.
B. Challenged with E. ictaluri then treated with FFC at the
listed dose rate for 5 consecutive days.
Gross examination revealed external
and internal lesions characteristic
of E. ictaluri infection
in 99% of fish that were culture-positive
for the organism. External lesions
included inflammation through the
sutra fontanel of the skull (“hole
in the head”) and hemorrhages
on the skin and fins. Exophthalmia
was also observed. Internal lesions
included hemorrhages on the gastrointestinal
tract and liver, as well as a congested
spleen, ascites and swollen kidneys.
The incidence of these lesions was
greatest in the challenged, untreated
fish (Group 2).
Histopathologic examination showed an increased degree of inflammatory
cell infiltrate in the liver, heart, gills, anterior kidney and spleen
of challenged, untreated fish (Group 2), and a paucity of lesions in
unchallenged fish and FFC-treated fish. No treatment-related changes
were noted on either gross or microscopic examination.
Conclusions: Florfenicol at a daily dose rate of 10–40
mg/kg body weight for 5 consecutive days was safe, palatable and highly
effective in reducing mortality and infection rates following E.
ictaluri challenge in channel catfish fingerlings. Treatment at
all dose rates increased survival and weight gain, and no adverse treatment-related
pathologic changes were found.
Comments: In this study,
a dosage of 10 mg FFC/kg daily for
5 consecutive days was effective
in reducing mortality in catfish
fingerlings exposed to E. ictaluri.
However, a longer duration of treatment—10
consecutive days—is recommended
for commercial situations, when conditions
are less controlled than in an experimental
setting. Back
to top
Dose Titration—Study
2
The following dose titration study was conducted to assess the safety,
palatability, and efficacy of Aquaflor-medicated
feed and to determine the optimal dose rate in channel catfish exposed
to a pathogenic strain of E. ictaluri.
Study Design: Six hundred
(600) laboratory-reared, 4- to 5-month-old
channel catfish fingerlings with
no known history of exposure to E.
ictaluri were divided into 5
groups of 120 fish:
- Group 1—not challenged
with E. ictaluri and fed
unmedicated feed
- Group 2—challenged with E.
ictaluri and fed unmedicated
feed
- Group 3—challenged and
fed Aquaflorat
5 mg FFC/kg/day for 10 days
- Group 4—challenged and
fed Aquaflorat
10 mg FFC/kg/day for 10 days
- Group 5—challenged and
fed Aquaflorat
15 mg FFC/kg/day for 10 days
Sera from 50 additional fish from
the same source were evaluated using
a modified agglutination assay and
found to be negative for antibodies
to E. ictaluri.
The study fish were weighed in groups
and then exposed by immersion for
2 hours to approximately 8.8 x 10 colony-forming
units (CFU)/mL of a confirmed pathogenic
isolate of E. ictaluri,
obtained from a natural outbreak
of ESC. Treatment with Aquaflor-medicated
feed began in Groups 3–5 the
day after exposure and continued
for 10 consecutive days. All surviving
fish were then observed for an additional
14 days, during which time all groups
were fed an unmedicated ration.
Throughout the study, feeding activity
was subjectively graded each day,
based on the amount of feed consumed.
A score of 2 was assigned if 50–100%
of the food was consumed. A score
of 1 was given if <50% of the
food was consumed. A score of 0 was
given if little or no food was consumed.
The fish were also monitored daily
for abnormal behavior indicative
of morbidity (e.g., lethargy, abnormal
swimming patterns) and for adverse
events, such as piping at the water
surface or unexpected deaths.
Any fish that died during the study
were necropsied and isolation of E.
ictaluri was attempted. At the
end of the study period (day 25),
all surviving fish were counted,
weighed in groups, euthanized and
submitted for microbiologic and pathologic
examination.
Results: Cumulative mortality
rates over the 24-day observation
period are shown in Figure 4-3. Following
exposure, the first mortalities occurred
on day 5, in the challenged, unmedicated
fish (Group 2). Mortalities were
seen in this group from day 5 to
day 18, with the majority occurring
on days 6–8. The cumulative
mortality rate in this group was
60%, with individual tank mortality
rates ranging from 45–85%. One
of the unchallenged, unmedicated
fish (Group 1) died.
Only 9/360 fish in the Aquaflor-treated
groups died (Figure 4-3). Mortality rates in the Aquaflor-treated
fish (Groups 3–5) and in the unchallenged, untreated fish (Group
1) were significantly lower than the mortality rate in the challenged,
untreated fish (Group 2; p<0.0001 for each contrast). There were no
statistically significant differences in mortality rate between Aquaflor-treated
groups or between Aquaflor-treated
and unchallenged fish.
A. Total
number of deaths. Includes fish
that died naturally and fish that
were euthanized at the end of the
study.
B. Challenged with E. ictaluri then treated with FFC at the
listed dose rate for 10 consecutive days.
No adverse events were noted during
the study. Feeding activity in the
unchallenged, unmedicated fish (Group
1) was scored at 2 throughout the
study. The average feeding activity
score for the challenged, unmedicated
fish (Group 2) ranged from 0.33 to
1.67 from Day 3 to 13. From Day 3
onward, the average feeding score
did not return to 2 with the exception
of Day 23.
Feeding activity in all Aquaflor-medicated
groups was scored at 2 for all days
throughout the study, except day
18, in which one of the tanks in
Group 5 (15 mg FFC/kg) had a feeding
activity score of 1, dropping the
average score for Group 5 on day
19 to 1.83.
Bacteria characteristic of E.
ictaluri were cultured from
100% of fish that died naturally
during the study period. Infection
rates for all fish, including surviving
fish euthanized at the end of the
study period, are shown in Figure
4-4.
A.
Total number of infected fish.
Includes fish that died naturally
and fish that were euthanized at
the end of the study.
B. Challenged with E. ictaluri then treated with FFC at the
listed dose rate for 10 consecutive days.
Gross examination revealed external
and internal lesions characteristic
of E. ictaluri infection
in 81% of fish that were culture-positive
for the organism. External lesions
included inflammation through the
sutra fontanel of the skull (“hole
in the head”), hemorrhages
on the skin and fins, and exophthalmia.
Internal lesions included hemorrhages
on the gastrointestinal tract and
liver, as well as a congested spleen,
ascites and swollen kidneys. The
incidence of these lesions was greatest
in the challenged, untreated fish
(Group 2).
Conclusion: Aquaflor at
a daily dose rate of 5–15 mg FFC/kg body weight for 10
consecutive days was safe, palatable and highly effective in
reducing mortality and infection rates following E. ictaluri challenge
in channel catfish fingerlings. No adverse treatment-related
gross pathological effects were found at any dose rate.
Comments: While there were
no significant differences in mortality
or infection rates between the 5
mg/kg and 10 mg/kg Aquaflor groups,
a dosage of 10 mg FFC/kg/day for
10 consecutive days is recommended
to ensure adequate intake of FFC
by all fish in the tank or pond.
Feeding observations indicated excellent
consumption of medicated feed at
all dose rates. However, in other
fish species, it has been demonstrated
that the more aggressive eaters receive
proportionately more of the medicated
feed than the less aggressive eaters. Back
to top
Dose Confirmation
Study
The following study was conducted to confirm the efficacy
and palatability of Aquaflor-medicated
feed, at a dose rate of 10 mg FFC/kg/day for 10 consecutive days,
in channel catfish exposed to a pathogenic strain of E. ictaluri.
Study Design: Six hundred
(600) laboratory-reared, 4- to 5-month-old
channel catfish fingerlings with
no known history of exposure to E.
ictaluri were divided into 2
groups of 300 fish:
- Group 1—challenged with E.
ictaluri and fed unmedicated
feed
- Group 2—challenged and
fed Aquaflor 50%
Type A Medicated Article at 10
mg FFC/kg/day for 10 days
Sera from 50 additional fish from
the same source were evaluated using
a modified agglutination assay and
found to be negative for antibodies
to E. ictaluri.
The study fish were weighed in groups
and then exposed to approximately
9.5 x 105CFU/mL of a confirmed pathogenic
isolate of E. ictaluri,
obtained from a natural outbreak
of ESC. Both treatment groups received
commercial unmedicated feed on Day
1. In Group 2, treatment with Aquaflor-medicated
feed began 2 days after exposure
and continued for 10 consecutive
days. All surviving fish were then
observed for an additional 14 days,
during which time both groups were
fed an unmedicated ration.
Throughout the study, feeding activity
was subjectively graded each day,
based on the amount of feed consumed.
A score of 2 was assigned if 50–100%
of the food was consumed. A score
of 1 was given if <50% of the
food was consumed. A score of 0 was
given if little or no food was consumed.
The fish were also monitored daily
for abnormal behavior indicative
of morbidity (e.g., lethargy, abnormal
swimming patterns) and for adverse
events, such as piping at the water
surface or unexpected deaths.
Any fish that died during the study
were necropsied and isolation of E.
ictaluri was attempted. At the
end of the study period (day 25),
all surviving fish were counted,
weighed in groups, euthanized and
submitted for microbiologic and pathologic
examination.
Results: Cumulative mortality
rates are shown in Figure 4-5. Following
exposure, the first mortalities occurred
on day 3 in the challenged, unmedicated
fish (Group 1). The majority of deaths
in this group occurred between days
5 and 11, with the most occurring
on day 7. The cumulative mortality
rate in this group was 87.3%, with
individual tank mortality rates ranging
from 60–100%.
Only 42/300 (14%) of the Aquaflor-treated
fish died. The majority of these deaths occurred between days 5 and 7.
Individual tank mortality rates for this group ranged from 0–75%.
The cumulative mortality rate in the Aquaflor-treated
fish was significantly lower than that in untreated fish (p<0.0001).
The average feeding activity score
in the Aquaflor-treated
fish ranged from 1.7–2. The
lowest score in this group was recorded
on day 4, when 5/15 tanks had a feeding
activity score of 1. The remaining
10 tanks had a feeding score of 2,
yielding an average score for that
day of 1.7. From day 6 onward, at
least 14/15 tanks of Aquaflor-treated
fish maintained feeding scores of
2.
The lowest score for the untreated
fish (Group 1) was also recorded
on day 4, when the average for the
group was 0.13. The average feeding
activity score was <1.6 on 12
of 25 study days. The score was <1
on 6 of 25 days. From day 17 onward,
the feeding activity score in surviving
fish was 1.8–2.
E. ictaluri was cultured
from 251/300 fish (83.7%) in the
untreated group and only 28/300 fish
(9.3%) in the Aquaflor-treated
group (Figure 4-5).
A. Includes
fish that died naturally and fish
that were euthanized at the end
of the study.
B. Challenged with E. ictaluri then treated with FFC at the
listed dose rate for 10 consecutive days.
Gross examination revealed external
and internal lesions characteristic
of E. ictaluri infection
in 88% of fish that were culture-positive
for the organism. External lesions
included inflammation through the
sutra fontanel of the skull (“hole
in the head”), hemorrhages
on the skin and fins, and exophthalmia.
Internal lesions included hemorrhages
on the gastrointestinal tract and
liver, as well as a congested spleen,
ascites and swollen kidneys. The
incidence of these lesions was greatest
in the challenged, untreated fish
(Group 1).
Conclusions: Aquaflor at a daily
dose rate of 10 mg FFC/kg body weight for 10 consecutive days was palatable
and highly effective in reducing mortality and infection rates following E.
ictaluri challenge in channel catfish fingerlings. Back
to top
Clinical Efficacy—Pond
Trial
The following study was conducted to confirm the efficacy
of Aquaflor-medicated feed, at
a daily dose rate of 10 mg FFC/kg body weight for 10 consecutive days,
for the control of mortality associated with ESC caused by E. ictaluri in
catfish under commercial farming conditions.
Study Design: The study
was conducted using approximately
154,000 channel catfish fingerlings
(150–180 days of age, weighing
6.6–7.8 grams) from an ESC-free
facility. Fish were held in 14 ponds
of approximately 0.1 acre each, at
a stocking rate of approximately
11,000 fish/pond. Ponds were assigned
to 1 of 2 treatment groups:
- Group 1—fed unmedicated
feed
- Group 2—fed Aquaflor50%
Type A Medicated Article at a dose
rate of 10 mg FFC/kg/day for 10
days
Fish from both groups were challenged
with a pathogenic isolate of E.
ictaluri obtained from a natural
outbreak of ESC. All fish were fed
unmedicated feed, and ponds were
observed until the cumulative morbidity/mortality
rate attributable to ESC (based on
clinical signs and/or lesions) reached
0.3% per pond. At that point, fish
in ponds assigned to Group 2 received
Aquaflor-medicated
feed for 10 consecutive days. At
the end of the treatment period,
the fish were observed for a further
14 days.
During the 24-day study period,
all ponds were monitored daily for
morbidity and mortality. All dead
or moribund fish displaying clinical
signs of ESC (e.g., swimming in circles
at the pond surface, lethargy, “hole
in the head,” ascites) were
collected and examined by gross necropsy.
Samples were submitted for isolation
of E. ictaluri. In ponds
in which >5 moribund/dead fish
were found per week, only 5 moribund
or minimally autolyzed dead fish
were cultured per week from that
pond.
At the end of the study (day 25),
all fish were euthanized and 20 fish
from each pond were examined by gross
necropsy and cultured for E.
ictaluri. In samples positive
for E. ictaluri, MICs for
FFC were determined on up to 15 fish
from each pond. All carcasses were
subsequently incinerated or buried.
Results: For this analysis,
a fish's survival time was defined
as the number of days a fish lived
after the pond was admitted into
the study. The survival analysis
not only took into account a fish's
living status at the end of the study,
(alive or dead) but also utilized
the information of how long the fish
lived. This allowed the survival
rate of all the fish in the entire
study period to be estimated, and
therefore, provide for a better understanding
of the treatment effect over time.
Results
from the survival analysis based
on fish recovered, are shown in Figure
4-6. The two treatment groups were
significantly different (p<0.0001),
with the Aquaflor group
having a higher survival rate than
the control. The Aquaflor group
had statistically significant lower
cumulative mortality rate than the
control group (p=0.0397, one-sided
test). The parameter estimate of
0.7906 equates to an odds ratio of
2.20, indicating the odds of mortality
are 2.2 times the odds of mortality
in the Aquaflor-treated
group.
A. Vertical
bar separates treatment/post-treatment
periods.
B. Challenged with E. ictaluri then treated with FFC at the
listed dose rate for 10 consecutive days.
The MIC for all tested isolates
of E. ictaluri was 0.25 µg/mL.
The mean Disk Diffusion Assay zone
of inhibition was 36.8 mm (range:
32–50 mm) for all E. ictaluri isolates.
Conclusion: Aquaflor administered
to channel catfish under commercial farming conditions at a daily
dose rate of 10 mg FFC/kg body weight for 10 consecutive days
was effective in reducing mortality associated with infection
by E. ictaluri. Back
to top
Environment
An environmental risk assessment has been performed for the use of Aquaflor in
catfish production. This assessment included evaluation of all available
data for florfenicol and its metabolites, and generation of data specific
for the use of florfenicol in freshwater environments.
Environmental
Exposure Profile
The concentrations of florfenicol
and its metabolites in the receiving
environment are dependent on the
quantities of medicated feed administered
and consumed and their fate in the
environment.
Florfenicol has a molecular weight
of 358.21 daltons, water solubility
of 1.32 g/L at pH 7, and a log Kow value
(partition coefficient) of 0.37,
the latter indicating little potential
for bioaccumulation. A conservative
estimate of biodegradation half-life
in aquatic systems is 30 days. As
the physiochemical characteristics
of florfenicol and its major metabolites
show, accumulation of florfenicol
or its degradation products in sediments
or biota is unlikely.
Because nearly all catfish feed is formulated as floating extruded pellets,
this type of feed has high water stability and does not sink into sediments
where it becomes unavailable to fish. Virtually 100% of dispensed feed
is consumed by catfish and little (if any) reaches the sediments. Thus,
florfenicol and its metabolites enter the sediment only via excreta,
with the compounds then moving into the water column through leaching
from feces and by mixing of the aqueous phase of excreta into the water
column.
As an enclosed aquaculture system,
catfish ponds do not represent the
ambient environment and are not of
environmental concern. However, any
overflow or release of water (draining)
from the ponds into the general environment
needs to be assessed. Catfish are
typically reared in large ponds approximately
1 m in depth, with the water level
maintained below the surrounding
soil surface by a perimeter levee.
To reduce water loss via overflow
and the release of effluent from
the ponds, the water level is maintained
below the overflow structure of the
pond (i.e., a 20-cm storage capacity
below the overflow level). This strategy
normally prevents rainfall from causing
overflow. In addition, clay soil
types are often a predominant feature
of pond construction, so leakage
from ponds is minimal. These factors
serve to limit the release of pond
water and any florfenicol-related
residues into the environment.
Environmental
Exposure Concentrations
Studies investigating the concentrations
of florfenicol and metabolites released
into the environment have been conducted.
Assessments were based on hypothetical
drug-use scenarios that would release
the greatest possible amount of florfenicol
(and metabolites) into the environment.
The “worst-case” scenario
that was developed involved the complete
draining of a nursery pond (fingerlings)
42 days after completion of a 10-day
florfenicol treatment regimen.
This worst-case analysis yielded
a preliminary predicted environmental
concentration (PEC) in the pond of
0.067 mg/L, a calculation that assumed
100% of the florfenicol-related residues
were in the water column (none partitioned
to sediments, and none remained in
fish) and assumed no residue degradation
occurred in the pond. A more realistic
analysis that accounted for the 30-day
florfenicol half-life in water yielded
an estimated PEC of 0.0268 mg/L in
pond water. If this is then diluted
1:10 into receiving waters, the refined
PEC is estimated to be only 0.00268
mg/L.
Clearly, very low concentrations
of florfenicol or metabolites are
released into the environment, even
under worst-case conditions. Considering
the fact that drug degradation occurs
in water, and pond effluent is dispersed
and diluted in receiving waters,
the environmental risk potentially
posed by florfenicol-related residues
in waters outside catfish ponds is
very low. Sediments were not included
in this analysis because florfenicol
does not enter or remain in sediments
in significant amounts, but will
move into the water phase. Furthermore,
any florfenicol that is released
from ponds will remain in the water
phase and will not partition to sediments.
Environmental
Safety
Studies undertaken by Schering-Plough
Animal Health constitute a comprehensive
data set regarding the safety of
florfenicol for invertebrates, fish,
birds and mammals. To
establish the potential for an adverse
effect in the environment, the minimum
inhibitory concentration (MIC), lethal
concentration (LC50)
or effect concentration (EC50)
values, and no observable effect
concentration (NOEC) were compared
to the PEC in aquatic environments.
Ratios of the PEC to the predicted
no effect concentration (PNEC) were
established for a wide range of aquatic
life forms. The PNEC values were
derived by applying a 10- to 100-fold
safety factor to the L(E)C50 or
NOEC obtained. Details of the environmental
risk characterization data for selected
freshwater species are shown in Table
5-1.
The refined PEC is far less than the calculated PNEC values.
The very low PEC/PNEC proportions reflect the lack of toxicity
of florfenicol to aquatic organisms. Since only very small amounts
of florfenicol can enter the aquatic environment, and the drug
and its metabolites are then rapidly degraded and dissipated,
florfenicol poses little if any risk to aquatic ecosystems. Existing
toxicity data indicate that florfenicol is, in general, more
active against prokaryotic than eukaryotic organisms. However,
the likelihood of environmental effects are very limited given
the low PEC:PNEC ratios, the drug’s intended use patterns,
its fate in the receiving environments and its low toxicity.
These data indicate that florfenicol administration via the feed to catfish
reared in commercial production facilities will not adversely affect
the aquatic environment. Back to top
A. Safety
factor of 100 applied (10X to account
for intraspecies variation; 10X
for extrapolation from acute to
chronic data).
B. Refined PEC of 0.00268 mg/L for florfenicol (pond water released into
environment).
C. MIC (µg/L).
References
1. Anon. (2000) Catfish Health and
Production Practices, United States
Department of Agriculture (USDA)
Animal and Plant Health Inspection
Service (APHIS) National Animal Health
Monitoring System (NAHMS) Report.
2. MacMillan, J.R. (1985) Infectious
diseases. In Channel Catfish
Culture. Tucker, CS (ed.), Elsevier,
New York, NY, pp. 405–496.
3. Thune, R.L. (1991) Major infectious
and parasitic diseases of channel
catfish. Veterinary and Human
Toxicology, 33 (supplement 1):
14–18.
4. Gaunt, P.S., Endris, R.G., Khoo,
L., McGinnis, A., Santucci, T., Leard,
T., Jack, S., Katz, T., Radecki,
S.V., Simmons, R. (2003) Preliminary
assessment of the tolerance and efficacy
of florfenicol against Edwardsiella
ictaluri administered in feed
to channel catfish. Journal of
Aquatic Animal Health, 15: 239–247.
5. Gaunt, P.S., Endris, R.G., Khoo,
L., Howard, R., McGinnis, A., Santucci,
T., Katz, T., (2004) Determination
of dose rate of florfenicol in feed
for control of mortality in channel
catfish, Ictalurus punctatus (Rafinesque),
infected with Edwardsiella ictaluri,
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