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Appl Environ Microbiol, June 1998, p. 2318-2322, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Bioencapsulation of Two Different Vibrio
Species in Nauplii of the Brine Shrimp (Artemia
franciscana)
Bruno
Gomez-Gil,1,*
Maria A.
Herrera-Vega,2
F. Alberto
Abreu-Grobois,2 and
Ana
Roque1
Department of Pathology, CIAD/Mazatlán
Unit for Aquaculture and Environmental
Management,1 and
Laboratorio de
Conservación y Manejo de Recursos Bióticos, Estación
Mazatlán, Instituto de Ciencias del Mar y Limnología,
UNAM,2 Mazatlán, Sinaloa 82000, México
Received 22 September 1997/Accepted 18 March 1998
 |
ABSTRACT |
Two groups of nauplii from the brine shrimp (Artemia
franciscana) were enriched with different bacteria, and the
dynamics of bacterial uptake by the nauplii were observed. This study
showed that the efficiency of Artemia nauplii in
bioencapsulating bacteria strongly depends on the type of bacteria
used, time of exposure, and status (live or dead) of the bacteria.
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TEXT |
Live nauplii of the brine shrimp
(Artemia spp.) have been used as vectors for delivering
compounds of diverse nutritional (10, 26) and/or therapeutic
(5, 6, 23, 27) value to larval stages of aquatic animals, a
process known as bioencapsulation. Inoculating the digestive tracts of
target organisms with probiotic bacteria through bioencapsulation and
feeding is another alternative use for Artemia nauplii.
Probionts can be defined as a live microbial feed supplement, which
beneficially affects the host animal by improving its intestinal
balance (12). Bioencapsulated lactic acid bacteria have been
successfully introduced into turbot larvae with significant
improvements in survival (13). Bacteria with various
characteristics have been incorporated into Artemia
nauplii to orally challenge turbot larvae with a pathogenic
Vibrio anguillarum strain (7, 16). This route has
also been used to vaccinate sea bass fry (8), juvenile carp
(17), and fish fry (4).
It has been suggested that bacterial infections are initiated through
the oral route in penaeid larvae and postlarvae (19). Therefore, an oral challenge should be a reliable method to reproduce actual infections and also to introduce probiotic strains. The aim of
this study was to evaluate the bioencapsulation of (i) a potential
pathogenic bacterium and (ii) a potential probiotic bacterium in
Artemia nauplii.
Artemia cyst hatching and disinfection.
Artemia
franciscana cysts from the Great Salt Lake, Utah, were employed
for this study. The corion of the cysts was chemically removed by
employing the methodology proposed by Sorgeloos et al. (25),
a process known as decapsulation. Hatching of the decapsulated cysts
was performed in a sealed flask with 200 ml of sterile seawater (3.5%
salinity). The cysts were stocked at a density of 5.0 g
liter
1 and incubated at 30°C with constant illumination
and oxygenated through mechanical agitation to prevent bacterial
contamination. To ensure a complete disinfection of the nauplii,
chloramphenicol (Chloromycetin; Parke-Davis) at 30.0 mg
liter1 and trimethoprim-sulfamethoxaxole (Bactrim;
Productos Roche) at 40.0 and 8.0 mg liter1, respectively,
were added to the hatching water. After 24 h, the recently hatched
nauplii were collected aseptically in a 120-µm-pore-size sieve
and washed thoroughly with sterile distilled water.
To evaluate if any antibiotic residue was left in the nauplii after
rinsing, a modified disk diffusion test (2) which permitted the growth of marine bacteria was performed. Nauplii were rinsed and
macerated with a tissue homogenizer, a Whatman no. 1 filter paper disc
(7.0 mm in diameter; Whatman, Inc., Clifton, N.J.) was impregnated with
20 µl of the liquid supernatant. As control, other discs were also
impregnated with samples from the hatching water and the antibiotic
solution. Muller-Hinton agar (Bioxon) was prepared with 2.5% NaCl and
dispensed in 10-mm-diameter petri dishes. The impregnated discs
were placed in duplicate on the agar plates inoculated with reference
bacteria as a lawn. The reference bacteria employed were
Escherichia coli (ATCC 25922) from the American Type
Culture Collection, Vibrio
parahaemolyticus (LMG 2850T) from the Microbiology
Laboratory, University of Gent, Vibrio harveyi (LMG 4044T),
Vibrio damsela (LMG 7892T), and isolates C7b and HL57. After
48 h of incubation, the inhibition halos were measured.
Bacterial culture and preservation.
The bacterium HL57
was isolated from the hemolymph of a farm-grown, diseased
juvenile shrimp caught in the state of Sinaloa, Mexico. The bacterium
C7b was collected from unpolluted seawater in the same state.
Thiosulfate-citrate-bile-sucrose agar (TCBS; Difco, Detroit, Mich.) was
employed to isolate and partially purify the bacteria.
For further analyses, the bacteria were grown and purified in tryptic
soy agar (TSA) or tryptic soy broth (TSB) (both from Bioxon) made with
distilled water and 2.0% (wt/vol) NaCl to obtain a final concentration
of 2.5%. Media and solutions were sterilized by autoclaving at 121°C
for 20 min. The cultures were incubated at 30°C for 20 to 24 h
or 48 h.
The isolates were preserved at
70°C in an ultralow mechanical
freezer (Revco Scientific, Asheville, N.C.) in cryovials filled
with
glass beads (
14). In order to recover strains from
cryopreservation,
a bead was obtained from the appropriate cryovial and
placed in
a test tube with TSB at 25 to 30°C and incubated overnight
at
30°C with constant agitation. A sample was then taken from the
test tube and streaked on TSA and incubated at 30°C for 20 to
24 h.
Bacterial inoculum.
Ten milliliters of a fresh bacterial
culture were centrifuged at 5,000 rpm for 10 min at 10°C (Beckman,
Instruments, Inc., Fullerton, Calif.), the liquid supernatant was then
discarded, and the pellet was suspended in sterile saline solution.
This process was repeated again, and the cell concentration in the suspension was adjusted to an optical density of 1.00 at 610 nm in a
spectrophotometer (model DR-2000; Hach, Loveland, Colo.). To estimate
the bacterial concentration achieved, the suspension was serially
diluted in sterile saline and spread plated in TSA or Marine agar
(Difco).
Bacterial characterization.
Tests were performed to
characterize the isolates by following the recommendations of Baumann
and Schubert (3) and Austin and Lee (1). The
bacterial isolates were analyzed for their reaction to the following
tests: Gram staining, oxidase production (Oxoid identification sticks;
Oxoid, Basingstoke, England), motility, oxidative-fermentative
metabolism of glucose, sensitivity to the vibriostatic agent O/129
(2,4-diamino-6,7-diisopropylpteridine phosphate; Oxoid), and swarming
in solid media. The Biolog-GN microplates (Biolog, Hayward, Calif.)
were employed to analyze the ability of the isolates to use
different carbon sources. The API 20NE system
(bioMérieux, Marcy l'Etoile, France) was also employed to
further characterize the isolates. Both systems were inoculated
according to the recommendations of the manufacturer, but the bacterial
suspensions were prepared as described above.
Bioencapsulation of bacterial isolates in the Artemia
nauplii.
Sterile nauplii were added to a 200-ml flask with
the bacterial suspension (sterile seawater and the desired
bacterium) at a density of 100 nauplii ml1 and
107 bacterial cells ml1. Controls were
bacteria only and nauplii only. Four replicates were prepared for
each treatment. Each experiment was repeated once for each isolate
(HL57 and C7B).
Two 1.0-ml samples from each replicate were collected at the following
intervals after the nauplii were placed in the flask:
at 15, 30, and 45 min and at 1, 2, 4, 8, and 24 h. The nauplii
from
each sample (except from the "bacteria only" control) were
collected in sterile conditions, thoroughly washed, and macerated
in a
tissue homogenizer. Serial dilutions of the supernatant fluid
(or from the seawater in the "bacteria only" control) were
prepared,
and 0.1 ml was spread plated in Marine and TCBS agars. The
petri
dishes were incubated at 30°C for 24 h, and the CFU were
counted.
To find out if the nauplii were digesting the bacteria, half of the
nauplii were removed after 30 min from the bacterial broth
under
sterile conditions, thoroughly washed, and transferred to
a flask with
sterile seawater. The nauplii were sampled with a
pipette and
counted at the same intervals and in the same manner
as the batch that
remained in the bacterial suspension.
The
Artemia nauplii were completely disinfected without
apparent damage to the organisms, as observed by Gorospe
(
15). No
antibiotic residue was microbiologically detected.
Inhibition
halos were observed in all tested bacteria for the
antibiotic
solution (15.0 to 30.0 mm) and for the hatching seawater
(11.0
to 20.0 mm), but no halos could be seen in the macerated
nauplii
after they were washed. Mohney et al. (
21) and
Dixon et al.
(
11) performed similar disk diffusion tests,
and their results
are comparable to those of this study. Cappellaro et
al. (
5)
proved that with
Artemia cysts hatched in
two antibiotic solutions,
sulfadimethoxine sodium salt and
enrofloxacin, the residual antibiotic
detected with high-pressure
liquid chromatography and UV was only
5.0 to 16.7% of the initial
solutions. It is possible that the
microbial bioassay was not sensitive
enough to detect small amounts
of residual antibiotics left in the
nauplii. Even when a low concentration
of residues of antibiotics
remains, it might not be enough to
cause any bacterial inhibition in
the experiments performed with
the nauplii.
The bacterial isolates were identified as belonging to the
Vibrio genus, as they were gram-negative short rods, oxidase
positive,
and motile, had fermentative metabolism of glucose, and were
sensitive
to the vibriostatic agent O/129 (
20). Isolate C7b
was able to
utilize sucrose,
D-mannitol, and
L-leucine but did not utilize
cellobiose, lactose,
L-arabinose,
D-melibiose,
L-rhamnose,
-hydroxybutyric
acid or
-aminobutyric acid; it was positive for
-galactosidase
(
o-nitrophenyl-
-
D-galactopyranoside
[ONPG]), nitrate reduction,
and gelatinase; it swarmed in solid
media and produced big smooth
yellow colonies in TCBS agar. With these
characteristics, it was
identified as
Vibrio alginolyticus.
The Biolog Microlog 2.0 identification
system also identified it as
V. alginolyticus, with a similarity
index of 0.897.
Isolate HL57 showed positive utilization of citrate,
D-mannitol, and
L-arabinose but negative
utilization of sucrose. It was
positive for
-galactosidase (ONPG),
nitrate reduction, and gelatinase,
no swarming in solid media was
observed, and it grew as 3- to
4-mm blue-green colonies in TCBS agar.
These characteristics permitted
its identification as
Vibrio
parahaemolyticus. The Biolog Microlog
2.0 system also gave the
same identification, with a similarity
index of 0.589. A similarity
index of 0.5 is the minimum value
for a reliable identification
computed with the Microlog 2.0 software.
Optical densities of the inocula and their corresponding CFU
milliliter
1 for the experiments with the two species are
presented in Table
1. Significant
differences among the values of the bacterial
suspension inoculated
were observed (
P = 0.004, Kruskal-Wallis
one-way
analysis of variance). At the same optical density, isolate
HL57 gave a
concentration almost 10-fold higher than C7b, 1.50
× 10
8 and 3.17 × 10
7 CFU ml
1,
respectively. Even within the C7b isolate, differences were
found
at two similar optical densities (Table
1). In another
study,
V. anguillarum was found to have a concentration of 2.5
× 10
8 CFU ml
1 at an optical density at 610 nm of 1.0 (
4), while Roque (
24)
found 4.5 × 10
7 CFU ml
1 for
Vibrio vulnificus
at the same optical density.
Bacterial isolates were successfully incorporated into the
Artemia nauplii. In both experiments with isolate HL57,
when nauplii
were maintained in the bacterial broth, the quantity
of bacteria
bioencapsulated rapidly increased after 30 min (Fig.
1) with a
sustained level above 2,000 CFU
nauplius
1, followed by a slight decline after 8 h.
At 24 h, the bacterial
level increased again, but all the
nauplii died. In the treatment
where the nauplii were removed
after 30 min from the bacterial
broth and placed in sterile saline, the
bacterial level in the
nauplii started to decrease immediately,
reaching a minimal level
at 8 h. The concentrations increased
again at 24 h, although all
nauplii died. The experiments
conducted with isolate C7b (Fig.
2)
showed a different pattern from that of isolate HL57. In this
set of
experiments, the C7b isolate was rapidly bioencapsulated,
reaching a
peak at 45 min. The concentration of bacteria per nauplius
decreased
slowly to reach a minimum at 24 h. By the end of both
experiments,
no dead nauplii were observed. After the nauplii
were removed
from the bacterial broth (at 30 min), the bacterial
concentration
declined dramatically, 10-fold at the next measurement
(45 min). They
continued to decline to a minimum at 8 h, with
a slight increment
observed at 24 h (Fig.
2).
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FIG. 1.
Concentration of isolate HL57 (V. parahaemolyticus) bioencapsulated in Artemia
nauplii. The counts were done on Marine agar, and data are from two
experiments. The mean ± 95% confidence interval (error bar)
(n = 4) is shown for each point. Note the change of
scale.
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FIG. 2.
Concentration of isolate C7b (V. alginolyticus) bioencapsulated in Artemia nauplii.
The counts were done on Marine agar, and data are from two experiments.
The mean ± 95% confidence interval (error bar)
(n = 4) is shown for each point. Note the change of
scale.
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|
Results observed by Campbell et al. (
4) with formalin-killed
bacteria showed that maximum uptake of
V. anguillarum
occurred
at 60 min in a bacterial concentration of 1.5 × 10
7 CFU ml
1, while at a lower concentration
of 1.5 × 10
6, a peak was observed after 120 min. A
similar pattern was observed
with
Brachionus plicantilis
when challenged with a
V. anguillarum vaccine
(
18). The
Artemia nauplii ingested the
maximum quantity
of isolate HL57 cells after 2 h of contact with
the bacterial
broth (Fig.
1 and Table
1). No statistical difference was
encountered
between both experiments at 2 h of bioencapsulation
(Student's
t test = 2.10,
P = 0.080).
The difference between the values at
1 and 2 h was highly
statistically significant (
t test = 7.61
and
P = 0.0003 for experiment 1;
t test = 7.59 and
P = 0.0003
for experiment 2). There was no
significant difference between
the 2- and 4-h bacterial counts
(
t test = 0.77 and
P = 0.4680
for
experiment 1;
t test = 0.40 and
P = 0.7049 for experiment
2). With these data, it was concluded that 2 h is the optimum
exposure time when the largest number of isolate HL57
can be bioencapsulated
in the least time.
The nauplii bioencapsulated the maximum number of C7b bacteria
after 45 min of contact with the bacterial broth in both experiments
(Fig.
2 and Table
1). No difference was encountered between either
experiment at 45 min (
t test = 2.091,
P = 0.0815). The difference
between values at 30 and 45 min was highly
significant (
t test
= 6.197 and
P = 0.0008 for experiment 1;
t test = 7.59 and
P =
0.0003 for experiment 2). The difference between
values at 45
min and 1 h was also significantly different, with a
higher count
at 45 min (
t test = 4.431 and
P = 0.0044 for experiment 1;
t test
= 10.29 and
P < 0.0001 for experiment 2). Therefore, 45 min was
the minimum time when the most cells of isolate C7b could be
bioencapsulated.
In this study,
Artemia nauplii could bioencapsulate from
2.42 × 10
3 to 4.77 × 10
3 CFU
nauplii
1 of live bacteria (Table
1). Different
results were found by
Campbell et al. (
4) (10
5
formalin-killed CFU nauplii
1) with dead
V. anguillarum cells, but Chair et al. (
7) obtained
results similar to this study (8.0 × 10
3 CFU
nauplii
1) with live
V. anguillarum.
Analysis of the results suggest the
following: if the bacteria are
dead, the concentration is an important
factor in the uptake of
bacteria by
Artemia nauplii, but if the
bacteria are
alive, the species or strain is also a significant
factor. Although the
HL57 isolate was at a concentration almost
10-fold higher than that of
the C7b isolate, the latter reached
an intake peak in less than half
the time. One explanation is
that isolate C7b had the ability to more
rapidly colonize the
Artemia nauplii.
The control (bacteria without nauplii) maintained a constant level
of bacteria (10
8 CFU ml
1) throughout the
course of experiments with both isolates (Fig.
1 and
2). The
bacterial levels in the controls and in the treatments
did not show a
positive correlation in either experiment with
isolate HL57 (for
experiment 1, Spearman correlation test,
r =
0.180,
P = 0.619,
n = 8; for experiment 2, Pearson correlation
test,
r =
0.289,
P = 0.487,
n = 8). The same correlation
was
observed in experiments with isolate C7b (Pearson correlation
test,
r = 0.357,
P = 0.385,
n = 8 for experiment 1; and
r =
0.321,
P = 0.439,
n = 8 for experiment 2).
These negative correlations
indicate that the change in the
nauplius bacterial levels is not
due to the quantity of bacteria
available in the broth but to
whether the bacteria were ingested or
firmly attached to the nauplii.
In the control where nauplii
remained in sterile seawater only,
no bacteria were registered during
the course of all experiments.
Bacterial colonization of the nauplii could occur externally, via
attachment to the body surfaces or internally by ingestion
(
16). After the nauplii were removed from the bacterial
suspension,
the bacterial content decreased rapidly. This decrease
might be
due to the removal of the external bacteria after the
nauplii
were washed and placed in sterile seawater. The bacteria
still
detected could be the ones colonizing the interior or firmly
attached
to the external surfaces. Similar trends were observed with
rotifers
after they were removed from a bacterial suspension
(
18). It
should be emphasized that the bacterial counts in
the nauplii
placed in the bacterial solution had no correlation
with the bacterial
counts of the solution, which suggests active uptake
by the nauplii.
Rico-Mora and Voltolina (
22) challenged
Artemia
nauplii with
V. alginolyticus and
V. parahaemolyticus isolates and obtained
almost 100% mortality
after 24 h for the first species and 48
h for the second
species. In our work,
V. alginolyticus isolated
from
seawater caused no mortalities after 24 h, while the
V. parahaemolyticus isolated from diseased shrimp caused almost 100%
mortality. In
peneid shrimp, differences in the pathogenicity of
bacteria could
depend on the species tested (
28) and on the
strain characteristics
(
9); a similar case could be presumed
for
Artemia nauplii.
| |
ACKNOWLEDGMENTS |
We appreciate the kind cooperation of Jean Swings and Johan
Vanderberghe, Microbiology Laboratory, University of Gent, for providing reference bacteria.
This work was supported in part by the International Foundation for
Science grant A/2203-1.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, CIAD/Mazatlán Unit for Aquaculture and Environmental
Management, AP. 711, Mazatlán, Sinaloa 82000, México.
Phone: (69) 880157. Fax: (69) 880159. E-mail:
bruno{at}servidor.unam.mx.
| |
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0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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