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Laboratory Efficacy of Amoxicillin for the Control of
Streptococcus iniae Infection in Sunshine Bass
Ahmed M. Darwish
a
& Adnan A. Ismaiel
a
a
Harry K. DupreeStuttgart National Aquaculture Research Center, U.S. Department of
Agriculture , Agricultural Research Service , Post Office Box 1050, Stuttgart, Arkansas,
72160, USA
Published online: 09 Jan 2011.
To cite this article: Ahmed M. Darwish & Adnan A. Ismaiel (2003) Laboratory Efficacy of Amoxicillin for the Control of
Streptococcus iniae Infection in Sunshine Bass, Journal of Aquatic Animal Health, 15:3, 209-214, DOI: 10.1577/H03-016
To link to this article: http://dx.doi.org/10.1577/H03-016
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209
Journal of Aquatic Animal Health 15:209214, 2003
Copyright by the American Fisheries Society 2003
Laboratory Efcacy of Amoxicillin for the Control of
Streptococcus iniae Infection in Sunshine Bass
AHMED M. DARWISH* AND ADNAN A. ISMAIEL
Harry K. DupreeStuttgart National Aquaculture Research Center,
U.S. Department of Agriculture, Agricultural Research Service,
Post Ofce Box 1050, Stuttgart, Arkansas 72160, USA
Abstract.An experimental trial was performed to evaluate the efcacy of amoxicillin in con-
trolling Streptococcus iniae infection in sunshine bass (a hybrid of female white bass Morone
chrysops male striped bass M. saxatilis). Minimum-inhibitory-concentration studies of amoxi-
cillin against multiple S. iniae isolates showed a sensitivity range of 0.01560.5 g/mL. The
amoxicillin dose levels tested were 30, 50, 80, and 120 mg of active ingredient per kilogram of
sh body weight per day. Administration of medicated feed started 1 d after infection by immersion
exposure to S. iniae and continued for eight consecutive days; this was followed by an observation
at 15 d posttreatment. Amoxicillin increased the survival rate from 1% in the infected, nonmed-
icated group to an average of 95% in the infected groups receiving the four doses; there were no
signicant differences among the medicated groups. Survivors of the infection were not found to
be carriers of the bacteria (i.e., there was negative bacterial isolation).
Streptococcosis is a disease caused by bacteria
in the genus Streptococcus that are gram positive,
nonmotile, catalase negative, fermentative in glu-
cose, and nonspore forming. The disease affects
more than 20 species of sh (Kitao 1993; Stoffre-
gen et al. 1996a; Plumb 1999a) and causes eco-
nomic losses in the culture of striped bass Morone
saxatilis (Plumb 1999a).
Streptococcus iniae has been implicated in the
infection of sunshine bass (a hybrid of female
white bass M. chrysops male striped bass) in the
United States (Stoffregen et al. 1996a). Although
the bacterium causes zoonotic infections in hu-
mans (Centers for Disease Control 1996; Fish fan
beware 1996; Weinstein et al. 1997), current in-
formation suggests that it represents a risk only to
older and immunocompromised people who suffer
puncture wounds while handling infected sh
(Shoemaker et al. 2001).
The U.S. Food and Drug Administration (FDA)
has approved the antibacterials oxytetracycline
and Romet (a 1:5 combination of ormetoprim and
sulfadimethoxine) in food sh, but their use is lim-
ited to catsh and salmonids. Presently there are
no approved antibacterial agents for use with
striped bass and their hybrids. Amoxicillin has
been registered for use in food sh in the United
Kingdom since 1990 and is a candidate for treating
streptococcosis in hybrid striped bass in the United
States. Amoxicillin is a penicillin derivative with
* Corresponding author: adarwish@spa.ars.usda.gov
Received April 15, 2003; accepted October 29, 2003
a broad spectrum of antibacterial activity that func-
tions by inhibiting bacterial cell wall synthesis,
which leads to death by osmotic rupture. It can be
administered enterally or parenterally and has a
moderate tissue distribution (Stoffregen et al.
1996b).
There is currently no published information on
the efcacy of amoxicillin in controlling S. iniae
infection in hybrid striped bass. The objective of
this study was to determine the in vitro sensitivity
of S. iniae to amoxicillin and the drugs efcacy
in controlling S. iniae infection in sunshine bass.
Methods
In Vitro Sensitivity
Antimicrobial agent.A stock solution of
amoxicillin (Gurvey and Berry, Inc., Toronto, On-
tario) was made by dissolving 160 mg of amoxi-
cillin (163.2653 mg of the stock powder to account
for its 98% purity) in 100 mL of de-ionized water.
The stock solution was lter- sterilized by passing
it through a nylon membrane with a pore size of
0.45 m (Corning Laboratory Science Company,
Corning, New York). An amoxicillin solution of
10 g/mL was prepared by adding 0.1 mL of stock
solution to 15.9 mL of MuellerHinton broth
(MHB).
Bacteria.The isolates of S. iniae used in the
present study were identied and provided by John
Maurer of the University of Georgia, College of
Veterinary Medicine, and John Hawke of Louisi-
ana State University, College of Veterinary Med-
icine (Table 1). Upon receipt, the isolates were
cultured on brainheart infusion agar (BHIA) sup-
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210 DARWISH AND ISMAIEL
TABLE 1.Results of minimum inhibitory concentration (MIC) assays of amoxicillin against Streptococcus iniae.
Isolate
a
MIC (g/mL) Host
SI ATCC
SI 2032-96H
SI 2378-96H
SI 2030-96H
SI 2031-96H
Isolate 10
LA 99-301G
LA 94-093A
LA 95-290
LA 94-142B
0.0312
0.0156
0.0312
0.0312
0.0156
0.0312
0.0312
0.5
0.0156
0.0312
Amazon river dolphin Inia geoffrensis
Human
Human
Human
Human
Human
Tilapia Tilapia spp.
Tilapia
Sunshine bass
Tilapia
LA 93-331
LA 97-003
LA 94-426
K 122-00 bB 6P
K 014-01 aB
K 315-00 aB
K 092-01 bK
K 446-01 cK
K 420-01 cB
0.0312
0.25
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
Sunshine bass
Tilapia
Tilapia
Sunshine bass
Sunshine bass
Sunshine bass
Sunshine bass
Sunshine bass
Sunshine bass
a
The rst six isolates were provided by John Maurer of the University of Georgia, those starting
with LA were provided by John Hawke of Louisiana State University, and the rest were from
our collection.
plemented with 5% sheep blood for 24 h at 30C.
Purity was tested by streaking the sheep blood agar
for isolated colonies and gram staining; the iden-
tity was conrmed biochemically (Pier and Madin
1976) and by polymerase chain reaction ampli-
cation (Berridge et al. 1998). The bacteria were
suspended in sterile phosphate- buffered saline at
a concentration of 1.2 10
7
colony-forming units
(CFU) per milliliter, which was determined by the
optical density (Darwish et al. 2000). The suspen-
sion was divided in half and either inactivated by
incubation at 60C for 1.5 h (negative control) or
not treated.
Minimum inhibitory concentration (MIC).The
tube titration method was used for MIC assay of
amoxicillin against 19 isolates of S. iniae (Stokes
1975). The working solution was used to prepare
duplicate test tubes of MHB containing doubling
dilutions of amoxicillin. These test tubes were in-
oculated with the bacterial suspension at a nal
concentration of 3 10
5
CFU/mL (Darwish et al.
2000). The doubling dilutions ranged from 8 to
0.0078 g/mL. Positive controls contained live
bacteria, MHB, and no amoxicillin, while the neg-
ative controls had inactivated bacteria, MHB, and
no amoxicillin. The test tubes were capped and
examined for bacterial growth after 48 h of in-
cubation at 30C. The lowest concentration with-
out visible bacterial growth was considered to be
the minimum inhibitory concentration.
Laboratory Efcacy Studies
Preparation of amoxicillin diet.Amoxicillin
was incorporated into a commercial feed ration
(Nelson and Sons, Inc., Murray, Utah) to provide
30, 50, 80, and 120 mg of amoxicillin per kilogram
of sh per day when sh are fed 2% of their body
weight per day. The four diets contained 1.5, 2.5,
4, and 6 mg of amoxicillin per gram of feed, re-
spectively.
The commercial feed was ground to less than
0.5 mm in diameter in a hammer mill (Model
F21M, W-W Grinder Corp., Wichita, Kansas). Pul-
verized ration with antibiotic added was thor-
oughly blended in a V-mixer (Blendmaster Lab-
oratory, Patterson-Kelly, Stroudsberg, Pennsyl-
vania) for 15 min. To avoid heating vitamins and
antibiotic during V-mixing, the mixing program
included three 3-min periods of intensier bar ro-
tation separated by two 3-min periods during
which the V-mixer shell rotated without intensier
bar operation. The dry ingredients were then
placed in a commercial food mixer (Model A-200,
Hobart, Troy, Ohio) and an appropriate amount of
distilled water added until a uniform mixture was
obtained. The moistened mixture was passed
through a meat grinder equipped with a 3-mm die
to obtain uniform pellets. Pelleted diets were air
dried for 2436 h in a temperature-controlled room
at 20 2C and then frozen in air-tight containers
at 18C until needed. Small quantities of the diet
were thawed and refrigerated at 4 C until used.
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211 USE OF AMOXICILLIN AGAINST STREPTOCOCCOSIS IN BASS
The amoxicillin level in three 0.5-g samples of
each diet was analyzed by high performance liquid
chromatography following the method of Luo and
Ang (2000). The 0-, 30-, 50-, 80-, and 120-mg
diets contained the following levels of amoxicillin
per gram of diet: 1.11 0.01, 1.65 0.05, 3.02
0.16, 4.46 0.01, and 6 mg (mean SE),
respectively. These data represent a recovery rate
of 72.5 1.52%.
Challenge protocol.Streptococcus iniae iso-
late LA 94-426 was passed four times in hybrid
striped bass to maximize its virulence, and aliquots
of the culture were stored at 80 C in brainheart
infusion broth containing 25% glycerin until re-
quired. Prior to exposure, each group of sh in an
aquarium was weighed to calculate the amount of
diet (2%) to be administered daily. The weight of
the sh to be challenged was 35.8 0.22 g. In
the challenge treatments, the sh from each aquar-
ium were placed in 10 L of aerated water con-
taining S. iniae at a concentration of 3.6 10
7
CFU/mL (Darwish et al. 2000) for 10 min and then
returned to their home aquarium. The negative
controls were similarly handled but not exposed
to S. iniae. Fish were observed twice daily and
clinical signs recorded. Dead and moribund sh
were necropsied, and bacterial isolation was at-
tempted from the trunk kidney, brain, and eye on
BHIA supplemented with 5% sheep blood. Mor-
ibund sh were euthanized by immersion in 300
mg of methanesulfonate 3-aminobenzoic ethyl
ether per liter before the necropsy and bacterial
isolation were performed. At the end of the trial,
four sh from each aquarium (all of those re-
maining if fewer than four survived) were sacri-
ced and necropsied, and the trunk kidney, brain,
eye, and liver were cultured to detect the presence
of S. iniae. Cultured bacteria were identied bio-
chemically (Pier and Madin 1976).
Experimental design.Sunshine bass were pro-
vided courtesy of Keo Fish Farm, Keo, Arkansas.
Fish were randomly distributed into thirty 40-L
ow-through aquaria, with 20 sh per aquarium.
The sh in each aquarium were collectively
weighed to adjust daily rations and fed the control
(nonmedicated) diet for 5 d to acclimate them to
experimental conditions. The body weight at the
beginning of the acclimation period was 33.44
0.27 g per sh. The feeding rate was maintained
at 2% of body weight per day, which was divided
into two equal feedings (morning and afternoon).
The ow-through aquarium system provided well
water at a constant ow rate (0.60.7 L/min) and
temperature (27.6 0.14C). Supplementary aer-
ation in all aquaria maintained oxygen concentra-
tions at or near saturation. Dissolved oxygen and
total ammonia nitrogen were measured every day
(Hach DR/2010, Hach Co., Loveland, Colorado)
and maintained at levels of more than 5.5 mg/L
and less than 0.3 mg/L, respectively.
After the 5-d acclimation period, feed was with-
held for 24 h and the sh in each aquarium were
collectively weighed. Six treatments were then
randomly assigned to aquaria, each treatment
group consisting of ve replicate aquaria. The six
treatments consisted of four groups of sh that
were challenged with S. iniae and then offered
medicated feed (30, 50, 80, or 120 mg/kg), one
group that was challenged with S. iniae and then
offered nonmedicated feed (positive control), and
one group that was not challenged and offered non-
medicated feed (negative control). Feeding of the
appropriate medicated or nonmedicated diets be-
gan 2224 h after the bacterial challenge and con-
tinued for 8 d. All groups of sh were then fed
the control diet for an additional 15-d withdrawal
period.
Statistical analysis.Survival rates within
aquaria were subjected to one-way analysis of var-
iance (Zar 1984) with the MINITAB program
(MINITAB, Inc. 2000), and differences among
treatment means were determined using Tukeys
procedure (Tukey 1953). Treatment effects were
considered signicant at P 0.05.
Results
In Vitro Sensitivity
The bacterial isolates were identied as S. iniae.
The MIC assays of 17 of the 19 (89%) isolates
yielded values between 0.0156 and 0.0312 g/mL,
whereas the other 2 isolates yielded values of 0.25
and 0.5 g/mL (Table 1).
Efcacy Trials
Efcacy.Amoxicillin was highly effective in
reducing mortality in sunshine bass infected with
S. iniae (Figure 1). There was a signicant differ-
ence between the survival rates of the amoxicillin-
medicated treatments and the challenged, non-
medicated treatment (Table 2). Average survival
in the four medicated treatments was 95 1.9%,
with no statistical differences between any of them
and the uninfected negative control, which had a
100% survival rate. Severe infection was achieved,
as evidenced by the 99 1% mortality in the
infected, nonmedicated treatment and the 100%
positive bacterial isolation from moribund or dead
sh.
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212 DARWISH AND ISMAIEL
FIGURE 1.Cumulative mortality of sunshine bass in-
fected by immersion exposure to S. iniae and fed various
amounts of amoxicillin per kilogram of body weight per
day for 8 d. Each treatment had 100 sh equally divided
among ve tanks.
TABLE 2.Percent survival of sunshine bass infected with Streptococcus iniae (LA 94-426) and fed different amox-
icillin-medicated diets for 8 d. Values are means SEs; means followed by different letters were signicantly different
(P 0.05). Each treatment had ve tanks with 20 sh in each tank.
Amoxicillin dose (mg/kg body weight/d)
0 30 50 80 120 Control
a
1 1 z 99 1 y 95 2.2 y 96 1 y 90 5.2 y 100 0 y
a
Control tanks were neither infected nor medicated.
Mortalities started 48 h post infection (PI) and
continued for 16 d PI. At the conclusion of the
experiment (24 d PI), attempts to isolate S. iniae
from all sacriced sh yielded negative results (20
from each of the challenged treatments) and no
clinical signs or gross pathology were observed.
No challenged, nonmedicated sh were available
for necropsy except for one individual. Fish in the
negative control group did not exhibit clinical
signs or mortalities, and S. iniae was not recovered
from any of these 20 sh.
Clinical signs and gross pathology.No clinical
signs or gross pathology were observed in the neg-
ative control group. Clinical signs or gross pa-
thology were observed primarily in the challenged,
nonmedicated group and in the groups of chal-
lenged, medicated sh. Moribund sh were le-
thargic as well as anorexic at 2 d PI and swam
erratically in random directions or on their sides.
Externally the sh had hemorrhages, erythema,
and various degrees of dark skin pigmentation. The
hemorrhages and erythema involved the skin, ns,
gills, opercula, and vents (Figure 2A). Eye lesions
were observed as early as 24 d PI and consisted
of bilateral or unilateral exophthalmia, corneal
opacity, and hemorrhages (Figure 2B). Internally,
hemorrhages were observed in the liver, gastro-
intestinal tract, peritoneum, and muscles (Figure
2C). The spleen was dark in color and swollen,
while the gastrointestinal tract was devoid of feed
and contained a yellowish mucoid uid.
At the conclusion of the experiment, sh in all
treatments were feeding and no longer exhibiting
clinical signs or internal lesions.
Discussion
This is the rst study to evaluate the efcacy of
amoxicillin in controlling a bacterial infection in
sunshine bass. Oral administration of amoxicillin
at a dose of 30 mg per kilogram of body weight
per day or more for 8 d was effective in increasing
the survival of sh challenged with S. iniae to 90%
or more, compared with 1% for challenged, non-
medicated sh. The MIC assay yielded values be-
tween 0.0156 and 0.0312 g/mL for 17 isolates
and 0.25 and 0.5 g/mL for 2 other isolates. The
relative consistency of the MIC values should
make the development of a treatment protocol for
S. iniae infection in hybrid striped bass less com-
plicated.
The signicant reduction in the mortality of
challenged, medicated sunshine bass suggests that
the serum amoxicillin concentration exceeded the
MIC of the pathogen by a factor of 24, which is
the margin necessary for an antibacterial to be ef-
fective in controlling a systemic infection (Blood
et al. 1979). Amoxicillin signicantly reduced
mortality (average, 1.5% at 48 h PI in the medi-
cated groups) when it was administered 24 h be-
fore the rst mortality in the challenged, nonme-
dicated control (51% at 48 h PI). There are cur-
rently no pharmacokinetic studies of amoxicillin
of any kind in hybrid striped bass. However, it was
shown in Atlantic salmon Salmo salar that serum
amoxicillin peaks about 2 h after oral administra-
tion at 16C (Stoffregen et al. 1996b). In mono-
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213 USE OF AMOXICILLIN AGAINST STREPTOCOCCOSIS IN BASS
FIGURE 2.Sunshine bass infected by immersion exposure to S. iniae that show (A) skin hemorrhages at 2 d
post infection (PI), (B) bilateral exophthalmia at 4 d PI, and (C) severe muscle hemorrhages at 4 d PI.
gastric animals, 7292% of the amoxicillin is
absorbed (Plumb 1999b). Amoxicillin has wide
tissue distribution, and when the meninges are in-
amed it will cross the blood brain barrier into
the cerebrospinal uid in concentrations ranging
from 10% to 60% of that found in the serum
(Plumb 1999b). Although the distribution of
amoxicillin in the tissues of hybrid striped bass is
unknown, if it is similar to that reported for other
animals, that could explain the drugs efcacy in
this study (particularly since streptococcal infec-
tion is known to produce mingoencephalitis; Stof-
fregen et al. 1996a).
The efcacious levels of amoxicillin in this
study are similar to those reported in salmon
against Aeromonas salmonicida (40120 mg per
kilogram of sh; Inglis et al. 1992; Inglis et al.
1993; Roberts and Shepherd 1997). The wide
range of amoxicillin regimens reported in the lit-
erature could be partly explained by several fac-
tors, including the sensitivity of the target path-
ogen, the concentration of amoxicillin ingested
and absorbed by the host, and the stage of the
infection (Inglis et al. 1993; Plumb 1999a). In this
study, the bacterial isolate used to produce the in-
fection was relatively sensitive to amoxicillin and
the medicated diet was immediately ingested upon
administration, thus minimizing the chance of the
drugs leaching into the water.
The clinical signs and lesions produced in this
experiment are similar to those reported in the lit-
erature (Stoffregen et al. 1996a; Plumb 1999a;
Evans et al. 2000). The reluctance of sh to feed
2 d post infection emphasizes the critical impor-
tance of monitoring and early intervention with
oral medicated diet. If inappetance begins, oral
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214 DARWISH AND ISMAIEL
antibiotic therapy will be ineffective in reducing
sh losses. The infection in this study was sys-
temic, as shown by the positive isolation of S. iniae
from the brain, kidney, and eye. At the conclusion
of the experiment no carriers were detected in any
treatment group receiving a medicated diet.
Although amoxicillin appears to be effective
against S. iniae infection in sunshine bass, con-
trolled eld trials and target animal safety and tis-
sue residue studies will be required for the FDA
to consider its approval.
Acknowledgments
We acknowledge Steven Rawles for allowing us
to use the nutrition laboratory facilities, John
Hawke and John Maurer for donating the bacterial
isolates, and Vaughn Ostland for conrming the
identity of the bacterial isolates by PCR. Technical
reviews by John A. Plumb, Andrew Michel, and
Andrew Goodwin were also greatly appreciated.
Mention of trade names is solely for the purpose
of providing specic information and does not im-
ply recommendation or endorsement by the U.S.
Department of Agriculture.
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