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Fish & Shellfish Immunology 28 (2010) 320e325

Contents lists available at ScienceDirect

Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Parenteral immunization of fish, Labeo rohita with Poly D, L-lactide-co-glycolic

acid (PLGA) encapsulated antigen microparticles promotes innate
and adaptive immune responses
T. Behera a, P.K. Nanda a, C. Mohanty b, D. Mohapatra a, P. Swain a, *, B.K. Das a,
P. Routray a, B.K. Mishra a, S.K. Sahoo b
a
Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751002, India
b
Nanomedicine Laboratory, Institute of Life Sciences, Bhubaneswar-751023, India

a r t i c l e i n f o a b s t r a c t

Article history: Immunogenicity of different antigen preparations of outer membrane proteins (OMP) of Aeromonas
Received 27 August 2009 hydrophila such as Poly D, L-lactide-co-glycolic acid (PLGA) microparticles, oil emulsion, neat OMP and
Received in revised form bacterial whole cells were compared through intra-peritoneal injection in fish, Labeo rohita. Among these
5 November 2009
preparations, PLGA encapsulated antigen stimulated both innate and adaptive immune parameters and
Accepted 9 November 2009
Available online 14 November 2009
the immunogenicity exhibited by PLGA microparticles was significantly higher (p < 0.05) at both 21 and
42 days post-immunization suggesting that the above delivery system would be a novel antigen carrier
for parenteral immunization in fish, Labeo rohita
Keywords:
Aeromonas hydrophila Ó 2009 Elsevier Ltd. All rights reserved.
Immune response
Microparticles
Outer membrane proteins
PLGA

Vaccination is one of the methods for prevention of infectious
diseases of human and other animals, including fish [1]. Inefficiency
of currently used conventional vaccine is due to lack of appropriate
adjuvants and/or suitable vaccine carrier. The new generation of
vaccines mainly contain purified proteins (isolated from microor-
ganisms or recombinant proteins) and peptides (by direct chemical
peptide synthesis), which are poorly immunogenic. Therefore, the
search for harmless and effective adjuvants is urgently needed in
modern vaccinology. Use of several adjuvants like particulates
(aluminium salts, ISCOMS, virosomes, chitosan, QS-21 and lipo-
somes); microbial products (microbial toxins, live viral and bacterial
delivery vectors, monophosphoryl lipid A); and oil emulsions
{Freund's incomplete adjuvant (FIA), Freund's complete adjuvant
and MF-59} have been extensively reviewed [2]. Although each one
creates its own type of immune modulation, they also exert some
side effects [3]. Further, their ineffectiveness to certain antigens and
their inability to elicit cell-mediated immune responses, particularly
cytotoxic T-cell responses, limits their application against intracel-
lular parasites and viral infections. When new vaccine formulations
* Corresponding author.
E-mail address: pswainy2k@yahoo.co.in (P. Swain).
are being sought, there are many aspects to consider such as effec-
tiveness in inducing the correct immune response, stability in the
host, toxicity and economic aspects. One such highly promising
technology is based on polymeric microparticles, which permit
a sustained or pulsed release of encapsulated antigen thus mini-
mizing the requirements for repeated administration and dose of
antigen ensuring long-term protection.
In contrast to other carriers, microparticles are more stable and
elicit both humoral as well as cellular immunity [4]. Evidence from
mammalian studies indicate that biodegradable microparticles
may act as efficient antigen delivery vehicles due to their potential
advantages of reducing the number of injections, enhancing the
immune response and reducing the total antigen dose needed to
achieve protection [5e7]. Among the polymeric systems developed,
PLGA microspheres have been widely used for controlled delivery
of peptides [8], native and synthetic proteins [9] and nucleic acids
[10] because of their excellent tissue compatibility, biodegrad-
ability, non-toxic nature and their approval by the Food and Drug
Administration for safe use in human [6]. The PLGA can also be
made into any micro- and nano sizes, with a capability of encap-
sulating almost any molecule [11]. In vivo, the polymer undergoes
random non-enzymatic hydrolysis and forms the endogenous
metabolites, lactic and glycolic acids [7], which are innocuous to the

1050-4648/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2009.11.009
 Author's personal copy
T. Behera et al. / Fish & Shellfish Immunology 28 (2010) 320e325
body and are eliminated by the Kreb's cycle as carbon dioxide and
in urine [12]. Several encouraging trials have been conducted
providing data to confirm the superior efficacy of this system over
other adjuvants in several vaccines [13e15]. Today PLGA particles
are used in different marketed products, medical and pharmaceu-
tical fields, and are capable of releasing peptides and proteins
slowly and continuously from 1 to 4 months [11]. Further, PLGA
particles have been shown to be taken up in vivo by the main
antigen presenting cells (APC) in mammals, dendritic cells, and
enhance antigen-presentation efficacy by 10e100 fold [16e18]. The
easy manufacture of this microparticle and the possibility of
administration by different routes offered the additional advan-
tages for its use as a vaccine carrier [19].
Fish vaccinology faces similar problems that are encountered in
vaccine design for human and mammals, as they are considered to be
very important in reducing economic losses in aquaculture caused by
some diseases [20e22]. This must be considered for developing
effective vaccines to face old challenges with new possibilities in the
21st century. In this regard, the use of biodegradable microparticles
as an antigen delivery system in fish is a relatively new area of
research. Among the biodegradable microparticles, PLGA micropar-
ticles as a vaccine carrier in fish have only been investigated through
oral vaccination in rainbow trout [23]. Moreover, PLGA microparti-
cles alone also stimulate certain non-specific immune parameters
and pro-inflammatory cytokine production through intraperitonial
injection in fish [24], but there is no report for PLGA as an antigen
carrier in fish through parenteral administration.
Hence the present study was undertaken to evaluate PLGA
microparticles as an antigen carrier using low molecular weight
protein antigen such as outer membrane proteins (OMP) of Aero-
monas hydrophila, a known bacterial pathogen of fish and the
results obtained herewith found that it stimulated both specific and
innate immune parameters.
1. Materials and methods
1.1. Bacteria
Aeromonas hydrophila strain (Ahv), isolated from Channa striatus
showing dropsy conditions was preserved in a lyophilized condi-
tion in our laboratory and was used throughout the study.
1.2. Isolation of outer membrane proteins
The OMP of A. hydrophila (Ahv) were prepared according to the
method of Nikaido [25] with minor modifications. The pellet
obtained from 1 L culture of A. hydrophila was washed twice in 40 ml
of 0.15 M phosphate buffered saline (PBS, pH 7.2), once in 40 ml of
20 mM TriseHCl (pH 7.5) and centrifuged at 13,000
g for 10 min.
The cells were then resuspended in 20 ml TriseHCl and disrupted by
sonication for 30 min at 10 W at an interval of 30 s. Unbroken cells
and cellular debris were removed by centrifugation at 4000
g for
15 min. The supernatant was then further centrifuged at 10,000
g
for 1 h at 4  C. The pellet was suspended in 20 ml of 2% (w/v) sar-
cosine and incubated at room temperature for 30 min to solubilize
the inner membrane. The solution was then centrifuged at
10,000
g for 1 h at 4  C. The resultant pellet of sarcosine-insoluble
components was freeze-dried and stored at 20  C until use. Protein
concentration of OMP was determined using Genei TM protein esti-
mation kit (Bangalore genei, India) by the BCA method.
1.3. Antigen formulations
Three forms of antigen preparations were done as described
below.
321
1.3.1. Preparation of PLGA microparticle encapsulated OMP
PLGA (copolymer ratio 50:50, I.V ¼ 0.76) was purchased from Bir-
mingham polymers, Inc. (Birmingham, AL). Polyvinyl alcohol (PVA,
average MW 30,000e70,000) was purchased from SigmaeAldrich Co.
(St Louis, MO 63195, USA). All reagents used were of analytical grade
from E-merck, India and organic solvents used were of HPLC grade.
Microparticles containing different antigens were formulated using
a double emulsion-solvent evaporation technique [26] with little
modification. In this method, aqueous solution of antigen dissolved in
300 ml of distilled water was emulsified with 100 mg of PLGA in
chloroform solution (3% w/v) followed by vortexing for 2 min to get
a primary emulsion. The primary emulsion was further emulsified in
an aqueous PVA solution (12 ml, 5% w/v) to form an oil-in-water
emulsion. For preparation of microparticles, the emulsion was
homogenized for 2 min and stirred for overnight at room temperature
to allow the evaporation of organic solvent. Microparticles were
recovered by normal centrifugation at 5000
g for 10 min using
SIGMA 3K30 (Germany) centrifuge. The process of centrifugation was
repeated three times to remove excess PVA and un-encapsulated
antigens. The recovered microparticle suspensions were lyophilized
for two days (80  C and <10 mm mercury pressure, LYPHLOCK,
Labconco, Kansas City, MO) to get lyophilized powder for further use.
1.3.2. Particle size and zeta potential analysis
Dynamic light scattering (DLS) was used to measure the
hydrodynamic diameter along with size distribution
(polydispersity index, PDI). The DLS analysis was performed using
a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The DLS
measurements were done with a wavelength of 532 nm at 25  C
with an angle detection of 90 . Approximately, 1 mg of the
lyophilized samples was dissolved in 1 ml MilliQ water and 100 mL
of these solutions were further diluted to 1 ml for the measure-
ments of particle size. All measurements were performed in
triplicate.
1.3.3. Scanning electron microscope (SEM) studies
The surface morphology of microparticles was characterized by
scanning electron microscopy (SEM) [JEOL JSM-T220A, MA], oper-
ating at an accelerating voltage of 10e30 kV. The microparticles
were sputtered with gold to make them conductive and placed on
a copper stub prior to the acquisition of SEM images.
1.3.4. Encapsulation efficiency measurement
The antigen loading in microparticles was determined from the
total amount of antigen added in the formulation and the antigen
amount that was not encapsulated. For this, the concentration of
antigen in the supernatant was analyzed by micro BCA protein
assay to determine free antigen concentration [27]. Briefly, 500 ml of
the supernatant obtained from centrifugation was mixed with
500 ml of PBS (0.02M, pH 7.4) followed by the addition of 1 ml of the
working solution of micro BCA assay. The solution was then incu-
bated for 60 min at 60  C. The samples were later cooled and read at
562 nm. Calibration curves were made with corresponding solu-
tions of blank microparticles. Thus, the antigen encapsulation
efficiency (EE) of the microparticles was calculated with reference
to the standard plot of antigen plotted with absorbance against
concentration of antigen (mg/ml).
1.3.5. Preparation of bacterial whole cell antigen
The A. hydrophila strain (Ahv) was cultured in brain heart
infusion (Hi Media, India) broth for 24 h at 30  C. After incu-
bation, the total bacterial count was enumerated by standard
plate count method. The bacterial culture was inactivated with
1% formalin at 4  C overnight. The bacteria were then centri-
fuged at 10,000
g for 10 min and washed twice in phosphate
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322 T. Behera et al. / Fish & Shellfish Immunology 28 (2010) 320e325
buffered saline (PBS, pH 7.2). Finally, the bacteria were sus-
pended at 109 cells ml1 in PBS.
1.4. Emulsification with FIA
OMP (20 mg) in 1 ml PBS was emulsified with an equal volume of
FIA and stored at 4  C until further use.
1.5. Immunization protocol
Indian major carp, Labeo rohita (rohu), juveniles of average
weight ranging from 30 to 40 g were acclimatized in the wet
laboratory of Fish Health Management Division of Central Institute
of Freshwater Aquaculture (CIFA), Kausalyaganga, India, 15 days
prior to the start of the experiment. The fish were fed with artificial
carp diet with constant aeration and daily one-third water
exchange. Water temperature of the experimental tanks was 27  C
to 30  C. Fish were separated into 5 groups, 10 fish in each group
were immunized separately with 0.1 ml of different preparations @
20 mg of OMP and 0.1 ml (109 cells ml1) bacterial whole cell
antigen (WC), whereas controls were injected with PBS. The fish of
all the treated groups including the control group were bled at an
interval of 3-weeks (at 21 and 42 days) post-injection to study
various immune parameters.
1.6. Preparation of anti-rohu-globulin rabbit serum
The rabbit anti-rohu globulin was prepared as per a standard
method [28] using sera obtained from healthy adult rohu of
average weight 250e300 g. Briefly, serum was collected from
healthy rohu and pooled to 10e15 ml. An equal volume of satu-
rated ammonium sulphate solution was mixed with the pooled
sera drop by drop and then placed on a magnetic stirrer overnight
at 4  C. The sample mixture was centrifuged at 10,000
g for
10 min at 4  C and the precipitate was dissolved with 5 ml car-
bonateebicarbonate buffer (pH 9.6). The sample was then centri-
fuged at 10,000
g for 10 min at 4  C. The pellet was collected and
the volume was made to 2 ml with carbonateebicarbonate buffer
(pH 9.6). The globulin solution was dialyzed using dialysis
membrane (Snakeskin, Pierce Chemical Company, USA) with 7000
molecular weight cut off against PBS (pH 7.2) for 72 h at 4  C, after
which the globulin was collected. The anti-rohu globulin sera were
raised in a New Zealand white rabbit as per the method of Lund
et al. [29].
1.7. Immunoresponse studies
1.7.1. Myeloperoxidase activity
For determination of myeloperoxidase activity, 15 ml of serum
was diluted in 135 ml of Hank's balanced salt solution (Ca2þ, Mg2þ
free) and then 50 ml of 20 mM, TMB (3, 30,5,50-tetra methyl
benzidine) and 5 mM H2O2 were added. The reaction was stopped
after 2 min by adding 50 ml of 4 M sulphuric acid and the optical
density (O.D) was read at 450 nm [30] using UVeVIS spectropho-
tometer, Thermo Spectronic, UK.
1.7.2. Respiratory burst assay
The respiratory burst activity was measured by the reduction of
nitro blue tetrazolium (NBT) by intracellular superoxide radicals
[31]. Briefly, 100 ml of heparinised blood from fish of each group was
mixed with 100 ml of 0.2% NBT (Sigma, USA) solution and incubated
for 30 min at 25  C. After incubation, 50 ml from the above was
mixed with 1 ml of N, N diethylmethyl formamide (Qualigens,
India) and then centrifuged at 6000
g for 5 min. The O.D of the
supernatant was measured at 540 nm.
1.7.3. Bacterial agglutination activity
The agglutination test was conducted in ‘U’-shaped microtitre
plates. Two-fold serial dilution of 25 ml fish serum was made with
an equal volume of PBS in each well, to which 25 ml of formalin-
killed A. hydrophila (107 cells ml1) suspension was added. The
plates were incubated overnight at room temperature. The titre
was calculated as the reciprocal of the highest dilution of serum
showing complete agglutination of the bacterial cells.
1.7.4. Haemagglutination activity
The haemagglutination activity of serum samples was carried
out using a standard method [32]. This assay was done in
‘U’-shaped microtitre plates by serial two-fold dilution of 50 ml
serum with PBS (pH 7.2). Then 50 ml of freshly prepared 1% New
Zealand white rabbit red blood cell (RBC) suspension was added
to each well. The plates were kept at room temperature
(28e30  C) for 2 h or overnight at 4  C if agglutination was not
visible within 2 h. The titre was calculated as the reciprocal of
the highest dilution of serum showing complete agglutination
of RBC.
1.7.5. Haemolytic activity
The haemolytic titre of serum was determined in a similar
manner as described for HA titre [32] by using fresh sera from
all the groups. Titre was expressed as the reciprocal of the
highest dilution of serum showing complete haemolysis of the
rabbit RBC.
1.8. Triple antibody indirect enzyme linked immunosorbent assays
The triple antibody indirect ELISA was conducted as per the
method of Swain et al. [28] with slight modifications using 96
well microtitre polystyrene plates (Nunc, Denmark). The wells
were separately coated with 50 ml of purified outer membrane
proteins from A. hydrophila (Ahv) (1e2 mg/well) diluted in car-
bonateebicarbonate buffer (pH 9.6) overnight at 4  C. The plates
were then washed in PBS containing Tween-20 (PBS-T, pH 7.2)
and blocked with 100 ml of 3% skim milk powder for 2 h at 37  C.
The wells were further washed in PBS-T. The fish sera raised
against several antigens was two fold diluted after initial dilu-
tion of 1:10 with PBS (pH 7.2) as first antibody and added to
homologous antigen-coated wells in duplicate per serum dilu-
tion. The plates were incubated at 37  C for 45 min and washed
thrice in PBS-T. Rabbit anti-rohu sera (the second antibody) at
a dilution of 1:20 was added to each well and incubated at 37  C
for 45 min. Then the anti-rabbit-HRPO conjugated goat serum
(the third antibody) was added and incubated for 45 min. The
wells were then thoroughly washed and added with 50 ml of
substrate solution (5 mg of O-phenylene diamine tetra hydro-
chloride and 10 ml of H2O2 (38%, v/v) in 5 ml of acetate buffer,
pH 5.0). The plates were incubated at 37  C for 5 min in a dark
chamber and finally O.D was recorded at 450/655 nm in
a microplate reader (BIO-RAD, USA). The antibody activity was
expressed in terms of O.D value after subtracting the values
obtained by unimmunized healthy sera.
1.9. Statistical analysis
The statistical analysis system (SAS) software (version 6.12) was
used to analyse all the data [33]. One-way analysis of variance
followed by Duncan's multiple range tests were done to compare
the variations in various immune parameters at significance level of
difference (p < 0.05) in different injected groups. The mean stan-
dard error (S.E) of assayed parameters was calculated for each
group of fish.
 Author's personal copy

T. Behera et al. / Fish & Shellfish Immunology 28 (2010) 320e325 323

40 b
0.8
Intensity (%)

30
0.7 c c

OD value at 540 nm
20 0.6 a c
0.5
10 21 days
0.4
42 days
0 0.3
1 10 100 1000 10000
0.2
Size (d.nm)
0.1
Fig. 1. Typical size distribution of PLGA microparticles as determined by dynamic light 0
scattering. CONTROL PLGA- FIA-OMP OMP WC
OMP
Treatment
2. Results
Fig. 3. The respiratory burst activity (measured by NBT assays) of blood of Labeo rohita
in different treated groups at 21 and 42 days post-immunization (Values are mean OD
2.1. Physico-chemical characterization of antigen
values  S.E.). Mean values bearing same superscript are not statistically significant
loaded microparticles (p > 0.05) at 21 and 42 days post-immunization.

Antigen loaded microparticles were prepared from the PLGA

polymer using double emulsion method. Antigen was efficiently
loaded in the PLGA microparticles, reaching encapsulation effi-
ciency of 25  1.3%. DLS analysis revealed that the formulated
microparticles had an average diameter of 1121  10.6 nm (Fig. 1).
Topology and size of the microparticle as observed by SEM analysis
confirmed the smooth and spherical nature of OMP-loaded PLGA
microparticles (Fig. 2). The average size of these microparticles was
in the range of 1 mm.
2.2. Immune response studies
2.2.1. Non-specific immune responses
The non-specific immune parameters of fish following the
injection with different antigen preparations(PLGA-OMP, FIA-OMP,
OMP and WC), at 21 and 42 days post-immunization are presented
in Figs. 3e7. All these parameters i.e. myeloperoxidase, respiratory
burst activity, haemagglutination, hemolytic and bacterial aggluti-
nation titre were significantly higher (p < 0.05) in all treated groups
than the control. The PLGA-OMP treated group showed signifi-
cantly higher (p < 0.05) titre than other groups (FIA-OMP, OMP and
WC). These parameters did not vary much in all treated groups at
21 and 42 days post-immunization.
2.2.2. Specific immune responses
The serum antibody titre, as measured by indirect ELISA, was
expressed in terms of mean OD values (after subtracting the values
obtained by unimmunized healthy sera) (S.E.) and presented in
Fig. 8. The antibody titres at 21 and 42 days post-immunization
were significantly higher (p < 0.05) in the PLGA- encapsulated
antigen and FIA treated groups than the OMP and WC treated
groups. However, no significant difference (p > 0.05) in the anti-
body level at 21 and 42 days post-immunization was recorded
between these two groups.

3. Discussion

PLGA has been proven to be a very useful antigen delivery

system in mammals since it provides long lasting immunity
[34e36]. So, we attempted to evaluate the potential use of PLGA
microparticles as antigen carrier in fish using the OMP of Gram-
negative bacteria, A. hydrophila.
The isolated OMP of A. hydrophila was encapsulated in PLGA
microparticles and the resulted microspheres were tested for
immunogenicity in fish along with other antigenic preparations.

0.2 b 21 days
42 days
OD value at 450 nm

0.15 c
c
c
0.1
a
0.05

0
CONTROL PLGA- FIA-OMP OMP WC
OMP
Treatment

Fig. 4. The myeloperoxidase activity of sera of Labeo rohita in different treated groups
at 21 and 42 days post-immunization (Values are mean OD values  S.E.). Mean values
Fig. 2. Scanning electron micrograph of outer membrane proteins loaded PLGA bearing same superscript are not statistically significant (p > 0.05) at 21 and 42 days
microparticles (bar ¼ 1 mm). post-immunization.
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324 T. Behera et al. / Fish & Shellfish Immunology 28 (2010) 320e325

9 b 8 b
8 7
7 c
6 c
6 c
Titer (log2)

Titer (l og2)
5
5 c
21 days 21 days
4
4 c 42 days 42 days
c 3 a
3
2 a 2
1 1
0 0
Control PLGA- FIA-OMP OMP WC control PLGA- FIA-OMP OMP WC
OMP OMP
Treatment Treatment

Fig. 5. The haemagglutinating activity of sera of Labeo rohita in different treated Fig. 7. The bacterial agglutination activity of sera in different treated groups of Labeo
groups at 21 and 42 days post-immunization (Values are mean log2 titre values  S.E). rohita at 21 and 42 days post-immunization (Values are mean log2 titre values  S.E).
Mean values bearing same superscript are not statistically significant (p > 0.05) at Mean values bearing same superscript are not statistically significant (p > 0.05) at
21 and 42 days post-immunization. 21 and 42 days post-immunization.

7 0.12
b a
6
Mean OD value at 450nm 0.1 a
5 c
Titer (log2)

0.08
4 c 21 days b b
c 21 DAYS
3 42 days 0.06
a 42 DAYS
2 0.04
1
0.02
0
Control PLGA- FIA-OMP OMP WC 0
OMP PLGA-OMP FIA-OMP OMP WC
Treatment Treatment

Fig. 6. The heamolysin titre of sera of Labeo rohita in different treated groups at 21 and Fig. 8. The mean OD values (S.E.) of specific antibody level in different treated groups
42 days post-immunization (Values are mean log2 titre values  S.E). Mean values of Labeo rohita detected through indirect ELISA at 21 and 42 days post-immunization
bearing same superscript are not statistically significant (p > 0.05) at 21 and 42 days Mean values bearing same superscript are not statistically significant (p > 0.05) at
post-immunization. 21 and 42 days post-immunization.

A successful micro particulate system has a high loading capacity to
reduce the quantity of the carrier required for administration. In
this study, antigen was efficiently loaded in PLGA microparticles,
reaching encapsulation efficiency of 25  1.3% with average size
diameter of 1121  10.6 nm. In an earlier study, similar results e.g.
size range w1080 nm with encapsulation efficiency of w25% were
obtained by using bovine serum albumin as a model protein
antigen in PLGA microparticles [37].
Both specific and non-specific immune parameters were studied
and all four preparations of vaccines used (PLGA-OMP, FIA-OMP,
OMP and WC) elicited immune responses in the fish at varying
levels as compared to the control. The PLGA-OMP treated group
showed significantly higher immune responses than the other
groups at both 21 and 42 days post-immunization. PLGA micro-
particles have been found to enhance phagocytosis by macro-
phages [38], and neutrophils [18] in mouse and human,
respectively. Comparable results were also found by the use of
PLGA alone in mammal [16] as well as in fish, rainbow trout [24].
The specific antibody titres of the sera were higher in both FIA-
OMP and PLGA-OMP treated groups with no significant difference
(p < 0.05) between them. Similar results were also found when
PLGA was used as carrier for peptide vaccine in mammals [39] and
in mice through subcutaneous route using BSA as a model antigen
[40]. Moreover, the superiority of PLGA microspheres over alum
adjuvant in eliciting high antibody responses was seen in mice
through subcutaneous administration [41]. In this study the supe-
riority of PLGA microparticles was recorded without any side
effects, which is usually noticed in FIA formulation [3]. The serum
antibody titres detected at 21 days persisted for at least 42 days.
According to O'Hagan et al. [42], the level of antibody remained
high even one year after injection through subcutaneous route in
mice which indicates the injectable PLGA microparticles control the
release of antigen over a period of several weeks. So, the use of
PLGA microparticles as a vaccine carrier can reduce the number of
administrations and induce both innate and adaptive immunity.
Among these four preparations of OMP of A. hydrophila, PLGA
microparticles showed encouraging results without any adverse
effects on fish health. Therefore, it can be considered to be a useful
antigen carrier for inducing both cellular and humoral immune
response in fish through parenteral immunization.
Acknowledgements
The authors are thankful to the Director, Central Institute of
Freshwater Aquaculture, Bhubaneswar and the Director, Institute of
Life Sciences, Bhubaneswar for providing all necessary facilities to
carry out the above study.
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