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Federico J. Rotman , Marty Riche , Peter Van Wyk & Daniel D. Benetti
Harbor Branch Oceanographic Institute, 5600 U.S. Highway 1 North, Fort Pierce, Florida,
34946, USA
Available online: 04 Aug 2011
To cite this article: Federico J. Rotman, Marty Riche, Peter Van Wyk & Daniel D. Benetti (2011): Efficacy of a Commercial
Probiotic Relative to Oxytetracycline as Gram-Negative Bacterial Control Agents in a Rotifer (Brachionus plicatilis) Batch
Culture, North American Journal of Aquaculture, 73:3, 343-349
To link to this article: http://dx.doi.org/10.1080/15222055.2011.603968
ARTICLE
Federico J. Rotman
Rosenstiel School of Marine and Atmospheric Science, University of Miami,
4600 Rickenbacker Causeway, Miami, Florida 33149, USA
Marty Riche*
U.S. Department of Agriculture, Agricultural Research Service,
Sustainable Marine Aquaculture Systems, 5600 U.S. Highway 1 North, Fort Pierce, Florida 34946, USA
Daniel D. Benetti
Rosenstiel School of Marine and Atmospheric Science, University of Miami,
4600 Rickenbacker Causeway, Miami, Florida 33149, USA
Abstract
Two trials were conducted to evaluate two gram-negative bacterial control strategies in batch cultures of the
rotifer Brachionus plicatilis. In the first trial, rotifers at an initial density of 47/mL were cultured for 5 d and dosed
with a 10-mg/L solution of either oxytetracycline or a commercial probiotic consisting principally of Bacillus and
Lactobacillus. In the second trial, rotifers at an initial density of 140/mL were cultured for 4 d and dosed with 20-mg/L
solutions of oxytetracycline or the probiotic. Rotifer density, egg counts, and egg : female ratios were determined daily.
At the termination of trial 1 and on days 0, 2, and 4 of trial 2, counts of bacterial colonies were conducted to estimate
bacterial loads in the rotifer culture water. Oxytetracycline was an effective antimicrobial and enhanced rotifer
production in the higher-density culture but had only a marginal effect on production in the lower-density culture.
Conversely, inoculation of the low-density culture with the probiotic had no effect on rotifer production. Inoculation
of the high-density culture adversely affected rotifer production by increasing mortality and reproductive inhibition.
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ROTMAN ET AL.
METHODS
Cultures and measurements.Two trials were conducted to
evaluate the efficacy of the commercial probiotic Epicin relative to oxytetracycline-inoculated and untreated rotifer batch
cultures. The first trial examined a rotifer culture initially inoculated with 47 1.9 (mean SE) rotifers/mL (low density);
the culture in the second trial was initially inoculated with 140
0.9 rotifers/mL (high density). Rotifers were obtained from a
cultured stock at Harbor Branch Oceanographic Institution, Fort
Pierce, Florida. Batch cultures used to draw rotifers for all treatments in both trials were inoculated with Epicin probiotics 9 d
before the trials started. To reduce possible cross-contamination
of the oxytetracycline treatment, rotifers were rinsed for 30 min
before the trials began.
Rotifers were cultured in a batch culture system in fifteen 60L circular tanks. Before each trial, the tanks were disinfected
with a 100-L/L solution of chlorine for 12 h, then neutralized
with sodium thiosulfate. For both trials the rotifer cultures were
initiated (day 0) by placing rotifer inoculums in 20 L of live
Nannochloropsis oculata. All rotifer cultures were fed daily an
additional 10 L of algae originating from the same batch of algal
culture to remove cell density and differential bacterial loads as
confounding effects between treatments within each trial. An
additional 0.06 g/L supplementation with Culture Selco 3000
(INVE Aquaculture, Inc., Salt Lake City, Utah) was added twice
daily to each rotifer culture. A single air stone was supplied to
each culture to maintain dissolved oxygen levels above 4 mg/L.
A completely randomized design with repeated measures
was employed with microbial control strategy as the fixed factor effect. Batch rotifer cultures were inoculated with Epicin,
oxytetracycline (Terramycin), or neither, as a control. The batch
culture tanks served as the experimental unit. Treatments were
run with five replicates.
Probiotic inoculants were prepared according to the manufacturers specifications in the manner of Benetti et al. (2001).
Oxytetracycline was obtained from a local feed manufacturer
(Bonney, Laramore, and Hopkins, Inc., Fort Pierce, Florida).
Both oxytetracycline treatments and probiotic solutions were
prepared with sterile seawater and administered to the rotifer
cultures before feeding. The low-density experimental cultures
were each treated with 10 mg/L solutions of oxytetracycline or
probiotics upon culture startup and again on culture days 2 and
4. High-density cultures were treated with 20 mg/L solutions of
oxytetracycline or probiotics upon startup and with an additional
20 mg/L treatment on days 2 and 3 of the trial.
Rotifer density and percent egg-carrying females were quantified daily during both trials. Temperature and dissolved
oxygen were recorded at sampling. The low-density experiment was terminated at day 5, the high-density experiment at
day 4.
At the end of the low-density trial, a pooled water sample
of equal aliquots from each tank was obtained (consisting of
six vertical samples per tank) and passed through a 5-m poresize sterile screen. A sterile 10-L inoculation loop was used
to apply three streaks from each pooled water sample to marine
agar (MA) plates (Difco, Fisher Scientific, Suwanee, Georgia)
to determine total bacterial count and to thiosulfate-citrate-bile
salts-sucrose agar (TCBS) plates (Difco) to quantify Vibrio spp.
Before quantification, MA plates were incubated at 28 C for
45 d; TCBS plates were incubated at 28 C for 24 h. For each
replicate, the number of individual colony forming units (CFU)
in each of the three streaks was counted. The mean of these
three counts was used for statistical analyses.
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FIGURE 1. Mean (A) rotifer density and (B) egg production after treatment
with oxytetracycline, probiotics, or neither (control) during a 5-d batch culture. Error bars are SDs; N = 5. Arrows indicate treatment applications. The
initial inoculation was 47 rotifers/mL. Daily means with different letters are
significantly different (P < 0.05).
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ROTMAN ET AL.
FIGURE 2. Mean colony-forming units (CFU) grown on thiosulfate-citratebile salts-sucrose agar (TCBS) and marine agar corrected for gram-negative
colonies (Corr-MA) from water samples collected at the termination of trial
1. Rotifer cultures were treated with oxytetracycline, probiotics, or neither
(control) during a 5-d batch culture. Error bars are SEs; N = 5. Within agar
types, treatments with different labels are significantly different (P < 0.05).
FIGURE 3. Mean (A) rotifer density and (B) egg production after treatment
with oxytetracycline, probiotics, or neither (control) during a 4-d batch culture. Error bars are SDs; N = 5. Arrows indicate treatment applications. The
initial inoculation was 140 rotifers/mL. Daily means with different letters are
significantly different (P < 0.05).
trial. Conversely, egg production in the oxytetracycline treatment remained elevated from day 1 through day 3 before decreasing on day 4.
The TCBS bacterial counts were not different at the initiation
of trial 2; however, by day 2, these counts had nearly doubled in
the control, tripled in the probiotics treatment, and decreased by
an order of magnitude with oxytetracycline (Table 1). By day 2
the bacterial counts in the probiotic treatment were significantly
higher than in the control. Bacterial counts continued to increase
in the control and probiotic treatments and but decreased in the
oxytetracycline treatment at day 4. All treatment results were
significantly different at termination of the trial (day 4) than
initiation of the trial (day 0).
The mean CFU values on the MA following correction for
gram-negative bacteria also were not different among the treatments at the initiation of the trial. Counts in the control treatment continued to increase throughout the trial, and by day 4
were too numerous to quantify (Table 1). Counts in the cultures
inoculated with probiotics were too numerous to quantify by
day 2.
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TABLE 1. Mean colony-forming units (103/mL) grown on thiosulfate-citrate-bile salts-sucrose agar (TCBS) and marine agar corrected for gram-negative
colonies (Corr-MA) from water samples collected during trial 2. Rotifer cultures were treated with oxytetracycline, probiotics, or neither (control) on days 0, 2,
and 4 during a 4-d batch culture. Standard errors are given in parentheses; N = 5. Within days and agar types, values with different letters are significantly different
(P < 0.05).
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Treatment
TCBS
Control
Probiotic
Oxytetracycline
Corr-MA
Control
Probiotic
Oxytetracycline
Day 0
Day 2
Day 4
P-value,
Day 0 vs. day 4
48.1 (2.1) z
48.6 (1.2) z
42.2 (1.3) z
77.0 (7.8) y
139.9 (4.3) z
4.1 (1.0) x
120.9 (10.7) y
191.1 (3.7) z
2.7 (0.6) x
< 0.0001
< 0.0001
< 0.0001
93.0 (7.9) z
96.8 (5.1) z
97.6 (6.2) z
138.6 (9.1) z
>300
4.8 (1.8) y
>300
>300
3.4 (1.1)
< 0.0001
< 0.0001
< 0.0001
DISCUSSION
One function of probiotics in aquaculture is to reduce
the incidence of Vibrio and other gram-negative heterotrophic
pathogens. However, gram-negative bacteria apparently were
not inhibited by the addition of Epicin. Similarly, Gatesoupe
(1994) noted that lactic acid bacteria used as a probiotic in
rotifer cultures did not affect total counts of Vibrio growing on
TCBS agar. Abasolo-Pacheco et al. (2009) also reported elevated
Vibrio and Pseudomonas in Epicin-treated seawater relative to
amounts in untreated seawater.
Rotifer densities for all treatments increased over the course
of trial 1. Although the oxytetracycline treatment had a significantly higher final density, no difference was detected in final
densities between the control and probiotic treatment. This was
probably the result of a decrease in egg production on day 3
observed in the control and probiotic treatments but not in the
oxytetracycline treatment. The explanation for this decrease is
unclear, but it is probably independent of density or treatment
effect, given that egg production was significantly higher on day
5 than day 4 despite the addition of treatments on day 4. Nevertheless, no differences in egg production between the probiotic
and control treatments were observed in the low-density culture.
Interestingly, despite the much higher microbial count in
the probiotic treatment, there was no benefit from its addition
to a low-density short-term rotifer batch culture. Similar results were reported for rotifers grown with Lactococcus lactis
AR21, despite its demonstrable efficacy in inhibiting V. anguillarum (Harzevili et al. 1998). Gatesoupe (1991) reported that
two strains of lactic bacteria increased rotifer production, but
a third, Streptococcus thermophilus, had no effect. It has been
proposed that efficacy depends on utilization of strains optimized for the environmental conditions of the system (Douillet
2000; Planas et al. 2004).
In contrast to trial 1, rotifer densities during trial 2 were
significantly different throughout. Densities in the oxytetracycline treatment were much higher than in the control, and those
receiving the probiotic treatment were significantly lower than
Dependent variable
Egg number, control
Egg number, treated
a
Not significant.
Independent
variable
Rotifer density
Egg : female ratio
Rotifer density
Egg : female ratio
r2
Pvalue
a
0.060
0.816 < 0.001
0.650 < 0.001
0.633 < 0.001
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ROTMAN ET AL.
1.0
0.9
0.8
Control
Probiotic
0.7
0.6
0.5
0.4
0.3
0.2
0.1
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0.0
+0
+1
+2
Day
+3
+4
ACKNOWLEDGMENTS
The authors thank the staff of Harbor Branch Oceanographic
Institute for providing support and the facilities necessary for
this study. This project was funded in part by USDA Agricultural Research Service Project Number 6225-63000-002-00D.
A special thanks to Megan Davis, Susan Allen, and Hassan Sebti
for technical support and assistance. Mention of trade names or
commercial products in this article is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. All
programs and services of the U.S. Department of Agriculture
are offered on a nondiscriminatory basis without regard to race,
color, national origin, religion, sex, marital status, or handicap.
FIGURE 4. Mean egg : female ratios in rotifer batch cultures receiving the
control and probiotic treatments during trial 2. Error bars are SDs; N = 5.
REFERENCES
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