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Bac te ri al D i sease i n Warm wate r F i sh :
New Strategies for Sustainable Control
N A T A L, B R A Z I L
foreWord
W
orldwide, production of farmed fish has skyrocketed from less than 1 million
tons per year in the early 1950s to nearly 52 million tons with a value of US $80
billion — an annual growth rate of nearly 7%.
Tilapia production, in particular, has markedly increased because of the fish’s
large size, rapid growth and palatability.
Not surprisingly, greater demands on production systems have also increased the
level of disease challenge. The tilapia industry must, therefore, find and implement
new ways to manage costly diseases and maintain efficient production.
To help with this effort, MSD Animal Health organized an educational symposium,
“Bacterial Disease in Warmwater Fish: New Strategies for Sustainable Control.”
Held in conjunction with the 2011 World Aquaculture Society conference in
Natal, Brazil, the symposium gave recognized experts the opportunity to share
their latest research and insights with production managers of the world’s top
tilapia farms.
We are grateful to the presenters for preparing these valuable papers. For
additional copies of this booklet or more information, please contact your
MSD Animal Health representative or visit us at the website listed below.
PALMA JORDAN
Marketing director
global Aquatic Animal Health
Msd Animal Health
http://aqua.merck-animal-health.com
p r o c e e d i n g s
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
tABLe of contents
4
Presenter biographies
6
The impact of streptococcosis on tilapia in Brazil and efficacy
of AquaVac® Strep Sa for managing the disease under
controlled conditions
11
ROGÉRIO SALVADOR, PhD
Agrarian science center, north paraná state
University, campus Luiz Meneghel, Brazil
AquaVac® Strep Sa vaccine efficacy for controlling streptococcosis
RODRIGO ZANOLO, MV, MSc
in intensively reared Nile tilapia (Oreochromis niloticus) in Brazil:
Msd Animal Health, Brazil
increased performance and profits
18
Streptococcus in tilapia: implications for vaccine
development and field experiences from Asia
25
The responsible use of veterinary medicines in aquatic
food production
28
Best treatment practices of warmwater fish pathogens
using feed medicated with Aquaflor® (florfenicol)
35
Depletion of florfenicol in water and florfenicol amine
from fillet tissue after feeding Aquaflor® (florfenicol) to
tilapia in a recirculating aquaculture system
NEIL WENDOVER, BSc
Msd Animal Health, singapore
MELBA B. REANTASO, PhD
Aquaculture service, fisheries and Aquaculture
department, food and Agriculture organization
of the United nations, italy
PATRICIA S. GAUNT, DVM, PhD, DABVT
department of pathobiology and population
Medicine, Mississippi state University,
college of Veterinary Medicine, UsA
MARK P. GAIKOWSKI , MA
Us geological survey, Upper Midwest
environmental sciences center, UsA
BiogrApHies
Rogério Salvador is a professor at the
Rodrigo Zanolo is an aquaculture
Neil Wendover works for the global Aquatic
Agrarian science center, north paraná state
marketing manager for Msd Animal Health,
Animal Health Business Unit of Msd Animal
University, campus Luiz Meneghel, Brazil; he is
Brazil. Zanolo holds a degree in veterinary
Health. for the past 4 years, he has been based
also director of the university’s Veterinary Hospital
medicine from Londrina state University and a
at the company’s vaccine laboratory in singapore.
and coordinator of the fish immunopathology
Master of science in animal (fish) pathology
As a technical manager, one of his major
Laboratory. He graduated as a veterinarian
and an MBA in marketing from getúlio Vargas
activities is the investigation and epidemiological
and has a master’s degree in animal science
foundation in são paulo. He has extensive
study of fish diseases throughout Asia. Wendover
from Londrina state University and a phd in
experience in aquaculture, where he has worked
provides regular training and technical support
aquaculture from são paulo state University Júlio
as a consultant in the production of fish and
to the aquaculture industry in the areas of
de Mesquita filho. salvador has broad experience
shrimp in the states of paraná and santa catarina.
disease diagnosis, health management and
in the areas of freshwater fish health and applied
microbiology in finfish aquaculture. currently he
vaccination strategies. He has extensive
e-mail: [email protected]
conducts research and provides orientation to
experience in tilapia production in both Asia
and Africa.
students in the area of aquaculture vaccines
and immunogens.
e-mail: [email protected]
e-mail: [email protected]
rodrigo Zanolo, MV, Msc
Msd Animal Health
cotia, são paulo, Brazil
neil Wendover, Bsc
global Aquatic Animal Health
Msd Animal Health, singapore
rogério salvador, phd
Universidade estadual do norte do paraná
(north paraná state University)
campus Luiz Meneghel
Bandeirantes, paraná, Brazil
B a c t e r i a l D i s e a s e i n Wa r m wa t e r F i s h :
4
Melba B. Reantaso is an aquaculture
Patricia Simmons Gaunt is an associate
Mark P. Gaikowski is a supervisory
officer with the Aquaculture service, fisheries
professor of aquatic animal health in the
biologist at the Us geological survey’s Upper
and Aquaculture department, food and
department of pathobiology and population
Midwest environmental sciences center,
Agriculture organization of the United nations.
Medicine, Mississippi state University, college
La crosse, Wisconsin, UsA. His specialty is aquatic
she is based in rome, italy, and has nearly 30
of Veterinary Medicine. she earned her dVM and
toxicology and his research has included target
years’ experience in many aspects of aquaculture
a phd in veterinary toxicology at Louisiana state
animal safety studies, development of an
and aquatic animal health, including research,
University. she was the principal investigator
external columnaris infection model in fish and
training, diagnostics, international aid and
for efficacy and residue depletion studies that
environmental safety studies. gaikowski holds
project development. she also has authored
were necessary for the approval of Aquaflor®
several awards and honors and has been
or co-authored more than 80 scientific and
(florfenicol) for use in catfish in the Us and
published extensively in aquaculture journals.
technical publications. reantaso has a phd and
was the principal investigator of a study on the
He holds Bs and MA degrees in biology from the
post-doctoral qualifications from the University
efficacy of Aquaflor against Streptococcus iniae
University of south dakota.
of tokyo and the nippon Veterinary and Animal
in tilapia.
e-mail: [email protected]
science University, Japan.
e-mail: [email protected]
e-mail: [email protected]
Melba B. reantaso, phd
Aquaculture officer
Aquaculture service (firA)
fisheries and Aquaculture resources Use and
conservation division (fiM)
fisheries and Aquaculture department
food and Agriculture organization of the
United nations (fAo)
Viale terme di caracalla, rome, italy
patricia simmons gaunt, dVM, phd,
diplomate, American Board of Veterinary toxicology
Mississippi state University college of
Veterinary Medicine
stoneville, Mississippi, UsA
Mark p. gaikowski, MA
Us geological survey
Upper Midwest environmental sciences center
La crosse, Wisconsin, UsA
New Strategies for Sustainable Control
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The impact of streptococcosis on tilapia in Brazil
and efficacy of AquaVac® Strep Sa for managing
the disease under controlled conditions
Rogério Salvador, PhD; Rodrigo Zanolo, MV, MSc; Leonardo Cericato, PhD
Key points
INTroDUCTIoN
*
*
*
Streptococcus agalactiae Biotype
II infection in tilapia has become
a serious problem in several
countries, resulting in economic
losses; vaccines to control the
disease represent a promising
new tool.
A study was conducted to
evaluate the efficacy of the
vaccine AquaVac® Strep Sa
against experimental challenge
with S. agalactiae Biotype II.
Vaccinated fish were
protected against challenge
and their mortality was
significantly lower compared
to unvaccinated controls; in
addition, the vaccine was
found to be safe for fish.
Aquaculture is one of world's growing
animal production segments.1 In Brazil,
however, commercial fish production in
net pens is only just starting, despite its
great potential represented by 6 million
hectares of water contained in weirs
and reservoirs constructed mainly for
generating hydroelectric energy. In the
future, Brazil is likely to become one of the
world's largest aquaculture producers.2
Among fish species showing potential for
cage farming is Nile tilapia (Oreochromis
niloticus). Over the past decade, it has
become the species with the largest
production volume in Brazil, representing
nearly 40% of the country's aquaculture.
Tilapia cultivation has been developed
primarily in Brazil's South, Southeast and
Northeast regions, the latter representing
the highest production volume. In 2004,
41% of total domestic tilapia production
occurred in this region. The Northeast
region, in fact, has led the country's
tilapia production since 2003, with a clear,
growing trend due to its climate and
technological development, enabling it
to serve the growing demand for tilapia
both regionally and nationwide.3
In recent decades, improved technology in
fish production units has become a major
competitive advantage. Efforts to survive
and withstand an increasingly globalized
market have become an evident need. In
this context, and despite efforts to improve
fish quality through the implementation of
health programs and novel technologies,
tilapia production success depends on
innovative tools.
A future challenge can now be foreseen:
Mankind demands more and more
products that are not only nutritious
but are wholesome and pathogen-free,
that promote health and that are
environmentally friendly and socially fair
— in perfect harmony with the globalized
world we live in today.4
Intensive fish production, however, brings
about stress, resulting in the emergence of
diseases and, therefore, mortality.5 Among
the major maladies affecting tilapia,
Streptococcus agalactiae infection, resulting
in streptococcosis, plays a very important
role worldwide.6 Epidemiological studies
sponsored by MSD Animal Health around
the world have shown the presence of two
different S. agalactiae groups or biotypes
(i.e., I and II). Tilapia isolates from different
regions in the world show that 26% of
streptococci were identified as S. agalactiae
Biotype I, while 56% were S. agalactiae
Biotype II. S. agalactiae Biotype II is the
world's most prevalent biotype, found
mostly in China, Indonesia, Vietnam, the
Philippines and Latin America. In Brazil,
serological studies show 100% positive
serology to S. agalactiae Biotype II.
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The largest economic impact due to
S. agalactiae in freshwater-farmed fish
species occurs in Nile tilapia. The
geographic distribution of S. agalactiae
includes regions with temperate, tropical
weather, where warmwater fish are
cultivated. So far, outbreaks have been
reported in several countries including:
the US, Japan, Kuwait, Israel, Thailand
and Brazil.
S. agalactiae field strains have already developed resistance to antimicrobials.9 Evaluation of the vaccine AquaVac® Strep Sa
under experimental conditions has demonstrated significantly decreased
mortality in vaccinated fish, illustrating
the efficacy of the vaccine for the
prevention and control of streptococcosis
in Nile tilapia.
(Unidade de Infecção Experimental de Organismos Aquáticos do Laboratório
de Imunopatologia de Peixes, LIPPE) and
conditioned in 12, 80-liter aquariums
(n = 15). Aquariums received UV-sterilized,
dechlorinated, running water from an
artesian well. Fish acclimation lasted 7
days, which was time enough for plasma
cortisol concentrations and osmolality
to return to baseline levels.
This pathogen is responsible for high
economic losses; mortality on a farm can
reach 90%, typically at the pre-market
age when substantial feed volumes
have already been consumed. We must
remember that feed is the largest
component of production costs and that
much has been invested in the fish by
this time.7
M AT E r I A l S A N D M E T H o D S
Water temperature was measured daily
(27° C/81° F ± 1.5°). Hydrogen ion potential
(7.1 ± 0.3) and dissolved oxygen (5.5 mg/L
± 1 mg/L) were measured weekly. All
values remained within welfarerecommended levels.10
Infection occurs when infected fish —
dead or alive, moribund or apparently
healthy — release the bacterium into
the water, allowing it to colonize the skin
of other fish. Invasive infections can also
occur, resulting in high mortality. In
addition, the bacterium can survive for
long periods of time in water, mud or
pond/greenhouse substrates, and
even on pieces of equipment used in
routine operations.8
If tilapia culture is to continue and proliferate, the industry will need strategies to
minimize the effects of disease. The advent
of S. agalactiae vaccines has brought
about a new, promising tool, since some
In the study, 180 Nile tilapia (Oreochromis
niloticus) juveniles from the same spawn
and weighing ~35 g were used. Prior to
the start of the study, the fish were
maintained in quarantine and subjected
to a prophylactic antimicrobial bath;
they were then conditioned in 250-liter
cages under constant dechlorinated
water exchange.
Upon the completion of quarantine,
fish were moved to the Aquatic
Organism Experimental Infection Unit,
Fish Immunopathology Laboratory
Throughout the experiment, ad libitum
feeding was provided twice daily (09:00h
and 17:00h) at the rate of 5% of biomass.
Experimental design
Fish in the 12 aquariums represented
three repetitions for each treatment group
(Table 1).
continued
tABLe 1
Fish distribution per treatment group
T1
Vaccinated with AquaVac Strep Sa / S. agalactiae-challenged
T2
Vaccinated with AquaVac Strep Sa / S. agalactiae-unchallenged
T3
Not vaccinated with AquaVac Strep Sa / S. agalactiae-challenged
T4
Not vaccinated with AquaVac Strep Sa / S. agalactiae-unchallenged
7
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tABLe 2
Vaccination
Vaccination against S. agalactiae was
performed at the end of the acclimatization
period at the laboratory. Fish weighing
~35 g (medium bodyweight) were
submitted to the vaccination process as
recommended by MSD Animal Health.
The fish were anesthetized by immersion
in 1% eugenol (Biodinamica®) containing
50 mg/L water. A single 0.05 mL dose
of AquaVac Strep Sa was injected
intraperitoneally in the anterior-medial
aspect, using a sterilized insulin
syringe/needle.
Survival of Nile tilapia (Oreochromis niloticus) vaccinated with AquaVac Strep Sa and
unvaccinated Nile tilapia 15 days after challenge with Streptococcus agalactiae (n = 15)
TREATMENT GROUP
T1*
T2
T3*
NUMBER OF SURVIVORS
(after 15 days)
AquaVac Strep Sa/Challenged
13
AquaVac Strep Sa/Challenged
14
AquaVac Strep Sa/Challenged
14
AquaVac Strep Sa/Unchallenged
15
AquaVac Strep Sa/Unchallenged
14
AquaVac Strep Sa/Unchallenged
14
Not vaccinated/Challenged
6
Not vaccinated/Challenged
8
Not vaccinated/Challenged
7
Not vaccinated/Unchallenged
14
Not vaccinated/Unchallenged
14
Not vaccinated/Unchallenged
14
Challenge
Challenge was performed 25 days
after vaccination. For inoculum
preparation, live streptococcus isolates
from naturally infected tilapia were
used. Isolates were previously classified
as Lancefield´s B group, using the
Slidex® Strepto Kit latex agglutination
test (BioMerieux, France), then
characterized as S. agalactiae based
on phenotype characteristics as
determined by the ApI® 20 Strep
Microtest System (BioMerieux, France.)11
The selected S. agalactiae strain was
seeded in brain/heart infusion broth,
incubated for 24 hours at 29° C (84.2° F)
T4
* Values were considered significantly different (P < 0.05) comparing group T1 to group T3.
under aerobic conditions. The challenge
dose (106 colony-forming units/mL) was
calculated on the basis of a lethal dose
killing 50% of the fish population (LD50).
Fish were observed for 15 consecutive
days after challenge for clinical signs and
death; the findings were recorded daily
and subjected to statistical analysis
(Tukey's test), with a 5% level
of significance.
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figUre 1
r E S U lT S
Animals in group T3 (not vaccinated/
challenged) that died showed clinical signs
compatible with streptococcosis, mostly
after 7 days post-challenge. Clinical signs
included lethargy, reduced appetite,
body-color darkening, uni/bilateral
exophthalmia, abdominal distention and
erratic/circular swimming. Necropsy
findings among these fish included ascites,
enlarged livers and diffused hemorrhages
in the central nervous system. Upon
microbiological analysis, S. agalactiae was
re-isolated, particularly from the brain,
which means generalized infection.
The appearance of clinical signs after 7
days post-challenge represents the natural
evolution of the infection. Neurological
signs suggest meningoencephalitis, and it
Mean number of survivors
Table 2 shows survival among fish in
various treatment groups. No significant
mortality occurred when comparing
groups T1 (vaccinated/challenged) and
T2 (vaccinated/unchallenged) with group
T4 (not vaccinated/unchallenged),
demonstrating the vaccine’s safety. None
of the fish that died in those groups
showed clinical signs compatible with
streptococcosis, but deaths occurred
within 48 hours after challenge, suggesting
that the cause of death was handlingassociated stress.
Mean number of survivors in Nile tilapia (Oreochromis niloticus) vaccinated with
AquaVac Strep Sa then challenged with Streptococcus agalactiae
18
16
14
12
10
8
6
4
2
0
a
a
a
b
Vaccinated/
Vaccinated/
Challenged (T1) Unchallenged (T2)
Not vaccinated/ Not vaccinated/
Challenged (T3) Unchallenged (T4)
Treatments
The letters “a” versus “b” denote statistical significance. Values were considered significantly
different (P < 0.05) when comparing T1 (vaccinated/challenged) to T3 (not vaccinated/
challenged) and demonstrate vaccine efficacy. Values were not considered to be significantly
different (P > 0.05) when comparing T2 (vaccinated/unchallenged) to T4 (not vaccinated/
unchallenged), which demonstrates vaccine safety.
Note: experiments were undertaken in triplicate (n = 15).
represents a clinical sign consistent with
streptococcosis. The mean number of
deaths among repetitions supports the
LD50 calculated for this particular study.
Variations in the numbers of dead fish
among repetitions can be the result of
innate, individual resistance variability.
Vaccination with AquaVac Strep Sa
protected animals against experimental
challenge with S. agalactiae, since mortality
in group T1 (vaccinated/challenged) was
significantly lower (P < 0.05) than mortality
in the T3 (not vaccinated/challenged) fish
(Figure 1). In this context, the relative
protection percent (RPP) was 84%. RPP was
determined using the following equation:
RPP = (1- (vaccinated fish deaths/control
fish deaths)) x 100.
continued
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The impact of streptococcosis on tilapia in Brazil
and efficacy of AquaVac® Strep Sa for managing
the disease under controlled conditions
CoNClUSIoN
rEFErENCES
Results from this study demonstrate
that AquaVac Strep Sa induced effective
protection in Nile tilapia experimentally
challenged with S. agalactiae. We, therefore, conclude that AquaVac Strep Sa
(MSD Animal Health) is a safe and highly
efficacious vaccine against the disease
caused by S. agalactiae Biotype II. A single
dose of AquaVac Strep Sa, administered
as directed, can be an important tool
in the prevention and control of
streptococcosis in Brazil, since serological
results so far show only the presence of
Biotype II in this country.
1
Crepaldi DV, et al. 2006. A situação da
aquacultura e da pesca no Brasil e no mundo.
Revista Brasileira de Reprodução Animal. 30:
81-85.
6
2
Marengoni NG. 2006. Produção de tilápia
do Nilo Oreochromis niloticus (Linhagem
chitralada), cultivada em tanques rede,
sob diferentes densidades de estocagem.
Archivos de Zootecnia. 55:127-138.
7
3
Ostrensky A, et al. 2008. Principais
problemas enfrentados atualmente pela
aqüicultura brasileira. In: Ostrensky A, et al.
Aqüicultura no Brasil: o desafio é crescer.
Brasília: Secretaria Especial de Aqüicultura
e Pesca/FAO. 135-158.
8
4
10
Medri V, et al. 2005. Desempenho das tilápias
nilóticas (Oreochromis niloticus) alimentadas
com diferentes níveis de proteínas de
levedura de destilaria em tanques-rede.
Acta Scientiarum (Animal Sciences). 27(2):
221-227.
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5
10
Vandenberg GW. 2004. Oral vaccines for
finfish: academic theory or commercial
reality? Animal Health Research Reviews.
52:301-304.
Pasnik DJ, et al. 2008. Antigenicity of
Streptococcus agalactiae extracellular
products and vaccine efficacy. Journal of
Fish Diseases. 28:205-212.
Evans JJ, et al. 2004. Efficacy of
Streptococcus agalactiae (group B)
vaccine in tilapia (Oreochromis niloticus)
by intraperitoneal and bath immersion
administration. Vaccine. 22:3769-3773.
Suresh AV. 1998. Tilapia update 1998. World
Aquaculture. 30:8-68.
9
Lim C, et al. 2006. Tilapia: biology, culture
and nutrition. An Imprint of the Haworth
Press, New York, United States: 678.
Sipaúba-Tavares LH, et al. 1994. Variação
dos parâmetros limnológicos em um viveiro
de piscicultura nos períodos de seca e chuva.
Revista UNIMAR. 16:229-242.
11
Salvador R, et al. 2005. Isolation and
characterization of group B Streptococcus
spp. from Nile tilapia (Oreochromis niloticus)
reared in hapas nets and in earth nurseries
in the north region of Paraná State, Brazil.
Ciência Rural. 35:1374-1378.
Zanolo
AquaVac® Strep Sa vaccine efficacy for controlling
streptococcosis in intensively reared Nile tilapia
(Oreochromis niloticus) in Brazil: increased
performance and profits
Rodrigo Zanolo, MV, MSc; Leonardo Cericato, PhD; Rogério Salvador, PhD;
Eduardo Yamashita; Gláucio Dorelles
Key points
INTroDUCTIoN
*
*
*
Streptococcus due to
Streptococcus agalactiae is
a significant problem in
Brazilian tilapia that tends to
strike well into the grow-out
period, after considerable
resources have been invested
in fish.
In two controlled trials
conducted at separate
commercial tilapia-production
sites with a history of acute
mortality and a positive
diagnosis of S. agalactiae
Biotype II, use of the vaccine
AquaVac® Strep Sa proved to
be safe for fish, reduced
mortality and improved
fish performance.
The return on investment
(roI) in Trial 1 was 7.4 times
higher when compared to
the investment in the vaccine.
In Trial 2, the roI for vaccination
was 5.3 times higher.
Brazilian fish farming has grown
exponentially over the last 20 years,
thanks to abundant water resources as
well as increased demand for fish protein.
Since much of this growth comes from intensive farming, particularly the production
of tilapia in floating cages, good health
management practices are crucial to the
success of the aquaculture industry.
Of the many bacterial diseases that can
affect cultivated fish around the world,
streptococcosis due to Streptococcus spp.
is the one with the greatest economic
impact on farmed tilapia.1 Streptococci are
widely distributed, opportunistic bacteria
found in the aquatic environment; their
pathogenicity is associated with stress
linked to poor water quality, ineffective
management and other conditions typical
of intensive aquaculture.2
In Brazil, early studies demonstrated the
presence of Group B Streptococcus, later
identified as Streptococcus agalactiae,
associated with acute mortality episodes in
farmed tilapia in the State of Paraná.3 More
recently, new characterization studies have
further confirmed S. agalactiae as the main
species responsible for streptococcosis in
intensive tilapia operations in Brazil.4
Risk factors such as high temperatures,
combined with other stressors faced by
caged tilapia including competition for
food, intense fish-to-fish contact resulting
in more physical injuries and potential
nutritional imbalance, have been shown
to be responsible for the increased
incidence of Streptococcus spp. septicemia.5
Diagnostic studies sponsored by MSD
Animal Health, and undertaken by
AQUAVET, UFLA (an aquatic-animal disease
laboratory), LIPPE and UENP (a fish
immunopathology laboratory), have
demonstrated that the presence of the
organism in several Brazilian states is
increasing, and there is a strong tendency
for rapid spread due to extensive transport
of animals.
Clinical signs of streptococcosis include
prominent eyes (exophthalmia), swimming
in circles (encephalitis), a swollen belly
(ascites) and nodular lesions in the muscles
of necrotized areas, which result in fillet
condemnations at the processing plant.
High mortality (3% to 20%) due to
streptococcosis has been observed in
intensive tilapia operations, depending
on the severity of the disease. When
poor conditions exist, mortality can be
even greater.
The high economic impact of streptococcosis is due to the fact that the disease affects
mostly fish between 300 g and 600 g in
bodyweight — well into the growout
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In addition, serology has been up to
100% positive for S. agalactiae Biotype II
(serotype Ib). This underscored the need
for a new preventative approach such as
vaccination. AquaVac Strep® Sa was
recently developed by MSD Animal Health
for control of S. agalactiae Biotytpe II
infections in farmed tilapia.
figUre 1
Epidemiological studies found approximately 150 isolates of S. agalactiae in
multiple Brazilian regions.
São Francisco river basin
and Northeast Big Weir
Epidemiological studies funded by
MSD Animal Health in Brazil found
approximately 150 isolates of
S. agalactiae from tilapia in multiple
Brazilian regions (Figure 1).
53 isolates positive for
S. agalactiae Biotype ii
(serotype ib)
Rio Grande basin
Paranaíba, Paraná, Tietê
and Paranapanema
97 isolates positive for
S. agalactiae Biotype ii
(serotype ib)
100% positive serology to S. agalactiae Biotype ii (serotype ib)
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period.4 By this time, large amounts of
money and time have been invested in the
fish, resulting in serious economic losses
due to mortality or, in fish that survive,
from decreased productivity and increased
feed-conversion rates (FCR.)
2 011
Zanolo
AquaVac® Strep Sa vaccine efficacy for controlling
streptococcosis in intensively reared Nile tilapia
(Oreochromis niloticus) in Brazil: increased
performance and profits
12
AquaVac Strep Sa is an oil-emulsion
vaccine containing killed S. agalactiae
Biotype II (serotype Ib) bacteria. The
vaccine is administered intraperitoneally
as a single dose of 0.05 mL per fish. Fish
to be vaccinated must weigh a minimum
of ~15 g. Laboratory studies have demonstrated that the onset of protection occurs
at approximately 28 days and lasts for up
to 7 months after vaccination.
M AT E r I A l S A N D M E T H o D S
To assess the safety, efficacy and economic
benefits of using AquaVac Strep Sa under
natural challenge conditions on Brazilian
tilapia farms, two trials were carried out in
different production sites in the Northwest
São Paulo state. In both trials, fish from the
same batch, weighing an average of 40 to
60 g, were vaccinated intraperitoneally
with a single 0.05 mL dose of AquaVac
Strep Sa, according to label directions.
Fish were sedated approximately 1 minute
prior to vaccination with a 50 ppm eugenol
(clove oil) solution. Vaccination in both
production units was performed between
July 20 and July 30, 2010. Water temperature was approximately 25° C (77° F) with a
dissolved oxygen concentration of 7 mg/L.
Both farms use similar production systems,
based on 18-m³ cages. In growout Phase I,
stocking density was 5,000 fish/cage, with
an average bodyweight of 40 to 60 g. Fish
were taken up to approximately 320 g
then classified on a per-size basis. In Phase
II, stocking density was 2,500 fish/cage,
fed to an average final bodyweight of
800 to 900 g.
p r o c e e d i n g s
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
Zanolo
TrIAl DESIgN
Farm 1: 30,000 fish divided in two
groups were used. One group included
15,000 vaccinated animals, while the
15,000 fish in the other group served
as unvaccinated controls.
For growout Phase I, the 15,000 fish in each
group were divided into three 18-m3 cages
(three repetitions of 5,000 fish each). At the
end of Phase I, all fish were classified by
size, then transferred to six cages of 2,500
fish each, for a total of six replicates.
Farm 2: 32,000 fish divided in two groups
were used. One group included 16,000
vaccinated fish, while the other group of
16,000 remained unvaccinated controls.
serological studies were performed with
samples from all experimental groups.
r E S U lT S A N D D I S C U S S I o N
AquaVac Strep Sa was highly effective in
both trials, significantly (p < 0.05) reducing
the S. agalactiae Biotype II (serotype Ib)associated mortality throughout the
production cycle. Product safety was
confirmed by comparing mortality among
vaccinates and controls after vaccination.
There were no great differences in mortality between the two groups immediately
following vaccination, before immunity in
vaccinates developed (Figure 2).
In both trials, outbreaks of acute mortality
were observed in all the experimental
cages (replicates), especially at the end of
Phase I. The effectiveness and benefits
of using AquaVac Strep Sa were evident,
however. Bacterial isolates were positively
identified as S. agalactiae Biotype II
(serotype Ib) and the results, which were
consistent at both experimental sites,
showed improved performance in
vaccinates after the development of
immunity as compared to controls.
Benefits including increased survival,
improved biomass gain, higher
performance and lower FCR were
measured (Table 1).
continued
figUre 2
For Phase I, fish in each experimental
group were placed in 18-m³ cages at the
rate of 5,250 fish each, to obtain three
repetitions. At Phase I completion, the fish
were classified by size, then transferred to
six cages of 2,625 fish each (six replicates).
Cumulative mortality in vaccinated and unvaccinated tilapia during the first 20 days
post-vaccination, before immunity developed in vaccinates
6.0%
6.0
5.5%
Vaccinated
Controls
After vaccination, fish were monitored daily
for behavior, daily mortality and growth
until harvest.
Both farms had a history of acute mortality
with a positive diagnosis of S. agalactiae
Biotype II (serotype Ib), which justified the
experimental vaccine program. During the
course of the trial, microbiological and
Mortality rate %
5.0
4.0
2.8%
3.0
2.0
2.0%
1.0
0
Farm 1
Farm 2
13
2 011
Farm 1: Daily mortality and final survival in the 15,000 vaccinated fish
Daily mortality
% Survival
100
80
95
90
Results – Farm 1:
Figures 3, 4 and 5 show the productive
performance results obtained on Farm 1.
Table 1 shows the best productive
results of the vaccinated group compared
to controls. It should be noted that the
highest final average bodyweight of fish
in the control group is due to the higher
mortality compared to the vaccinated
group, which reduced fish density
and enabled better growth among
surviving fish.
Daily mortality
60
6
JUNE
figUre 3
85
80
40
% Survival
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
Zanolo
AquaVac® Strep Sa vaccine efficacy for controlling
streptococcosis in intensively reared Nile tilapia
(Oreochromis niloticus) in Brazil: increased
performance and profits
75
20
70
65
0
23 31 08 16 24
JULY
AUGUST
01
09 17 25
SEPTEMBER
03
11 19
OCTOBER
27 04
12 20 28
NOVEMBER
Date
Vaccination
60
Harvest
figUre 4
Farm 1: Daily mortality and final survival in the 15,000 control fish
continued
Daily mortality
% Survival
100
N A T A L ,
14
95
80
Daily mortality
90
85
60
80
40
75
70
20
65
0
23 31 08 16 24
JULY
AUGUST
Vaccination
01
09 17 25
SEPTEMBER
Date
03
11 19
OCTOBER
27 04
12 20 28
NOVEMBER
Harvest
60
% Survival
B R A Z I L
100
p r o c e e d i n g s
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
Zanolo
figUre 5
Comparison of biomass gain (kg) between all vaccinated and unvaccinated fish on Farm 1
6.000
AquaVac Strep Sa
Controls
5.000
4283 kg
Running gain (kg)
3698 kg
4.000
585 kg
difference
3.000
2.000
1.000
23 31
JULY
08
13
20
AUGUST
27
03
10 17
24
SEPTEMBER
01
08
15
22
OCTOBER
29
05 12
19
NOVEMBER
Date
tABLe 1
Comparison of results in vaccinates and unvaccinated controls (average of three replicates)
on Farm 1
TREATMENT
VACCINATED GROUP
CONTROL GROUP
Initial weight (g)
51.7
51.0
Weight gain (g)
310.7
322.0
Final weight (g)
362.3
373.0
growout days
126
124
Biomass gain (kg)
1408.5
1173.3
Feed intake (kg)
2596.3
2450.1
Feed-conversion rate
1.85
2.10
Biomass (kg/m3)
83.36
71.41
Daily weight gain (g/day)
2.47
2.60
Survival (%)*
91.90a
76.57b
Total mortality (%)
8.10
23.43
Tukey´s test 5% (p < 5)
*The letters “a” and “b” denote statistical significance.
15
figUre 6
Daily mortality
Daily mortality
% Survival
100
120
80
100
80
60
60
40
40
20
0
2 011
JUNE
6
Results – Farm 2:
Figures 6, 7 and 8 show the
performance results obtained
on Farm 2, and Table 2
summarizes FCR and survival
in both farms.
Farm 2: Daily mortality and final survival in the 16,000 vaccinated fish
% Survival
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
Zanolo
AquaVac® Strep Sa vaccine efficacy for controlling
streptococcosis in intensively reared Nile tilapia
(Oreochromis niloticus) in Brazil: increased
performance and profits
20
29 06 14 22 30 07 15 23 01 09 17 25 02 10 18 26 04 12 20 28 05 13 21 29 06 14 22
JULY AUGUST
SEPTEMBER
OCTOBER
NOVEMBER
DECEMBER
JANUARY
FEBRUARY
Vaccination
Date
Harvest
figUre 7
Farm 2: Daily mortality and final survival in the 16,000 control fish
Highly significant differences
in performance were obtained
between the groups vaccinated with AquaVac Strep Sa
and controls in both trials.
Increased survival, thus
decreased FCR, resulted in
consistently higher economic
returns. The return on
investment (ROI) in Trial 1
was 7.4 times higher when
compared to the investment
in the vaccine. In Trial 2, the
ROI for vaccination was 5.3
times higher.
Daily mortality
% Survival
CoNClUSIoN
120
100
Daily mortality
80
80
60
60
40
N A T A L ,
40
16
20
0
20
29 06 14 22 30 07 15 23 01 09 17 25 02 10 18 26 04 12 20 28 05 13 21 29 06 14 22
JULY AUGUST
SEPTEMBER
OCTOBER
NOVEMBER
DECEMBER
JANUARY
FEBRUARY
Vaccination
Date
Harvest
% Survival
B R A Z I L
100
Under commercial production
conditions and in the face of
actual natural challenge,
AquaVac Strep Sa is highly
effective against streptococcal
infections (S. agalactiae
Biotype II, serotype Ib).
Productive parameters,
including survival/biomass
and FCR, were significantly
improved, resulting in an
excellent ROI in both trials.
p r o c e e d i n g s
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
Zanolo
figUre 8
rEFErENCES
Comparison of biomass gain (kg) between all vaccinated and unvaccinated fish on Farm 2
1
12.000
Suresh AV. 1998. Tilapia
Update 1998. World
Aquaculture. 30:8-68.
AquaVac Strep Sa
11.000
10822 kg
Controls
10.000
Running gain (kg)
9.000
8.000
9730 kg
2
1092 kg
difference
effect of environmental factors
7.000
Bunch EC, et al. 1997. The
on the susceptibility of hybrid
tilapia Oreochromis niloticus
6.000
x Oreochromis aureus to
5.000
streptococcosis. The Israeli
4.000
Journal of Aquaculture.
3.000
49:67-76.
2.000
1.000
3
Salvador R, et al. 2005.
Isolation and characterization
of group B Streptococcus
spp. from Nile tilapia
(Oreochromis niloticus) reared
in hapas nets and in earth
nurseries in the north region
of Paraná State, Brazil. Ciência
Rural. 35:1374-1378.
29 05 12 19 26 02 09 16 23 30 07 14 21 28 30 04 11 18 25 02 09 16 23 30 06 13 20 27 03 10 17
JULY AUGUST
SEPTEMBER
OCTOBER
NOVEMBER
DECEMBER
JANUARY FEBRUARY
Date
tABLe 2
Summary of FCR and survival results from both farms
FARM 1
Experimental groups
FARM 2
% Survival
FCr
% Survival
FCr
Controls
76.5%
2.10
78%
1.96
AquaVac Strep Sa
91.9%
1.85
87%
1.75
4
Figueiredo H, et al. 2007.
Estreptococose em Tilápia do
Nilo – Parte 1. Revista Panorama
da Aqüicultura. 17:103.
Improvement in vaccinated
fish compared to controls
15.4%
0.25
9%
0.21
5
Kubtiza F. 2002. Streptococcus
versus Tilápia: É preciso se
antecipar ao problema. Revista
Considering the tremendous economic
impact of streptococcosis on Brazilian
tilapia farms, AquaVac Strep Sa is a
valuable, highly effective vaccine.
AquaVac Strep Sa is an indispensable
tool for boosting the productivity,
performance and economic viability of
tilapia production in Brazil.
We must also consider that vaccination
results should be even more dramatic
under a mass vaccination scheme, since
protecting large fish populations will result
in decreased disease pressure in the field,
which can be reflected in overall benefits
for intensive tilapia farms.
Panorama da Aqüicultura. 7:65.
17
Wendover
Neil Wendover, BSc; Mario Aguirre; Rodrigo Zanolo, MV, MSc; Leonardo Cericato, PhD; Robin Wardle
Key points
INTroDUCTIoN
*
*
N A T A L ,
B R A Z I L
6
JUNE
2 011
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
Streptococcus in tilapia: implications for vaccine
development and field experiences from Asia
18
*
Epidemiological surveys
conducted by MSD Animal Health
in major tilapia-producing
countries demonstrate that
Streptococcus agalactiae
Biotype II is a globally significant
cause of streptococcosis,
resulting in chronic mortality
and economic losses.
In field trials, AquaVac® Strep Sa,
the first intraperitoneal vaccine
for tilapia, has proved to be safe
for fish and effective against
S. agalactiae Biotype II, while
laboratory studies have shown
it protects for at least 30 weeks.
The availability of AquaVac
Strep Sa could play a key role in
enabling the burgeoning tilapia
industry to sustain and continue
to grow.
Aquaculture is the world’s fastest growing
food sector and tilapia represents much
of that growth. The popularity of tilapia
continues to skyrocket. Global production
has nearly tripled since the beginning of
the decade with an estimated output of 3.7
million tons in 2010, according to the Food
and Agriculture Organization (FAO) of the
United Nations. No other fish species has
displayed such aggressive and sustainable
growth, year after year.
There are several reasons for increased
tilapia production. Tilapia can be produced
in versatile locations, water systems,
temperatures and salinities. They have
good performance characteristics such as
fast growth, a high fillet yield and low feedconversion ratio (FCR) as well as firm, white
fillet that makes them easy to market.
Tilapia can now be considered a commodity item with a stable supply, demand
and price. However, the cost of raw
materials is increasing, which is increasing
the cost of production and reducing
profit margins. Further advancements
in production efficiency are required to
improve profitability, and this trend is
continually driving the industry toward
consolidation and intensification. Indeed,
FAO reports that the overall number of
fish farms is decreasing, while the size of
individual farms is increasing, indicating
the concentration of fish farms into fewer
hands. This situation fosters the
emergence of production diseases —
and tilapia are no exception.
Experience has shown that most intensive
fish farming operations suffer from
between six and eight major production
diseases and that these must be prevented
or controlled before the industry can
become truly sustainable. In tilapia, we
have so far identified four major bacterial
diseases: Streptococcus agalactiae,
Streptococcus iniae, Flavobacterium
columnare and Francisella spp.; one viral
disease, iridovirus; and two major groups
of parasites: the monogeneans such as
Gyrodactylus and the protozoan such as
Trichodina (Figure 1). Their prevalence and
severity depend on many environmental
factors, such as geographical location,
culture system, farming intensity, water
salinity and temperature, and on several
biological factors such as age, genetics,
nutrition and stress.
STrEPToCoCCUS:
A N E S T A B l I S H E D P AT H o g E N
By far the most important diseases
economically are those due to
streptococcus. In many instances,
streptococcosis does not contribute the
highest mortality, but it kills large fish
and, as a consequence, heavily affects
the FCR, reduces marketable product
and damages production and processing
efficiency (Figure 2).
continued
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
Wendover
p r o c e e d i n g s
figUre 1
Major diseases affecting tilapia
PHASE
Hatchery sex-reversal
0g
PATHOGEN
Nursery
1g
Pre-growout
10 g
growout
100 g
1 kg
Trichodina; Dactylogyrus; Amyloodinium
Streptococcus spp. (Sa; Si)
Francisella spp.
Flavobacterium columnare
Edwardsiella tarda
Nocardia seriolae
Iridovirus
Saprolegnia; Branchiomyces
Note: Importance of the disease is roughly in proportion to the size of the arrow bars.
figUre 2
Some clinical signs of streptococcal diseases in farmed tilapia
LEFT: Typical pin-point petecchial
hemorrhaging caused by
Streptococcus agalactiae
MIDDLE: 3-gram juvenile tilapia
with bilateral exophthalmia
RIGHT: Late-stage bilateral
exophthalmia and corneal opacity
19
N A T A L ,
B R A Z I L
6
JUNE
2 011
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
Wendover
Streptococcus in tilapia: implications for vaccine
development and field experiences from Asia
20
tABLe 1
In 2000, MSD Animal Health initiated
extensive epidemiological surveys in the
major tilapia-producing regions of Asia
and Latin America. We have identified over
1,000 bacterial isolates from tilapia reared
at 74 sites in 14 countries to better
understand the relative importance of
streptococcal pathogens to the industry.
As other investigations have found, these
streptococcal species were the dominant
bacterial pathogens, accounting for more
than half of all bacteria identified. However,
it is interesting that while S. iniae is the
most commonly reported pathogen of fish,
our data show that S. agalactiae is the more
prevalent of the two in tilapia.
S . AG A L AC T I A E B I oT y P E P r E VA l E N C E
Detailed analysis of our isolates reveals
two distinct clusters that differ in a
variety of biochemical and phenotypic
characteristics. We refer to these clusters
as biotypes and differentiate between
typically beta-haemolytical “classical”
S. agalactiae (Biotype I) and typically
non-beta-haemolytical S. agalactiae
(Biotype II).
S. agalactiae Biotype II is considered the
most globally significant of the biotypes,
with chronic mortality in many Asian
Percent of total streptococcal isolations from tilapia reared in 14 countries
PREVALENCE
S. agalactiae Biotype I (Sa1)
26%
S. agalactiae Biotype II (Sa2)
56%
S. iniae
18%
and Latin American countries, whereas
S. agalactiae Biotype I is limited to Asia
and displays acute mortality peaks, often
associated with higher temperatures.
In our epidemiological surveys to date,
26% of all streptococcal isolates of tilapia
were found to be S. agalactiae Biotype I
and 56% were identified as S. agalactiae
Biotype II (Table 1).
VACC I N E D E V E lo P M E N T A N D
BIoTyPE SIgNIFICANCE
Vaccines to protect against S. agalactiae
have been described by various authors,
but it is difficult to conclude from these
studies if the vaccine and challenge strain
were from the same, or different, biotypes.
To determine if our classification of the fish
pathogenic S. agalactiae has consequences
for the development of vaccines to control
this devastating disease, we conducted
a laboratory challenge to determine
the ability of biotype-specific vaccines to
protect against lethal challenge with
S. agalactiae Biotype I or Biotype II strains.
Tilapia vaccinated with experimental
S. agalactiae Biotype I vaccines were
protected against lethal challenges with
virulent S. agalactiae Biotype I strains;
however, no protection was observed in
tilapia that received the Biotype I vaccine
when they were challenged with virulent
Biotype II strains. Similarly, fish vaccinated
with S. agalactiae Biotype II vaccines
were protected against lethal challenge
with Biotype II, but not with a virulent
Biotype I strain. Thus, vaccination with
biotype-specific bacterin vaccines induces
biotype-specific protection against
mortality caused by S. agalactiae.
Laboratory studies also demonstrated
that AquaVac® Strep Sa, a Biotype II vaccine
ultimately developed for commercial use,
protects against S. agalactiae Biotype II for
at least 30 weeks (Figure 3).
p r o c e e d i n g s
Wendover
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
figUre 3
Laboratory efficacy against S. agalactiae Biotype II 30 weeks after vaccination with
AquaVac Strep Sa
% Cumulative mortality
100
Vaccinates
80
Controls
60
RPP 85%
40
20
0
0
1
2
3
4
5
6
7
8
9
10
11 12
13
14
Days post-challenge
Experimental challenge with a virulent heterologous S. agalactiae Biotype II isolate
RPP = relative percent protection
FIElD ASSESSMENT oF
P r oToT y P E VACC I N E
Carefully controlled, field registration
trials were conducted to determine if
results in the lab would be the same in
the field with AquaVac Strep Sa, the
vaccine for S. agalactiae Biotype II.
The field trials were conducted in a lake
environment using square cages housing
~10,000 fish at a large Asian tilapia farm.
The trial was run in triplicate with three
test cohorts including AquaVac Strep Sa,
a placebo oil group and a negative,
unvaccinated control. The fish were
vaccinated at ~15 g after transfer to
growout cages from the nursery ponds.
Extensive bacteriological and virological
samplings were conducted before, during
and every month after vaccination and at
specific time points during the trial when
mortality was recorded above “normal.”
As per the routine growout strategy, the
trial was terminated and fish processed
after ~200 days (just under 7 months)
when they reached approxmately 1.2 kg.
Mortality, feed and water quality
parameters were recorded daily along
with the final harvest data.
Clear disease patterns emerged during the
trial in all cages, indicating that columnaris
disease or Flavobacterium columnare was
responsible for high initial mortality peaks
immediately after stocking. As the
columnaris or “saddle-back” disease
subsided, along with the initial transport
and stocking stress, then iridovirus (a
common viral disease of tilapia in this area)
resulted in three clear peaks of mortality
(Figure 4). This mortality pattern is often
indicative of the disease and losses can
routinely be as high as 30%. As the fish
got larger, S. agalactiae and, to a much
lesser degree, S. inae were increasingly
reisolated from moribund fish in all
three cohorts.
The mortality patterns indicate that
although the incidence of columnaris
disease and iridovirus was similar in all
groups, the degree of mortality from
S. agalactiae was relatively much lower in
the vaccine group when compared to the
placebo oil and negative controls. Data
gathered at harvest substantiated that
observation: Survival in vaccinates was
80% compared to 67% survival in the
placebo and negative control groups,
representing a 13% improvement in
vaccinated fish.
Correspondingly, FCR figures were 1.86 in
the vaccinates, compared to 2.06 and 2.05
in the placebo and negative control groups,
respectively. This was an approximate 10%
improvement in FCR (Table 2).
In this trial, vaccinated fish had improved
feed intake and feed utilization; consequently, they had improved production
efficiency performance, with 2.25
continued
21
figUre 4
CroSS-ProTECTIoN STUDIES
Daily mortality and cumulative survival in untreated controls
Daily mortality
Daily mortality
Flavobacterium
columnare
260
240
220
200
180
160
140
120
100
80
60
40
20
0
% Survival
120
100
S. agalactiae outbreak
Typical (three)
iridovirus peaks
80
60
Co-infection with
S. iniae and
S. agalactiae
% Survival
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
Wendover
Streptococcus in tilapia: implications for vaccine
development and field experiences from Asia
40
8 16 24 01 09 17 25 03 11 19 27 04 12 20 28 06 14 22 30 07 15 23 31 08 16 24 04 12
AUGUST SEPTEMBER
OCTOBER
NOVEMBER
DECEMBER
JANUARY
FEBRUARY MARCH
0
Date
2 011
JUNE
Results from a field trial with AquaVac Strep Sa
TREATMENT GROUP
% SURVIVAL
% IMPROVEMENT
IN SURVIVAL
13%
FCR*
Vaccinates
80%
1.86
Placebo-vaccinated
controls
67%
2.06
Untreated controls
67%
2.05
IMPROVEMENT
IN FCR*
ca 10%
6
B R A Z I L
N A T A L ,
C o M M E r C I A l V A C C I N AT I o N
ExPErIENCES
20
tABLe 2
22
Further controlled laboratory challenge
studies, using Indonesian, Malaysian,
Vietnamese, Honduran, Brazilian,
Mexican and Ecuadorian strains against
the relative commercial vaccine, indicate
that the vaccine cross-protects against
multiple, geographically diverse isolates
(Figure 5).
The success of the first large commercialscale injection vaccination programs in
tilapia to date has been variable, as
expected. The laboratory environment
demonstrates the efficacy of the vaccine
for protection against specific S. agalactiae
challenges, and well-controlled replicate
comparison trials have been conducted
comparing control versus vaccinated tanks.
In the lab, the fish are “clean” and free from
disease prior to, during and in general for
3 weeks after vaccination; furthermore,
there are no other diseases present other
than that for which the vaccine is being
tested. Although the numbers are
obviously smaller in the laboratory, another
important consideration is that 100% of
the housing unit is vaccinated.
FCR = feed-conversion ratio
metric tons more fish harvested in the
vaccinated population compared
to controls.
This well-controlled field trial clearly
demonstrates that AquaVac Strep Sa is
safe and efficacious when used under field
conditions. Significant improvements in
both survival and FCR mean an effective,
cost-efficient vaccine prevention strategy
is available for the industry to cope with
this devastating disease.
Since vaccine efficacy against S. agalactiae
and its ability to cross-protect against
multiple geographically diverse isolates
has already been clearly demonstrated
in the lab, the aim of a commercial
vaccination program is to discover the
best way to implement the vaccine
effectively under local conditions.
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
Wendover
p r o c e e d i n g s
figUre 5
The initial field trials and large-scale
vaccination programs have clearly taught
us lessons on some fundamental factors
to implement before any operation can or
should embark on a vaccination program.
Efficacy of AquaVac Strep Sa against local isolates / RPP = relative percent protection
ecUAdor
100
Vaccinates
% Cumulative mortality
1
83%
Controls
80
Correct diagnosis: Before a
commercial producer commits to a
vaccination program, the underlying
cause of mortality needs to be
determined by a fish health specialist.
rpp
60
40
20
2
Antimicrobial susceptibility
analysis: If a bacterial disease is
confirmed, a diagnostic laboratory should
perform an antimicrobial susceptibility test
to determine if the bacteria is susceptible
to therapeutic remediation and, if this is
the case, which antibiotic is most suitable
to control the outbreak.
S. agalactiae biotype confirmation:
If Streptococcus agalactiae is confirmed
as the primary disease, a more detailed
biotype analysis is necessary to ensure the
right vaccine is chosen for the right reason,
because we now know that immunity is
biotype-specific.
0
2
4
6
8
10
12
14
Days post-challenge
HondUrAs
100
rpp
Vaccinates
75%
Controls
80
% Cumulative mortality
3
0
60
40
20
0
4
The best results are obtained in operations
where the stages of production are
separated: The hatchery, nursery,
pre-growout and growout are located
0
2
4
6
8
10
12
14
Days post-challenge
Mexico
100
rpp
Vaccinates
100%
Controls
80
% Cumulative mortality
Healthy fish: Healthy fish means
that there is no sign of clinical or subclinical disease. Fish should be free of
disease and stress before, during and for
the first 2 to 3 weeks after vaccination.
That means climatic (water quality) and
housing conditions (biomass, handling,
etc.) at this stage are crucial.
60
40
20
0
continued
0
2
4
6
8
10
12
14
Days post-challenge
23
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Wendover
Streptococcus in tilapia: implications for vaccine
development and field experiences from Asia
in different areas with separated housing
unit employees and equipment. The
hatchery and nursery phases should,
preferably, be biosecure, well-controlled
and disease-free. This not only gives
juvenile fish the best start in life but also
allows vaccines to be administered in a
controlled environment.
There are four key components to the
vaccine program.
1
Correct administration: The aim is
to administer the vaccine in a consistent
and proper manner so there is minimal
stress on the animal and to ensure that
the correct dose is given to each and
every individual.
2
Sufficient immune response:
Correct vaccination and maintenance in
a healthy state under suitable housing
conditions prior to transfer will trigger
a sufficient immune response before
exposure to the “challenge environment.”
4
Shift the balance: It is important
to note that even if a fish has been
vaccinated, it may still be susceptible
to disease and infection depending on
its health, nutritional and stress status.
Therefore, a combination of good
management strategies, biosecurity,
housing, water quality, nutrition,
sanitation, immune stimulation and
vaccination will tip the balance toward
disease control.
A good vaccination program will result in a
rapid onset and full duration of protection.
Over time, the vaccination program will
have a self-perpetuating or ”snowball”
effect; with fewer sick and dying fish,
there will be less bacterial shedding in
the water. Less bacterial shedding in turn
lowers total challenge pressure, and
coupled with increasingly more protected
individuals, there will be overall better
population performance.
N A T A L ,
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CoNClUSIoN
24
3
Population protection: To achieve
population protection, it is important that
the entire population is vaccinated, not just
a fraction of that population. The aim is to
achieve blanket protection in the shortest
time frame possible; this is much easier in
an all-in/all-out production system, where
100% of stocked fish can be vaccinated, as
opposed to gradually replacing susceptible
fish with protected fish.
MSD Animal Health’s extensive epidemiological surveys of streptococcus
infections in tilapia have revealed some
startling insights about the complexity
of the disease.
Detailed analysis of our tilapia S. agalactiae
isolates suggests the presence of two
biotypes that have a variety of different
biochemical and phenotypic characteristics. These biotypes are geographically
diverse, with Biotype II being the most
prevalent in Asia and South America.
To our knowledge, there are no
obvious geographical, physiological
or environmental explanations for the
regional distribution of S. agalactiae
Biotypes I and II. Consequently, it would
be prudent to consider the possibility
that the distribution of the biotypes
may change over time, probably through
trade in live fish.
S. agalactiae vaccines are biotypespecific and do not cross-protect. One
commercially available vaccine, AquaVac
Strep Sa, has been developed to help
protect tilapia against S. agalactiae
Biotype II. The vaccine has been fully
tested in the lab and in field situations
and has clearly been demonstrated to
be safe and effective.
If all aspects of the production system are
aligned toward good biosecurity and there
is a specific health management plan with
vaccination as its cornerstone, tilapia farms
will be able to control streptococcosis and
attain consistent production volumes.
reantaso
The responsible use of veterinary medicines
in aquatic food production
Melba B. Reantaso, PhD
Key points
INTroDUCTIoN
*
*
*
globalization of the aquaculture industry has resulted
in intensified production,
pressure to improve production
performance and the widespread
movement of animals, which
have increased the risk for
disease and the need for
veterinary medicines.
Safe and effective veterinary
medicines are essential to
efficient commercial aquaculture
production, and animal health
product manufacturers play a
key role in the development of
such drugs.
The global trend is toward
increasingly stringent, uniform
standards, but more responsible
use of veterinary medicines
could be achieved by better
enforcement of current
regulations and improved
health extension support to
aquaculture farmers.
globalization of the aquatic animal
product trade and intensified aquatic
animal production are among the trends
contributing to new market opportunities
for the aquatic farming industry, including
warmwater fish. These trends, however,
have also contributed to the spread of
aquatic pathogens and diseases, which
are a primary constraint to the culture
of many aquatic species. Chronic diseases
hinder performance and profits by
resulting in reduced growth, feedconversion rates and survival, while acute
disease outbreaks have the potential to
cause mass mortality and devastate an
entire aquaculture enterprise.
Although the ability to manage
aquaculture health issues has increased
tremendously in the last 30 years, the
rapid development of the aquaculture
industry continuously generates new
challenges. The result is an increased
reliance on veterinary medicines1 to help
ensure successful production by preventing
and treating disease outbreaks.
USE oF VETErINAry MEDICINES
I N A q U A C U lT U r E
Without question, veterinary medicines
have many benefits if used responsibly.
They enable the development of industrialscale food production systems necessary
to feed society and improve financial gain
for investors.
Veterinary medicines make it possible
to increase production efficiency and
minimize land, water, feed and other
resources that are required to produce a
unit of aquatic food. They are essential to
modern aquaculture production because,
when used wisely, they can improve
on-farm biosecurity and husbandry,
and help sustain the industry.
On the other hand, the misuse of
veterinary medicines could have negative
effects on human food safety and free
trade. Some veterinary medicines used
in aquaculture, such as chloramphenicol,
have been shown to have potentially
harmful effects on human health and
have been banned, reducing the already
limited arsenal of drugs available for
disease treatment. The detection of
chloramphenicol in internationally traded
shrimp caused a slowdown of imports,
resulting in economic losses to producers
and their governments.
Other concerns regarding the inappropriate
or misuse of veterinary medicines include
the potential for drug residues in food, the
development of resistant pathogens and
negative environmental impact.
continued
25
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reantaso
The responsible use of veterinary medicines
in aquatic food production
26
These concerns have contributed to the
evolution of some profound changes in
the development and use of veterinary
medicines in aquaculture and other
food-producing industries.
SHArED rESPoNSIBIlITy
In general, it can be stated that the global
trend is toward more stringent and uniform
standards and a more prudent and
responsible use of veterinary medicines
by the aquaculture industry.
Most markets now have regulations
dictating acceptable residue levels that
also address environmental safety.
Improved surveillance and technology
have significantly increased the ability
to detect trace amounts of banned or
restricted substance residues, leading to
improved detection levels.
Animal health product manufacturers are
playing a key role in the responsible use
of veterinary medicines. One contribution
is the development of drugs that have
been specifically researched, developed
and approved for use exclusively in
veterinary medicine. An example is the
broad-spectrum antibiotic florfenicol.
The development of veterinary pharmaceuticals or vaccines for aquaculture and other
food-producing industries requires a
high level of investment, expertise and
documentation; a huge amount of work
and extensive testing goes into ensuring
that an active compound or vaccine
antigen is safe and efficacious for animals,
humans and the environment and that it
will meet all regulatory requirements.
The manufacturing process involves
heightened quality control checks for each
stage of manufacturing, and compliance
with the process and procedures is key to
ensuring the consistency and reliability of
the medicine being produced. Improved
quality control programs are critical for
on-farm performance but also help ensure
that fish products are safe and wholesome
for human consumption.
When a farm uses a registered medicine
in the correct way and follows guidelines
for withdrawal, it can be confident that use
of the product will not result in harmful
residues — or cause disruptions in the
trade of foods. This is why it is important
for aquaculture producers to use approved,
branded veterinary medicines instead of
raw drugs or chemicals.
The role of the animal health manufacturer
does not end with release of the product.
The company must also monitor any
unexpected problems such as adverse
reactions that may arise in the field.
Producers and aquatic animal health
professionals, of course, play a critical
role in the prudent and responsible use
of veterinary medicines in aquaculture.
They are increasingly aware of the
need to avoid using medicines, especially
antibiotics, that are essential to human
medicine. There is also increased awareness
about the benefits of susceptibility testing
to ensure the appropriate use of antibiotics.
Susceptibility testing demonstrates
whether or not a given antibiotic will be
effective against the pathogen causing
a disease outbreak.
Preventive health management is also
contributing to more prudent and
responsible use of veterinary medicines
in aquaculture. Consider the maturation
of the salmon industry in Norway or
yellowtail culture in Japan, where
vaccination and improved husbandry
have reduced the aquaculture industry’s
reliance on veterinary therapeutics
to achieve improved production
and profitability.
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Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
reantaso
CoNClUSIoN
rEFErENCES
In summation, the responsibility for the
prudent and responsible use of veterinary
medicines in aquaculture must be shared
by aquaculture stakeholders.
1
Governments have a key role to play, as do
producers, health professionals, product
manufacturers and consumers. A major
responsibility for government and the
public sector is to develop and implement
good aquaculture practices; laws and
regulations pertaining to the manufacture,
licensing and use of veterinary medicines
should be made in consultation with
relevant private-sector stakeholders in
a transparent manner and in line with
international standards and guidelines.
Rather than further restrictions, more
prudent and responsible use of veterinary
medicines could be achieved by better
enforcement of current regulations and
improved health extension support to
aquaculture farmers.
The responsible use of veterinary
medicines is not only essential to the
health and safety of animals, consumers
and the environment, but to the
sustainability of commercial aquaculture
production. The use of such medicines
should be part of national and on-farm
health and biosecurity plans, conducted
in accordance with an overall national
policy for aquatic animal health management and sustainable aquaculture.
Veterinary medicines: Any substance or
combination of substances presented for
treating or preventing disease in animals or
which may be administered to animals with
a view to making a medical diagnosis or
to restoring, correcting or modifying
physiological functions in animals. European
Union (EU). 2004. Directive 2001/82/EC of
the European Parliament and of the Council
of 6 November 2001 on the Community
Code relating to Veterinary Medicinal
Products. Official Journal L -311, 28/11/2004,
pp. 1– 66. as amended by Directive 2004/
28/EC of the European Parliament and the
Council of 31 March 2004 amending
Directive 2001/82/EC on the Community
Code relating to Veterinary Medicinal
Products. Official Journal L – 136, 30/04/
2004, pp. 58–84.
This article contains excerpts from an article
“Improving biosecurity through prudent
and responsible use of veterinary medicines
in aquatic food production” published at the
FAO Aquaculture Newsletter No. 45 (August
2010 issue) based on an FAO Expert Workshop
of the same title held in Bangkok, Thailand,
in December 2009.
27
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Best treatment practices of warmwater fish pathogens
using feed medicated with Aquaflor® (florfenicol)
Patricia Simmons Gaunt, DVM, PhD, Diplomate, American Board of Veterinary Toxicology
Key points
INTroDUCTIoN
*
N A T A L ,
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*
28
*
Aquaflor® (florfenicol), a
fast-acting, broad-spectrum
antibiotic provided as a feed
premix, has been shown to
be highly efficacious against
Edwardsiella ictaluri and
Flavobacterium columnare
when administered at 10 mg
florfenicol (FFC)/kg bodyweight/day for 10 days.
Aquaflor should be administered as soon as bacterial
disease is recognized in fish;
treatment days should never
be skipped and the full, 10-day
treatment with Aquaflor
should be provided to ensure
treatment success.
Used judiciously in conjunction
with good husbandry, vaccines
and improved genetic stock,
Aquaflor is a valuable tool for
controlling warmwater fish mortality from bacterial infections.
Aquaflor® (florfenicol) is a fast-acting,
palatable, broad-spectrum antibiotic
provided as a feed premix. It is proven to
be an effective and safe antibiotic for use
in fish. To maintain the efficacy of this
valuable antibiotic and obtain maximal
benefit from feed medicated with
Aquaflor, producers and diagnosticians
must understand the rationale behind
the label indications and they must
work cooperatively.
Aquaflor is approved for use in finfish in
over 20 countries. Indications differ from
country to country but include control
of mortality due to diseases associated
with the warmwater bacterial pathogens
Edwardsiella ictaluri, Streptococcus iniae,
Streptococcus agalactiae, Flavobacterium
columnare, Francisella asiatica and
Aeromonas hydrophila.
In the US, the approval of Aquaflor for use
in fish involved many experimental efficacy
and safety studies required by the United
States Food and Drug Administration, a
US regulatory body. In the US, Aquaflor is
currently approved for control of mortality
due to enteric septicemia of catfish,1
furunculosis and coldwater disease in
salmonids.2 Aquaflor®-CA1 (florfenicol)
is conditionally approved for control of
mortality due to columnaris disease
in catfish.3
The objective of this paper is to explain the
best practices for incorporating Aquaflor
into overall disease management programs
for warmwater fish production.
EFFICACy STUDIES
In vivo and in vitro efficacy studies with
Aquaflor have been conducted.
In vitro efficacy studies: The sensitivity
of bacterial pathogens to florfenicol (FFC)
has been assessed by determining the
minimal inhibitory concentrations (MIC)
of FFC for bacteria from both experimental
studies and from diagnostic specimens
obtained from disease outbreaks on farms.
This laboratory assay quickly determines
the susceptibility of bacteria to drug.4
MIC values for warmwater fish pathogens
of interest are presented in Table 1. The
values indicate that a wide variety of
warmwater bacterial pathogens are
susceptible to FFC. MIC values correlated
with data from pharmacokinetic studies
can be used to predict and validate the
clinical performance of Aquaflor on farms.5
In vivo studies: Experimental Aquaflor
efficacy studies in warmwater fish were
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tABLe 1
figUre 1
figUre 2
Minimal inhibitory concentration
(MIC) of florfenicol in bacteria
cultured from catfish and tilapia.
The values indicate that the bacteria
are susceptible to the antibiotic.
Efficacy of Aquaflor demonstrated by
reduced mortality in channel catfish
challenged with E. ictaluri, then fed
feed medicated with Aquaflor at 10
mg FFC/kg of fish bodyweight for
10 days. The feeding regimens were
initiated 24 hours after exposure
to E. ictaluri.
Efficacy of Aquaflor demonstrated by
reduced mortality in channel catfish
challenged with F. columnare, then
fed feed medicated with Aquaflor at
10 mg FFC/kg of fish bodyweight for
10 days. The feeding regimens were
initiated 24 hours after exposure
to F. columnare.
PATHOGEN
Susceptibility
(MIC) (µg/mL)
Edwardsiella ictaluri
Flavobacterium columnare
0.25
100
0.5-1.0
Unmedicated feed
Unmedicated feed
Aquaflor
Aquaflor
60
87.3% (p < 0.001)
Aeromonas hydrophila
0.5-4.0
Mortality rate %
2.0-4.0
Mortality rate %
80
Streptococcus iniae
Streptococcus agalactiae
60
40
20
9.3%
0
40
30
20
10
8%
0
Channel catfish
Francisella asiatica
54.2% (p < 0.001)
50
Channel catfish
2.0
conducted at a dose rate of 10 mg FFC/kg
bodyweight against the pathogens
E. ictaluri and F. columnare in catfish
(Ictalurus punctatus) fingerlings.6-8
Aquaflor was highly efficacious (p < 0.001)
when compared to controls (Figures 1
and 2).
The efficacy of Aquaflor in warmwater
fish has also been demonstrated in
experimental studies against the
pathogens S. iniae9 and F. asiatica.10
In 2009, high mortality in market-size
catfish (~2 lbs or 0.907 g) associated with
A. hydrophila occurred on several farms
in Alabama, USA.11 Aquaflor was used
to contain the outbreak. There were no
untreated controls in these studies for
both animal welfare and economic
reasons. Nonetheless, comparison of
mortality before and after treatment
with feed medicated with Aquaflor
showed a dramatic decrease and, in
some cases, complete cessation of
mortality (Figure 3).
PHArMACokINETIC AND
rESIDUE DEPlETIoN STUDIES
Pharmacokinetic studies were conducted
to determine the disposition of FFC in
warmwater fish (reared in freshwater) and
to confirm the dose rates determined by
efficacy studies. After oral dosing of catfish
at 10 mg/kg11 (Figure 4) and tilapia at 15
mg/kg,12 a high concentration of FFC was
absorbed quickly from the intestine, rapidly
continued
29
figUre 3
Mortality among fish treated at one of several farms in Alabama with an A. hydrophila
outbreak. Over 200,000 channel catfish received 10 mg FFC/kg fish/day for 10 days.
1.800
1.600
Treatment period
1.400
% Daily mortality
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Best treatment practices of warmwater fish pathogens
using feed medicated with Aquaflor® (florfenicol)
1.200
B E S T T r E AT M E N T P r A C T I C E S
1.000
0.800
0.600
0.400
0.200
9.4
9.7 9.10 9.13 9.16 9.19 9.22 9.25 9.28 10.1 10.4 10.7 10.10
Note: Data courtesy of US Fish and Wildlife Service, AADAP
B R A Z I L
disseminated throughout the body and
maintained at a steady concentration
during the 10-day dosing period.
N A T A L ,
Feed medicated with Aquaflor should
be used in conjunction with proper husbandry, available vaccines and genetically
improved fish. Good fish husbandry
includes environmental management,
attention to stocking density, biosecurity
and good recordkeeping.
Dates — 2009
6
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0.000
30
time, which is the time between the last
dose of FFC and the time when the drug
residue levels fall below the 1 µg/g
tolerance. The results support a withdrawal
time of 12 days for warmwater fish following treatment with Aquaflor administered
according to label directions.*13-14
Tissue concentrations of FFC in catfish
and tilapia during the 10-day treatment
under the specified experimental
conditions were greater than the MIC
values of five pathogenic bacteria studied
(Table 1). This tissue concentration was
considered sufficient to effectively
combat the pathogenic bacteria.
In a single oral-dose study conducted
with catfish, the mean concentration of
Aquaflor in plasma declined below
1 µg/mL by 36 hours; this concentration
is below the highest MIC efficacy value
for most catfish pathogens (Table 1).
Therefore, it is critical to continue daily
dosing (every 24 hours) for 10 days to
achieve and maintain an efficacious
dose at the rate necessary to combat
bacterial infections.
Residue depletion studies were conducted
to determine the calculated withdrawal
The ideal aquaculture environment is
maintained at optimal water temperatures,
dissolved oxygen and chemistries for each
warmwater species. Although emphasis
is placed on increased stocking rates to
maximize production, fish should be
stocked near, but not in excess of, the
maximal capacity to avoid poor water
quality, suboptimal dissolved oxygen
concentrations and traumatic injury.
Any of these factors could stress and
predispose fish to disease.
Biosecurity measures such as footbaths
and cleaning, disinfecting and rinsing
equipment will help prevent the spread
of infectious agents on the farm. Diseases
can be transmitted by humans, predators
* Withdrawal times may vary by market.
Check local package insert for details.
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figUre 4
and scavengers, equipment and water.
Although predators and scavengers are
difficult to deal with, the control of other
vectors is manageable. Newly introduced
fish should be quarantined (and treated
if necessary) for 3 to 6 weeks before
introduction into the growout facilities on
farms that share common water systems.15
FFC plasma concentration versus time in channel catfish (Ictalurus punctatus) at a
mean water temperature of 25.4° C (77.7° F) after medication with Aquaflor at a dose
rate of 10 mg FFC/kg bodyweight. FFC was rapidly absorbed and attained effective
plasma concentrations above the MIC of most catfish pathogens.11
Drug residue tolerance level
100
Proper farm management requires
managers to maintain fish health records
that help enable recognition of diseases
and their proactive management.
Important farm records include the
seasonality of outbreaks, fish eating
patterns and behavior. For example, if
fish become anorexic during summer
when water temperature ranges from
24° C to 28° C (75° F to 82° F), the problem
pathogen is more likely to be S. agalactiae
rather than S. iniae.16
Producers should submit live, sick fish
to a diagnostic laboratory for examination
of lesions and culture of the suspected
pathogenic bacterium. Dead fish often
have been contaminated with additional
bacteria that are not the source of a disease
outbreak, which is why live, sick fish
should be examined and lesions cultured;
the results must be carefully interpreted to
avoid an incorrect diagnosis.17
It is advantageous for producers to
stock robust fish strains bred for fast
growout and high survival rates.
µg/mL
10
1
0.1
0.01
0.001
0
48
96
144
192
240
288
336
Hours
Genetically improved stock fed optimal
diets yield high quality alevins resulting
in larger, more disease-resistant juveniles
that perform well in the field.
However, vaccinated fish can still succumb
to pathogens if they are stressed and if
husbandry is less than optimal.
Prevention of disease is increasingly
becoming a reality due to the availability
of warmwater fish vaccines, which vary
from country to country. Vaccines
against E. ictaluri and F. columnare
(AquaVac-ESC® and AquaVac-COL®) in
catfish are commercially available in the
US; AquaVac® Strep Sa is the first-ever
intraperitoneal vaccine for tilapia and
protects against S. agalactiae Biotype II,
an important cause of streptococcosis.
U S E A q U A F l o r AT T H E F I r S T
SIgN oF DISEASE, ACCorDINg To
lABEl DIrECTIoNS
Feed medicated with Aquaflor should be
administered as soon as bacterial disease
is recognized in fish.18 A delay of just a few
days after the onset of disease signs can
mean the difference between successful
therapy and treatment failure.
continued
31
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Best treatment practices of warmwater fish pathogens
using feed medicated with Aquaflor® (florfenicol)
32
figUre 5
Some diseases can be diagnosed from
the age and class of fish, early signs and
lesions, and dates of outbreaks, while
others require culture results. Vigilance will
alert producers to early signs of disease,
such as anorexia or lethargic swimming.
Corneal opacity with exophthalmia
(bulging eyes) seen in tilapia
with streptococcosis
Columnaris disease caused by F. columnare
in tilapia is characterized by external
lesions of ulceration and necrosis of the
gills, mouth, skin, muscle and fins, giving a
ragged appearance of the fin rays caused
by sloughing of the epithelium.
Important bacterial pathogens of freshwater tilapia include S. iniae, S. agalactiae,
A. hydrophila, F. asiatica, F. noatunensis
and F. columnare. Fish infected with
the first four of these pathogens will
quickly lose their appetites, which
decreases medicated feed intake.
Experimental studies of F. columnareinfected catfish demonstrate that the
bacterial infection does not cause
anorexia in catfish.8 However, anecdotal
reports indicate that appetite is affected
in columnaris disease under field
conditions when the mouth becomes
extremely necrotic. As stated previously,
FFC tissue concentration must exceed
its MIC value for the bacteria during the
treatment period. Fish that are not eating
or only eating small quantities will be
inadequately medicated.9
Lethargic fish will likely have lesions that
are more indicative of a given disease than
are clinical signs. Streptococcosis in tilapia
is most frequently caused by S. agalactiae
and S. iniae. External lesions include
exophthalmia — bulging eyes —
skin ulcerations (from secondary infections)
and enlarged internal organs, especially
of the spleen and kidney, with whitish
necrotic nodules (Figure 6).
A. hydrophila in tilapia is characterized by
external lesions of hemorrhage, ulceration
and necrosis of the skin, base of the fins
and occasionally the muscle. Exophthalmia
is occasionally reported with this disease.
Internal lesions include bloody fluid in
the coelomic cavity and hemorrhage in
the organs.
Because Aquaflor requires a prescription
in many markets, such as the US and
Brazil, a veterinarian or other fish health
specialist should examine affected fish
and determine that they are sick based
on the signs of disease, lesions or the
results of diagnostic tests such as bacterial
culture.18 If the fish have a bacterial
disease that is treatable with Aquaflor,
the veterinarian will issue a medicatedfeed order. Dosing should be followed
according to label directions, using the
effective approved dose rate for 10 days’
duration. A dose rate of 10 mg/kg bodyweight in catfish is efficacious against
E. ictaluri and F. columnare; in tilapia alevins
and juveniles, this dosage has been shown
to be efficacious against F. columnare,
A. hydrophila and S. agalactiae.
Francisellosis in tilapia is caused by
F. asiatica and F. noatunensis. Typical
lesions of Fransicella spp. infection include
exophthalmia, whitish nodules (which
represent areas of necrosis) in the gills,
Medicated feed should be purchased
from a feed mill that follows Good
Manufacturing Practices to ensure
quality, purity and the indicated label
concentration. For practical reasons, the
with corneal opacity (Figure 5),
darkening of the skin, necrosis of gills
and hemorrhage of the skin, opercula,
vent and muscles. Internal lesions include
fluid in the coelomic cavity, hemorrhage
and enlargement of internal organs as
well as inflammation in joints and
the heart.
p r o c e e d i n g s
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
gaunt
figUre 6
Enlarged internal organs, especially of
the spleen and kidney, seen in tilapia
infected with F. asiatica
Photo courtesy of Dr. Juan A. Morales, Escuela de
Medicina Veterinaria, Universidad Nacional de
Costa Rica; and Dr. Esteban Soto, Ross University
School of Veterinary Medicine, St. Kitts
Aquaflor feeding rate (calculated
as a percentage of bodyweight) for
medicated feed may be predetermined
by commercial feed mills on the basis
of local feeding practices.20 Custom
blends can be produced by farm-mixing
where permitted.
It is imperative that producers feed the full
regimen of feed medicated with Aquaflor
and that feed medicated with Aquaflor is
fed as the sole ration during the treatment
period. If the farmer is uncertain that the
medication will work, bacterial isolation
and susceptibility testing must be
performed at a diagnostic laboratory.
Skipping treatment days or administering
feed medicated with Aquaflor for fewer
days than recommended will lead to
wasted resources. In addition, lower tissue
concentrations of the drug in fish can lead
to the selection of resistant bacteria, and
resistance to one medicated feed can lead
to resistance against other medicated
feeds,21 eventually leaving the farmer with
fewer options for bacterial treatment.
When used in conjunction with good
husbandry, vaccines and improved
genetic stock, judicious use of Aquaflor
is a valuable tool for controlling warmwater
fish mortality from bacterial infections.
Alternatively, producers may be tempted
to administer medicated feed excessively
because Aquaflor is highly palatable,
but this will only lead to higher tissue
concentrations than necessary, which
wastes resources. Producers should
adhere to recommended dosages and
required withdrawal times to prevent
prohibitive residues.
1
rEFErENCES
US Food and Drug Administration (US FDA).
[Internet]. 2009. [cited 2011 March]. FDA
approves new antimicrobial for catfish.
Available from: fda.gov/.
2
US Food and Drug Administration (US FDA).
[Internet]. 2007. [cited 2011 March]. FDA
approval of new antimicrobial for salmonids.
Available from: fda.gov/.
3
CoNClUSIoN
Aquaflor is effective against aquatic
warmwater bacterial infections such
as F. columnare, S. iniae, S. agalactiae,
A. hydrophila and E. ictaluri. Fish showing
early signs of disease associated with
these pathogens should be examined
for lesions and cultured at a diagnostic
laboratory to identify the bacteria.
It is imperative that producers use feed
medicated with Aquaflor according to
label directions to ensure maximal
efficacy, preserve bacterial susceptibility
and prevent prohibited tissue residues.
US Food and Drug Administration (US FDA).
[Internet]. 2009. [cited 2011 March]. First FDA
conditionally approved new animal drug
for columnaris disease in catfish. Available
from: fda.gov/.
4
Gaunt P. 2010. Determining antimicrobial
MICs against aquaculture pathogens
using Sensititre® plates. Aqua Bulletin
Technical Service Update. Number 3.
MSD Animal Health.
5
Miller R, et al. 2006. Epidemiologic cutoff
values for antimicrobial agents against
Aeromonas salmonicida isolates determined
by frequency distributions of minimal
inhibitory concentration and diameter of
zone of inhibition data. American Journal of
Veterinary Research. 67:1837–1843.
continued
33
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
gaunt
Best treatment practices of warmwater fish pathogens
using feed medicated with Aquaflor® (florfenicol)
6
Gaunt P, et al. 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.
2 011
JUNE
6
B R A Z I L
N A T A L ,
Soto E, et al. 2010. Comparison of in vitro
and in vivo susceptibility of Francisella asiatica
to florfenicol. Antimicrobial Agents and
Chemotherapy. 54: 4664-4670.
11
7
Gaunt P, et al. 2004. Determination of
dose rate of florfenicol in feed for control
of mortality in channel catfish Ictalurus
punctatus (Rafinesque) infected with
Edwardsiella ictaluri, etiological agent of
enteric septicemia. Journal of the World
Aquaculture Society. 35:257-267.
8
34
10
Gaunt P, et al. 2010. Efficacy of florfenicol for
control of mortality caused by Flavobacterium
columnare infection in channel catfish,
Ictalurus punctatus (Rafinesque). Journal of
Aquatic Animal Health. 22:115-122.
Gaunt P, et al. 2011. Single intravenous and
oral dose pharmacokinetics of florfenicol in
channel catfish (Ictalurus punctatus). Journal
of Veterinary Pharmacology and Therapeutics.
doi: 10.1111/j.1365-2885.2011.01340.x.
Personal communication, Dr. Rodrigo
Zanolo, MSD Animal Health.
17
Plumb J. 1999. Disease recognition and
diagnosis. Health Maintenance and Principal
Microbial Diseases of Cultured Fishes. 34-40.
18
Gaunt P. 2006. Veterinarians’ role in the
use of veterinary feed directive drugs in
aquaculture. Journal of the American
Veterinary Medical Association. 229: 1-3.
12
Bowser P, et al. 2009. Florfenicol residues
in Nile tilapia after 10-d oral dosing in feed:
effect of fish size. Journal of Aquatic Animal
Health. 21:14-17.
13
Wrzesinski C, et al. 2006. Florfenicol
residue depletion in channel catfish,
Ictalurus punctatus (Rafinesque).
Aquaculture. 253: 309-316.
9
Gaunt P, et al. 2010. Determination of
florfenicol dose rate in feed for control of
mortality in Nile tilapia (Oreochromis nilotica)
infected with Streptococcus iniae. Journal of
Aquatic Animal Health. 22: 158-166.
16
19
AliAbadi F, et al. 2000. Antibiotic treatment
for animals: effect on bacterial population
and dosage regimen optimization.
International Journal of Antimicrobial
Agents. 14:307-313.
20
Robinson E, et al. 2004. Feeds and feeding
practices. Biology and Culture of Channel
Catfish. 324–348.
14
Gaikowski M, et al. 2010. Depletion of
florfenicol amine, marker residue of
florfenicol, from the edible fillet of tilapia
(Oreochromis niloticus x O. niloticus and
O. niloticus x O. aureus) following florfenicol
administration in feed. Aquaculture. 301: 1–6.
15
Losordo T. 1997. Tilapia culture in intensive
recirculating systems. Tilapia Aquaculture in
the Americas. 185-208.
21
Welch T, et al. 2009. IncA/C plasmidmediated florfenicol resistance in the catfish
pathogen Edwardsiella ictaluri. Antimicrobial
Agents and Chemotherapy. 53: 845-84.
gaikowski
Depletion of florfenicol in water and florfenicol amine
from fillet tissue after feeding Aquaflor® (florfenicol)
to tilapia in a recirculating aquaculture system
Mark P. Gaikowski, MA; Melissa K. Whitsel, BS; Shawn Charles, MS; Susan M. Schleis, BS;
Louis S. Crouch, PhD; Richard G. Endris, PhD
Key points
INTroDUCTIoN
*
A study was conducted to
determine the depletion of
florfenicol (FFC) in water and
florfenicol amine (FFA), a marker
for FFC residue, from the fillet
of market-weight tilapia in a
recirculating aquaculture
system (rAS) after administration of feed medicated with
Aquaflor® (florfenicol).
*
Maximum FFC concentration
in water occurred on the 10th
dosing day and decreased to
807 ng/ml by 240 hours after
dosing; FFA decreased from a
mean of 13.77 μg/g at 1 hour
after dosing to a mean of 0.39
μg/g by 240 hours after dosing.
*
The results indicate that in
tilapia the FFA decline in rAS
is similar to that found in
continuous-flow systems and
also support the approved
withdrawal time for feed
medicated with Aquaflor.*
Streptococcus iniae, a Gram-positive
bacterium, causes substantial mortality
in tilapia, Oreochromis spp., especially
among fish cultured in recirculating or
intensive flow-through systems. Worldwide
annual economic loss as a result of S. iniaeassociated mortality in tilapia has been
estimated to be about US $100 million.1
Consequently, MSD Animal Health is
seeking US approval of Aquaflor®, a feed
premix containing the broad-spectrum
antibacterial agent florfenicol (50% w/w;
Figure 1A), for treatment of S. iniae in
tilapia. Use of trade or product names
does not imply endorsement by the
US government.
Aquaflor was recently approved in the US
at a dose rate of 10 mg/kg bodyweight per
day (BW/day) administered in feed for 10
days to control mortality due to enteric
septicemia in catfish (2005) and coldwater
disease and furunculosis in trout (2007); it
was conditionally approved (Aquaflor-CA1)
to control mortality due to columnaris
disease in catfish (2007).
Globally, Aquaflor, which is also marketed
as Aquafen® and Florocol® in some regions,
is registered for use in more than 20
countries including Norway (1993); Chile
(1995); Canada (1997); the United Kingdom
(1999); Ecuador and Venezuela (2005);
Colombia (2006); and Brazil, Costa Rica,
Vietnam and China (2007) to control
various susceptible pathogens in a variety
of commercially important freshwater
and marine species.
continued
figUre 1A /1B
Chemical structures of florfenicol (1A) and florfenicol amine (1B)**
1A
1B
O
H 3C
OH
O
S
H3C
OH
S
O
F
NH
O
O
NH2
F
Cl
Cl
**1A: Florfenicol [R-(R*,S*)-2,2-dichloro-N-[1-fluoromethyl-2-hydroxy-2-(4-methylsulfonylphenyl)]
ethyl acetamide] is the active ingredient of Aquaflor.
1B: Florfenicol amine [R*,S*0]-α-(1-amino-2-fluoroethyl)-4-(methylsulfonyl)-benzenemethanol])
is the marker residue of florfenicol.
* Withdrawal times may vary by market.
Check local package insert for details.
35
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
gaikowski
Depletion of florfenicol in water and florfenicol amine
from fillet tissue after feeding Aquaflor® (florfenicol)
to tilapia in a recirculating aquaculture system
We evaluated the depletion of florfenicol
amine (FFA) — a marker of florfenicol (FFC)
residue — from tilapia fillet and the
decline of FFC following FFC-medicated
feed administration at a nominal dose rate
of 20 mg/kg BW/day to fish reared in a
recirculating aquaculture system (RAS).
The objective of the study was to develop
the marker residue depletion data needed
to allow FFC administration at a proposed
maximum dose of 15 mg/kg BW/day for
10 consecutive days.2
N A T A L ,
B R A Z I L
6
JUNE
2 011
FlorFENICol DEPlE TIoN
36
FFC distribution, metabolism and
depletion following dosing at 10 mg/kg
BW/day has been well characterized in a
variety of fish.3-10,17,19 FFC was similarly
distributed in freshwater- or seawateracclimated tilapia with the maximum
concentration occurring 2 to 24 hours
post-dosing, depending on the tissue. 10
In Atlantic salmon, FFA (Figure 1B) was
identified as the primary metabolite of FFC
in muscle. 11 Muscle (skin-on fillet), by
regulation, is considered the edible
tissue of most fish. FFA was subsequently
selected as the marker residue of FFC
administration because it is the primary
FFC metabolite, and other lesser
metabolites (and FFC) are converted to
FFA through acid hydrolysis. 12
Monitoring total FFA concentration (FFA +
acid-hydrolyzed FFC and metabolites) in
the target tissue thus provides a conservative estimate of FFC residues and enables
calculation of a conservative withdrawal
period. Although FFC metabolism data
were not available for tilapia, FFA was assumed to be the marker residue since it is
the marker residue in cattle, swine, sheep,
poultry, catfish, salmon and trout.13
Data from residue depletion studies are
used to calculate a withdrawal period for a
drug, which is the time required for the animal to deplete the drug residue to a level
that is considered safe for human consumption. Regulatory agencies estimate
the safe concentration or maximum residue
level (MRL) by combining an acceptable
daily intake (ADI) level (from toxicology
data) with a standard human mass estimate and a consumption factor (an estimate based on the mass of residue-bearing
tissue consumed).
For FFC, the 10 µg/kg ADI is multiplied
by a standard human weight (60 kg), then
divided by a consumption factor (in fish,
a standard mass [300 g] of skin-on fillet
[muscle] is used), resulting in a tolerance
of 2 µg/g; the European Agency for the
Evaluation of Medical Products and the
US Food and Drug Administration have
applied an additional safety factor and
established the MRL for Europe and the
US at 1 µg/g.13-14
M AT E r I A l S A N D M E T H o D S
Commercial tilapia culture is principally
focused on the rearing of phenotypic males
produced by the administration of feed
containing 17α-methyltestosterone (MT)
to tilapia fry. Tilapia used in the study
were a mix of MT gender-reversed females
(phenotypic males) and genetic males of
the two most commonly cultured tilapia
strains, pure Nile tilapia (O. niloticus x
O. niloticus) and hybrid tilapia (O. niloticus
x O. aureus).
Aquaflor-medicated premix was used
to prepare the medicated feeds. The
feeds used were assayed for FFC content
by high-performance liquid chromatography15 (HPLC) before and after dosing
(Table 1). The non-medicated feed was
analyzed to determine proximate nutrient
content as well as inorganic or organic
contaminants (Eurofins Scientific Inc., Des
Moines, Iowa, USA). Non-medicated feed
and feed medicated with Aquaflor (2.667 g
FFC/kg) for the residue depletion study
(extruded 4.8 mm floating pellets) were
prepared according to standard procedures
at Delta Western Research Center
(Indianola, Mississippi, USA). Aquaflor
premix was added to the medicated feed
during mixing, prior to extrusion.
Nile and hybrid tilapia (mean weight
= 447 ± 56 g) were obtained from a
commercial farm. Tilapia were held in a
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
gaikowski
p r o c e e d i n g s
tABLe 1
Mean florfenicol (FFC) concentration* in feed
STUDY
TYPE
Nominal dose
(mg FFC/kg
BW1/day)
Nominal feed
concentration
(g FFC/kg)
Mean concentration
(g FFC/kg)
Percent of
nominal
post-dosing periods. Three equal feed
portions were offered each day with ~4
hours between each feed administration.
Daily feed consumption was estimated
during the dosing period.
start of dosing end of dosing
residue
depletion
1
2
0
20
0
2.667
<loq2
2.638
<loq
2.560
NA
97.5 %
Bodyweight
LOQ = limit of quantitation (0.0002 g/kg)
*Determined by high-performance liquid chromatography in feed samples collected at the
start and end of dosing for residue depletion studies
commercial RAS (Aquatic Eco-Systems
Fish Farm™ II) consisting of twin ~1,900 L
(500 gal.) polyethylene tanks, mechanical
filters (clarifier and suspended solids filter)
and a biological filter; there was ~3,350 L
(885 gal.) total system water.
The RAS biofilter was inoculated with
commercial biofilter bacterial inoculum
and allowed to operate for ~6.5 months
with fish present before FFC administration.
These fish were removed, and the tilapia
used for testing were stocked into the
RAS 38 days before FFC administration.
Temperature was maintained at 27° C to
27.6° C (80.6° F to 81.7° F). Waste solids
were removed once daily ~1 hour before
feeding, and concurrent with solids
removal, a portion of the RAS water was
removed (acclimation — 6-11%; dosing
and post-dosing — 5-8%) and replaced
with temperature-adjusted well water
(at ~22° C/71.6° F). Water chemistry
(temperature, dissolved oxygen, pH,
total ammonia, nitrite and nitrate) was
determined once daily prior to tank
cleaning. Water hardness and alkalinity
were determined weekly. Alkalinity was
maintained at >150 mg/L as CaCO3 by
occasional addition of sodium bicarbonate.
A single water sample was analyzed for
metals and volatile and semi-volatile
organics (Davy Laboratories, La Crosse,
Wisconsin, USA). No contaminants at
levels of concern were identified.
Non-medicated feed was offered at a rate
of 0.25%-1% BW/day during the 38-day
acclimation period; the feed rate was 0.75%
BW/day for the last 11 acclimation days and
remained constant through the remainder
of the study, including the dosing and
Five fish from each tank were sampled
4 days prior to dosing to obtain control
fillet tissue. Fish were indiscriminately
removed and sacrificed; the fish were then
scaled and skin-on fillets were collected,
individually bagged and stored at <-70° C
(-94° F).
Tilapia (n = 209) were offered feed
medicated with Aquaflor at 0.75% BW/day
(nominal dose = 20 mg FFC/kg BW/day)
for 10 consecutive days. The estimated
delivered dose was calculated from the
estimated feed mass consumed, feed FFC
concentration and the total fish mass at
terminal sampling. Twenty fish (10 per
tank) were indiscriminately removed from
the RAS tanks on days 0.04, 0.5, 1, 1.5, 2, 3,
4, 5 and 10 post-dosing, and skin-on fillets
were collected as previously described.
Water samples for FFC analysis were
collected: (1) concurrent with the control
fillet collection, (2) prior to tank cleaning
during the dosing period (~1 hour prior to
the first daily feeding), (3) just prior to the
second and third daily feeding intervals,
continued
37
N A T A L ,
B R A Z I L
6
JUNE
2 011
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
gaikowski
Depletion of florfenicol in water and florfenicol amine
from fillet tissue after feeding Aquaflor® (florfenicol)
to tilapia in a recirculating aquaculture system
38
(4) 4 hours after the third daily feed
interval and (5) concurrent with tissue
collection during the post-dosing period,
except the 0.04-day post-dosing fillet
collection. At each collection interval,
one water sample (~50 mL) was taken
from the RAS clarifier and from the RAS
suspended solids filter. Each sample
was hand-mixed and syringe-filtered
(Durapore® [PVDF, 0.45 µm] membrane,
Millipore, Billerica, Massachusetts, USA)
in ~2-mL aliquots into HPLC vials then
stored at ≤-20° C (≤-4° F) until analyzed.
FFA concentrations were determined using
a method validated for FFA in tilapia fillet
tissue at MPI Research, Inc. (State College,
Pennsylvania, USA). The method involved
converting all FFC residues to FFA by
acid-catalyzed hydrolysis. Fillet tissue was
hydrolyzed by adding 6N hydrochloric acid
then held for approximately 2 hours at
95° C to 100° C (203° F to 212° F). The
tissue-hydrolysate was extracted with ethyl
acetate and centrifuged. The aqueous
hydrolysate was retained and adjusted to
pH 12.5 or greater with 30% (w/w) sodium
hydroxide solution. The pH-adjusted
solution was adsorbed from 45 to 60
minutes onto a Varian Chem Elut CE120
sorbent column (Varian, Inc., Palo Alto,
California, USA) then eluted with methylene chloride. The methylene chloride
eluates were evaporated to dryness,
dissolved in 10 mM potassium phosphate
buffer (pH 4.0, 1% [v/v] acetonitrile),
filtered (0.2 µm) and then analyzed by HPLC
using UV detection at 220 nm. The method
quantitation limit (LOQ) was 0.05 µg/g.
which the tolerance limit was less than 1
µg/g. Analyses were considered significant
if P < 0.05.
r E S U lT S
FFC concentration was determined in water
samples using a validated determinative
procedure capable of quantifying FFC from
10 to 5,000 ng/mL and up to 20,000 ng/mL
after dilution. FFC concentration was
determined by ultra-pressure liquid
chromatography with mass spectrometric
detection on an atmospheric pressure
ionization interface. Water samples
were fortified with FFC-d4 as an internal
standard and analyzed directly. Ionic
transitions of 356 to 185 m/z and 360 to
189 m/z were monitored for FFC and the
internal standard, respectively. The method
LOQ was 10 ng/mL.
The mean minimum daily delivered doses
were 19.4 mg/kg BW/day for tilapia in tank
1 (range 19.3 to 19.57) and 19.8 mg/kg
BW/day for tilapia in tank 2 (range 19.7 to
20.0) or 97% to 99% of the target dose. Fish
consumed 100% of the feed medicated
with Aquaflor that was offered during the
10-day dosing period, similar to those of
non-medicated feed during the acclimation
and post-dosing periods. FFA concentrations in tilapia fillets are summarized in
Table 2. Representative analytical standard,
control and treated tissue chromatograms
are presented in Figure 2.
The residue depletion profile of FFA in
skin-on fillet of tilapia following withdrawal
from the medicated diet was estimated by
log-linear regression.16 The fitted loglinear regression model ([Ln (ppm)] =
2.3215 Ln (ppm) + –0.0151 Ln (ppm)/hour
x hours post-dosing) R2 was 0.8271. The
withdrawal period was defined as the time
when the tolerance limit of the residue
concentration was at or below the 1 µg/g
MRL. The tolerance limit was set as the 99th
percentile of the potential residue level at
95% confidence.16 The withdrawal period
was, therefore, equivalent to the time at
Mean FFA concentration in tilapia fillets
exceeded the MRL during the post-dosing
period until the last post-dosing fillet
collection (10 days post-dosing) when all
fillet concentrations were below the MRL.
Mean FFA concentrations steadily declined
during the post-dosing period. Fillet FFA
concentrations were similar between the
two study tanks throughout the postdosing period. FFA level depleted rapidly
to below the MRL, and the calculated
tolerance limit was below the MRL at 10
days after withdrawal of the medicated
feed (Figure 3).
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
gaikowski
p r o c e e d i n g s
tABLe 2
Mean florfenicol amine (FFA) concentration* in fillet tissue following administration
of feed medicated with Aquaflor
Days
post-dosing
TANK 1
TANK 2
Mean FFA in Nile
and hybrid tilapia
(µg/g)
FFA
(µg/g)
N
FFA
(µg/g)
N
0.04
14.42
(6.61)
[3.51-27.78]
10
13.13
(3.54)
[7.54-18.51]
10
13.77
(5.21)
0.5
12.12
(3.62)
[9.21-21.15]
10
14.79
(4.86)
[6.94-21.63]
10
13.45
(4.39)
1
7.35
(3.01)
[3.49-12.41]
10
7.98
(3.64)
[2.80-12.65]
10
7.67
(3.27)
1.5
7.39
(2.47)
[4.49-12.46]
10
4.09
(1.40)
[2.46-6.41]
10
5.74
(2.58)
2
4.60
(1.51)
[2.50-7.46]
10
5.33
(1.08)
[3.59-7.11]
10
4.97
(1.33)
3
2.59
(1.02)
[0.86-4.26]
10
3.08
(2.15)
[0.64-7.16]
10
2.84
(1.66)
4
1.93
(0.68)
[0.83-3.05]
10
2.38
(0.85)
[1.31-3.50]
10
2.16
(0.78)
5
1.40
(0.66)
[0.42-2.32]
10
1.37
(0.41)
[0.93-2.11]
10
1.38
(0.53)
10
0.46
(0.20)
[0.31-0.98]
10
0.33
(0.09)
[0.18-0.50]
10
0.39
(0.16)
* FFA concentration in fillet tissue from Nile and hybrid tilapia following administration of feed
medicated with Aquaflor as the sole ration for 10 consecutive days. Standard deviations are in
parentheses and the range is in brackets. Only samples above the FFA quantitation limit of
0.05 µg/g were included in summary calculations.
FFC levels in RAS water before dosing were
<LOQ. FFC concentration in water increased
during the dosing and post-dosing period
to a maximum concentration of 1,430
ng/mL concurrent with the 12-hour fillet
collection (0.5 day post-dosing; Figure 4).
The maximum mean FFC concentration of
1,400 ng/mL occurred concurrent with the
24-hour fillet collection (1 day post-dosing;
Figure 5). The mean FFC concentration decreased during the post-dosing period to
847 ng/mL at 10 days post-dosing (Figure
4). FFC concentration was remarkably similar in the samples collected at sites 1 and 2
(Figure 4).
Unionized ammonia-nitrogen levels were
<0.02 mg/L NH3-N during the acclimation,
dosing and post-dosing periods. Nitratenitrogen levels fluctuated in RAS from
between 7 to 123 mg/L with mean
concentrations of 67, 81 and 75 mg/L
during the acclimation, dosing and postdosing periods. Nitrite-nitrogen levels
occasionally exceeded the safe upper limit
of 2.0 mg/L but only during the acclimation
period; nitrite levels were <0.9 mg/L during
the 8 days prior to dosing. Nitrite levels
steadily increased during the dosing
period until peaking on dosing day 8 at
1.76 mg/L (Figure 5). This concentration
increase was apparently not associated
with any effect on the RAS biofilter but
rather the inadvertent buildup of biofilm
material in the water supply lines to the
continued
39
figUre 2A / 2B / 2c
Representative chromatograms* of extracts from control and treated tilapia fillet tissue
Absorbance units
2A
0.110
0.100
0.090
0.080
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
biofilter from the RAS tanks as nitrite levels
rapidly decreased during the remainder
of the dosing period and during the postdosing period after the water supply lines
were flushed. Nitrite levels again spiked
during the post-dosing period then
dropped after the RAS biofilter supply
lines were flushed (Figure 5).
20.00
Minutes
2B
Florfenicol amine -11.022
0.18
Absorbance units
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
6
Minutes
N A T A L ,
0.050
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
Florfenicol amine -11.171
Absorbance units
B R A Z I L
2c
40
DISCUSSIoN
Fish readily consumed feed medicated
with Aquaflor. There was no reduction in
feed consumption during the dosing
periods. There do not appear to be any
palatability concerns regarding feed
medicated with Aquaflor.
0.02
JUNE
2 011
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
gaikowski
Depletion of florfenicol in water and florfenicol amine
from fillet tissue after feeding Aquaflor® (florfenicol)
to tilapia in a recirculating aquaculture system
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
Minutes
* 2A: Extract from control tilapia fillet tissue sample
2B: Extract from control tilapia fillet sample fortified with florfenicol amine (FFA) at 2 ug/g
2C: Extract (2.33 ug/g FFA found) from fillet tissue taken from a fish at 5 days post-dosing
FFC concentration increased in RAS water
during the dosing period then gradually
decreased during the post-dosing period.
The apparent concomitant increase in
nitrite concentration in the RAS does not
appear to correlate to RAS-water FFC
concentration. Rather, the increase in RAS
nitrite concentration was apparently due
to microbial growth in the biofilter water
supply lines restricting water flow to the
RAS biofilter, as flushing those supply lines
was followed by a rapid decline in RAS
nitrite concentration. Some antibiotics
(erythromycin, oxytetracycline) have been
found to negatively affect denitrification in
aquatic systems,20-21 albeit at levels much
higher than observed in the present study.
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
This rapid clearance indicates that there is
likely to be only a short post-treatment
therapeutic effect associated with Aquaflor
administration, such as when FFC levels
are at or above the bacteria MIC. Without
concomitant steps by the farmer to
reduce disease transmission (e.g., water
disinfection, improved husbandry,
vaccination), re-infection of treated fish
is possible.
As with other antibiotics, Aquaflor should
not be expected to eliminate the need for
proper husbandry practices but should
be regarded as one effective tool in the
management of disease in aquaculture.
our interpretation of published regulatory
guidance, FFA should deplete from tilapia
treated with Aquaflor at 1.31 times the
maximum proposed dosage (15 mg/kg
BW/day for 10 consecutive days) to a level
safe for human consumption 11 days
after treatment.*
CoNClUSIoNS
FFA, a marker for FFC residue, depletes
rapidly in the skin-on fillet tissue of
tilapia following the withdrawal of feed
medicated with Aquaflor. When dosed at
19.62 mg FFC/kg BW/day for 10 days, FFA
detected in the skin-on fillet of tilapia
depleted to less than the MRL in all
samples collected 10 days after the end
of dosing. Based on these residue data and
continued
* Withdrawal times may vary by market.
Check local package insert for details.
figUre 3
Florfenicol amine (FFA) concentrations* in fillet tissue of tilapia
*ffA concentrations are from
skin-on fillet of tilapia following
administration of feed medicated
with Aquaflor at a nominal dose
rate of 20 mg florfenicol/kg
bodyweight for 10 days at
~27° c (80.6° f).
natural log-transformed ffA levels were fit to a log-linear model ([Ln (ppm)]
= 2.3215 Ln (ppm) + –0.0151 Ln (ppm)/hour x hours post-dosing
r2 = 0.8271; r2 = 0.52; coefficient of variation = 39.45; model fit — f = 851.26,
df = 1, p < 0.01
the calculated tolerance limit (99th percentile ffA level at 95th confidence level)
Maximum residue level of 1 μg/g for consumption at 10 days post-dosing
4
Natural log transformed florfenicol amine (µg/g)
FFC is known to be rapidly and completely
distributed in fish4-5,7-8,10-11,18 during dosing
and to rapidly deplete from tissues6,9,17,19
after withdrawal from medication, which
are excellent traits for use in food fish to
control susceptible bacterial infections.
However, its rapid elimination means that
tissue FFC levels will rapidly decrease after
fish are withdrawn from the medicated
feed. While minor differences in study
design (e.g., feeding procedure, test
temperature, feed rates) preclude direct
comparison between FFA-residue depletion
studies conducted with tilapia fed FFCmedicated feed while held in flow-through
systems17,19 and this study, there do
not appear to be substantial differences
between the depletion of FFA from skinon fillet of tilapia fed FFC-medicated
feed whether fed in a RAS or in a flowthrough tank.
gaikowski
p r o c e e d i n g s
3
2
1
0
..........................................................................................
-1
-2
0
2
4
6
8
10
Days post-dosing
41
figUre 4
Observed, system mean and theoretical florfenicol (FFC) concentrations in
recirculating aquaculture system water*
5000
Martinsen B, et al. 1993. Single dose
pharmacokinetic study of florfenicol in
Atlantic salmon (Salmo salar) in seawater
at 11° C. Aquaculture. 112:1-11.
Clarifier — observed FFC
Sediment filter
System mean
Theoretical
4000
5
Horsberg TE, et al. 1996. Pharmacokinetics
of florfenicol and its metabolite florfenicol
amine in Atlantic salmon. Journal of Aquatic
Animal Health. 8:292-301.
3000
2000
6
Pinault LP, et al. 1997. Absolute oral
bioavailability and residues of florfenicol in
the rainbow trout (Oncorhynchus mykiss).
Journal of Veterinary Pharmacology and
Therapeutics. 20:(Suppl. 1), 297-298.
1000
2 011
0
0
2
4
6
8
10
12
14
Days post-dosing initiation
16
18
20
*Concentrations were determined before, during and after FFC-medicated feed administration
at a nominal dose rate of 20 mg FFC/kg bodyweight/day for 10 consecutive days (days 0 to 9).
7
Samuelson OB, et al. 2003. Pharmacokinetics
of florfenicol in cod Gadus morhua and
in vitro antibacterial activity against Vibrio
anguillarum. Diseases of Aquatic Organisms.
56:127-133.
6
JUNE
assessment of efficacy against furunculosis.
Journal of Fish Diseases. 14:343-351.
4
Florfenicol concentration (ng/mL)
Held in conjunction with the W O R L D A Q U A C U LT U R E S O C I E T Y C O N F E R E N C E
gaikowski
Depletion of florfenicol in water and florfenicol amine
from fillet tissue after feeding Aquaflor® (florfenicol)
to tilapia in a recirculating aquaculture system
N A T A L ,
B R A Z I L
8
42
ACkNoWlEDgMENTS
rEFErENCES
The authors thank the staffs of the Thad
Cochran National Warmwater Aquaculture
Center, Delta Western Research Center, and
US Geological Survey Upper Midwest
Environmental Sciences Center, and Diane
Sweeney of Merck Animal Health* for her
statistical support to this study. The residue
depletion study was funded by Merck Animal
Health through a Cooperative Research and
Development Agreement with the US
Geological Survey. The data summarized are
presently in review by regulatory agencies
in several countries to satisfy the residue
depletion requirements to support regulatory
approval of Aquaflor in those countries.
1
* MSD Animal Health is known as
Merck Animal Health in North America.
Shoemaker CA, et al. 1997. Streptococcal
disease problems and control: a review. In:
Fitzsimmons K, editor, Tilapia Aquaculture.
Northeast Regional Aquacultural Engineering
Service. Ithaca, New York. 671-680.
2
Gaunt P, et al. 2010. Determination of
florfenicol dose rate in feed for control
of mortality in Nile tilapia infected with
Streptococcus iniae. Journal of Aquatic
Animal Health. 22:158-166.
3
Inglis V, et al. 1991. Florfenicol in Atlantic
salmon, Salmo salar L., parr: tolerance and
Yanong RP, et al. 2005. Pharmacokinetic
studies of florfenicol in Koi carp and
Threespot gourami Trichogaster trichopterus
after oral and intramuscular treatment.
Journal of Aquatic Animal Health. 17:129-137.
9
Wrzesinski C, et al. 2006. Florfenicol residue
depletion in channel catfish, Ictalurus
punctatus (Rafinesque). Aquaculture. 253:
309-316.
10
Feng JB, et al. 2008. Tissue distribution of
florfenicol in tilapia (Oreochromis niloticus x
O. aureus) after a single oral administration
in freshwater and seawater at 28° C.
Aquaculture. 276:29-35.
p r o c e e d i n g s
gaikowski
Bac te ri al D i sease i n Warm wate r F i sh : New Strategies for Sustainable Control
figUre 5
15
Nitrite concentration*
Nitrite (NO2-N-) concentration (mg/L)
2.0
Tank 1
Tank 2
10-day dosing period
1.5
1.0
0.5
Hayes JM. 2005. Determination of
florfenicol in fish feed by liquid chromatography. Journal of the Association of Official
Analytical Chemists. 88:1777-1783.
16
U.S. Food and Drug Administration (US
FDA) [Internet]. 2006. [cited 2008 April 28].
Guidance for Industry 3 - General Principles
for Evaluating the Safety of Compounds Used
in Food-Producing Animals. U.S. Department
of Health and Human Services. Available
from: www.fda.gov/cvm/Guidance/
published.htm.
17
0.0
-2
0
2
4
6
8
10
12
14
16
18
20
Days post-dosing initiation
Gaikowski MP, et al. 2010. Depletion of
florfenicol amine, marker residue of
florfenicol, from the edible fillet of tilapia
(Oreochromis niloticus x O. niloticus and O.
niloticus x O. aureus) following florfenicol
administration in feed. Aquaculture. 301:1-6.
18
* Determined in water samples removed from recirculating aquaculture system tanks 2 days
prior to the dosing period, during the 10-day dosing period and the 10-day post-dosing
period. Tilapia were offered florfenicol-medicated feed at a dose rate of 19.62 mg/kg
bodyweight (BW)/day during the dosing period; the daily feed mass offered during the 2 days
prior to dosing, the dosing and post-dosing periods was equivalent to 0.74-0.75, 0.73-0.76,
and 0.70-0.76% BW, respectively.
Lim JH, et al. 2010. Pharmacokinetics of
florfenicol following intramuscular and
intravenous administration in olive flounder
(Paralichthys olivaceus). Journal of Veterinary
Pharmacology and Therapeutics. 34:206-208.
19
11
Horsberg TE, et al. 1994. The disposition
of 14C-florfenicol in Atlantic salmon
(Salmo salar). Aquaculture. 122:97-106.
to fish), Summary Report 5.
EMEA/MRL/760/00-Final. Available from:
www.emea.europa.eu/pdfs/vet/mrls
/076000en.pdf.
20
12
Wrzesinski CL, et al. 2003. Determination
of florfenicol amine in channel catfish muscle
by liquid chromatography. Journal of the
Association of Official Analytical Chemists.
86:515-520.
13
European Agency for the Evaluation of
Medicinal Products (EMEA) [Internet]. 2000.
[cited 2008 July 17]. Committee for Veterinary
Medicinal Products, Florfenicol (extension
Bowser PR, et al. 2009. Florfenicol residues
in Nile tilapia after 10-d oral dosing in feed:
Effect of fish size. Journal of Aquatic Animal
Health. 21:14-17.
14
U.S. Food and Drug Administration (US
FDA), Center for Veterinary Medicine
[Internet]. 2005. [cited 2008 April 28]. Original
New Animal Drug Application, NADA 141246 (AQUAFLOR Type A Medicated Article
(florfenicol), an Antibiotic). U.S. Department
of Health and Human Services. Available
from: www.fda.gov/cvm/drugsuseaqua.htm.
Collins MT, et al. 1976. Effects of antibacterial agents on nitrification in an aquatic
recirculation system. Journal of the Fisheries
Research Board of Canada. 33:215-218.
21
Klaver AL, et al. 1994. Effects of
oxytetracycline on nitrification in a
model aquatic system. Aquaculture.
123:237-247.
43
AQUAFLOR®, AQUAVAC®, AQUAVAC-ESC® and AQUAVAC-COL® are
trademarks of Intervet International B.V. or affiliated companies or
licensors and are protected by copyrights, trademark and other
intellectual property laws. Copyright © 2012 Intervet International B.V.,
a subsidiary of Merck & Co., Inc., Whitehouse Station, NJ, USA.
All rights reserved.
This document contains information on veterinary products based
on international registration dossiers and may refer to products
that are either not available in your country or are marketed under
a different trade name. In addition, the approved indications as well
as safety and efficacy data for a specific product may be different
depending on local regulations and approvals. Not for use or
distribution in the United States. For more information, read the
product labeling that applies to your country or contact your local
Merck Animal Health/MSD Animal Health representative.
ISP-GA-23