Biofilms be the best alternative to combat

Biofilms are structured communities of
bacterial cells that are enclosed in a self-produced polymeric matrix and
adhere to biotic and abiotic surfaces, an interface or each other. Biofilm
formation commences when planktonic bacteria attaches irreversibly to a
surface. Free cell (FC) oral vaccines give poor and inconsistent immune
response and protection in fish mainly due to their destruction by enzymes in
stomach or foregut before reaching the immune responsive lymphoid organs.
Biofilms (BF) are known for their resistant nature to antibiotics, antibodies
and phagocytic cells due to a protective glycocalyx layer. This paper is
written to illustrate the biofilm oral vaccine which is believed to protect the
antigens against gastric destruction and found highly encouraging results
(antibody titre and protection upon challenge) in herbivore carps, omnivore
catfish (Clarias batrachus), carnivore
fish (Channa striatus) and tiger  shrimp (Penaeus



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Aquaculture is a steadily growing industry
world over. The number species and new technologies are increasing in
aquaculture industry for rising production. Intensification has come up as a
boon to meet the increasing food demand. However, diseases are stumbling block,
causing huge economic loss in millions each year world over. To control
bacterial and parasitic diseases, antibiotics and drugs were used
indiscriminately. Antibiotic has already raised lots of criticism over its
negative impact on living biota may lead to antibiotic-resistant pathogen. In this
present scenario, vaccination would be the best alternative to combat bacterial
and viral disease for the sustainable aquaculture.A1 
In this prospect, vaccinations against the bacterial and viral diseases play a
vital role in finfish and shellfish aquafarming.

of oral vaccines in aquaculture

Depending on the age and size of the fish,
commercial vaccines are administered either orally (by mixing with the feed),
by immersion (dip or bath) or by injection through the intraperitoneal (i.p.)
or intramuscular (i.m.) route (Embregts and Forlenza, 2016). Vaccine by
injection route although very effective in terms of immune response and long
term protection but it has some side effects including tissue inflammation,
adhesion and necrosis. On the other immersion vaccination required high
production costs.  Oral vaccines are an
attractive alternative to reduce the stress on the fish during immunization
(Gudding et. al., 1999). Due to the
ease, simplicity and practical applicability oral vaccination became the choice
of antigen delivery. However, attempts to orally vaccinate against different
bacterial diseases have either yielded mild and short lived or inadequate
responses. One of the important factors for the inconsistency and poor response
to oral vaccination is the digestive degradation of antigens in the foregut,
before the vaccine reaches immune-responsive areas in the hind-gut and other
lymphoid organs (Johnson and Amend, 1983; Rombout et. al., 1985).

for biofilm oral vaccines in aquaculture

Strategies evaluated for prevention of
vaccine destruction in gut, such as delivering vaccines in encapsulated antigen
microspheres (EAM), enteric coated vaccine and bioencapsulation of vaccine in
live feed which complex, costly and not practical. In this respect biofilm
vaccines would be the best alternative way for oral route of vaccination.
Biofilm cell produce adhesive exopolymeric substance, which is called
glycocalyx, offer protection to antigen from gastric destruction

Designing of biofilm-based vaccines

To mimic in vivo conditions, in
vitro bacteria have to be grown in liquid media by providing surface (such
as bentonite clay, microsperes, chitin flakes, etc.) depleting nutrients and
adding iron chelators and growing for longer periods. This leads to slow growth
of the organisms forming biofilms, which can express some novel proteins and
ensure maximum EPS production.

Efficacy of biofilm vaccines in aquaculture

Over the 20 years, the vaccine research has
been oriented towards safer and more effective vaccine preparation from a
normal inactivated bacterial vaccine to the biofilm vaccine (Azad, 1997). Various researches
conducted in the preparation of biofilm of Aeromonas
hydrophila and Vibrio alginolyticus
and its use as oral vaccine under the direct supervision of renowned Indian
scientist and ICAR (Indian Council of Agriculture Research) Emeritus Professor
Dr. K.M Shankar at the Laboratory of Aquatic Health Management, College of
Fisheries, Mangalore have shown promising outcome in teleost fishes like catla,
rohu, common carp (Azad, 1997,
1999, 2000), catfishes like C. batrachus
(Nayak et. al., 2004), C. striatus (Siriyappagouder et. al., 2014) and also from crustaceans
tiger shrimp (P. monodon) (Krupesh et. al., 2010).


The hypothesis was
proposed and substantiated first by Azad et al., (1997, 1999). They promoted a virulent A. hydrophila isolate to form biofilm on chitin flakes and
successfully utilized it as oral vaccine in catla, rohu and common carp.
Biofilm vaccinated carps had significantly higher antibody titre and protection
than free cell vaccinated fishes. Advantage 
of biofilm and free cell vaccines when studied by antigen localization
employing monoclonal antibody based immunefluorescence, indicated that biofilm
antigen compared to free cell antigen retained for longer time in larger
quantities in gut and other lymphoid organs like kidney and spleen (Azad et al., 2000). The protective nature of
glycocalx was believed to be protecting biofilm from gastric hydrolysis as free
cells showed a quicker clearance from lumen of hindgut within 6h of vaccine
uptake compared to biofilm, which remained for 48h following oral delivery. In
addition to that, biofilm might represent a more immunogenic material as seen
to have induced higher antibody and protective immunity (Azad et al., 1997, 1999) altogether making it
as a better vaccine carrier in oral immunization regime, keeping this
background biofilm of A.hydrophila was evaluated for
oral vaccination of walking catfish (Clarius batrachus) by Nayak et al., 2004. Serum agglutinating antibody titre and relative
percent survival (RPS) following challenge were found to be significantly
higher in catfish fed with BF vaccine
compared to that with free cell vaccine. Another study by Siriyappagouder et al., 2014, with C.
striatus, a carnivorous fish model, fed with biofilm (BF) and free cell
(FC) of A. hydrophila with the same
dose and duration of Nayak,
2004. They observed BF vaccinated fish upon challenge with A. hydrophila at 109 cfu/ml had significantly higher
relative per cent survival (88) than that with FC (29.6).  Krupesh
et al., (2010) used biofilm of V. alginolyticus to study preliminary
immune response in tiger shrimp (Penaeus
monodon) and the study showed that biofilm cells were superior to free cell
in stimulating the non-specific immune response of Penaeus monodon.


Biofilm cells probed to be effective as oral
vaccination of fin and shellfishes. It can be considered as simple, cheap and
ideal oral vaccination technique for bulk administration. Production of Biofilm
vaccines and its efficacy has been proven in institutional research experiments
and it is expected that the biofilm vaccine will available in the market by
2018 as commercial production was undertaken by the aqua drug company to
address the infectious diseases of Indian aquaculture.