180
(Schachte 1978 ; Acuigrup 1980 ; Lamers and de Haas 1983 ; Ruangpan et al. 1986 ;
Rahman and Kawai 2000 ; Chandran et al. 2002 ; da Silva et al. 2013 ; Das et al.
2013a ; Sen et al. 2014 ; Sirimanapong et al. 2014 ; Zhang et al. 2014a , b , c ) with the
host response including the production of superoxide anion by the head-kidney leucocytes (Basheera John et al. 2002 ). For example, use of ECPs achieved an RPS of
100 % in catfi sh, two weeks after vaccination (Zhang et al. 2014a , b , c ). Schachte
( 1978 ) recorded that the most convincing immune response, measured in terms of
antibody titre, was achieved after using injection techniques. A recombinant bivalent expressed OMP of V. vulnifi cus and Aer. hydrophila was used to vaccinate via
the i.p. route American eel ( Anguilla rostrata ) with challenge after 28-days leading
to RPS values of 50 % for both pathogens (Guo et al. 2015 ). Using formalised whole
cells applied by i.p. injection, Ruangpan et al. ( 1986 ) recorded complete protection
in Nile tilapia within only two weeks. Some protection, i.e. 53–61 %, occurred only
one week after vaccination. The importance of dose was highlighted by Dash et al.
( 2011 ), who administered 0.2 ml amounts of formalin-inactivated cells intraperitoneally, and determined that one month later the highest dose of 10
10 CFU/ml led to
the highest antibody titre and greatest protection after challenge. The lower doses of
10
7 and 10
5 CFU/ml gave correspondingly less antibody and protection (Dash et al.
2011 ). Incorporating purifi ed 43 kDa OMP of Aer. hydrophila in FCA and a booster
3 weeks later (without FCA) led to a demonstrable immune response and protection
in blue gourami (Trichogaster trichopterus) (Fang et al. 2000 ). The next most promising method of application was immersion vaccination, and thence oral methods of
administration, which were used successfully by Yasumoto et al. ( 2006 ), who
entrapped Aer. hydrophila antigens (protein concentration = 33 μg/ml) in liposomes.
The vaccine was fed to carp at doses of 30 μl/fi sh/day over 3 days leading to detectable humoral antibodies after 2 and 3 weeks (there was a decline in titre at 4 weeks)
and protection (at 22 days) after subcutaneous injection with Aer. hydrophila at
3.0 × 10
5 (RPS = 63.6 %) or 1.0 × 10
6 (RPS = 55 %) CFU/fi sh (Yasumoto et al. 2006 ).
OMPs with or without FCA were administered i.p. to rohu, and challenged after
2-months leading to RPS of 79–88 % (Das et al. 2013a , b ). Encapsulation of OMP
in biodegradable nanoparticles and administered by i.p. injection to rohu heightened
the immune response and improved protection against challenge (Rauta and Nayak
2015 ). A recombinant OMP with a modifi ed adjuvant was proposed as vaccine for
rohu, and was effi cacious in experiments both in terms of survival and immunostimulation, notably an increase in lysozyme and myeloperoxidase activities, serum
natural haemolysin titre and antibody titre, and upregulation of immune-related
genes, i.e. complement factor 4, IgM, interleukin-1 ß, lysozyme G, ß 2-microglobulin, and major histocompatibility complex I and II (Dash et al. 2014 ). The penaeidin
3–2 (these are antimicrobial peptides found in penaeids) gene was transformed into
rice using Agrobacterium . The transgenic rice bran was fed to tilapia, which were
protected against challenge (Liu et al. 2014 ). A recombinant haemolysin co-regulated protein, which is an important component of T6SS, was expressed, and administered to common carp leading to improved survival after challenge (survival = 46.67 %)
compared to the controls (survival = 7.14 %) (Wang et al. 2015a , b ).
4 Aeromonadaceae Representatives (Motile Aeromonads)
(Schachte 1978 ; Acuigrup 1980 ; Lamers and de Haas 1983 ; Ruangpan et al. 1986 ;
Rahman and Kawai 2000 ; Chandran et al. 2002 ; da Silva et al. 2013 ; Das et al.
2013a ; Sen et al. 2014 ; Sirimanapong et al. 2014 ; Zhang et al. 2014a , b , c ) with the
host response including the production of superoxide anion by the head-kidney leucocytes (Basheera John et al. 2002 ). For example, use of ECPs achieved an RPS of
100 % in catfi sh, two weeks after vaccination (Zhang et al. 2014a , b , c ). Schachte
( 1978 ) recorded that the most convincing immune response, measured in terms of
antibody titre, was achieved after using injection techniques. A recombinant bivalent expressed OMP of V. vulnifi cus and Aer. hydrophila was used to vaccinate via
the i.p. route American eel ( Anguilla rostrata ) with challenge after 28-days leading
to RPS values of 50 % for both pathogens (Guo et al. 2015 ). Using formalised whole
cells applied by i.p. injection, Ruangpan et al. ( 1986 ) recorded complete protection
in Nile tilapia within only two weeks. Some protection, i.e. 53–61 %, occurred only
one week after vaccination. The importance of dose was highlighted by Dash et al.
( 2011 ), who administered 0.2 ml amounts of formalin-inactivated cells intraperitoneally, and determined that one month later the highest dose of 10
10 CFU/ml led to
the highest antibody titre and greatest protection after challenge. The lower doses of
10
7 and 10
5 CFU/ml gave correspondingly less antibody and protection (Dash et al.
2011 ). Incorporating purifi ed 43 kDa OMP of Aer. hydrophila in FCA and a booster
3 weeks later (without FCA) led to a demonstrable immune response and protection
in blue gourami (Trichogaster trichopterus) (Fang et al. 2000 ). The next most promising method of application was immersion vaccination, and thence oral methods of
administration, which were used successfully by Yasumoto et al. ( 2006 ), who
entrapped Aer. hydrophila antigens (protein concentration = 33 μg/ml) in liposomes.
The vaccine was fed to carp at doses of 30 μl/fi sh/day over 3 days leading to detectable humoral antibodies after 2 and 3 weeks (there was a decline in titre at 4 weeks)
and protection (at 22 days) after subcutaneous injection with Aer. hydrophila at
3.0 × 10
5 (RPS = 63.6 %) or 1.0 × 10
6 (RPS = 55 %) CFU/fi sh (Yasumoto et al. 2006 ).
OMPs with or without FCA were administered i.p. to rohu, and challenged after
2-months leading to RPS of 79–88 % (Das et al. 2013a , b ). Encapsulation of OMP
in biodegradable nanoparticles and administered by i.p. injection to rohu heightened
the immune response and improved protection against challenge (Rauta and Nayak
2015 ). A recombinant OMP with a modifi ed adjuvant was proposed as vaccine for
rohu, and was effi cacious in experiments both in terms of survival and immunostimulation, notably an increase in lysozyme and myeloperoxidase activities, serum
natural haemolysin titre and antibody titre, and upregulation of immune-related
genes, i.e. complement factor 4, IgM, interleukin-1 ß, lysozyme G, ß 2-microglobulin, and major histocompatibility complex I and II (Dash et al. 2014 ). The penaeidin
3–2 (these are antimicrobial peptides found in penaeids) gene was transformed into
rice using Agrobacterium . The transgenic rice bran was fed to tilapia, which were
protected against challenge (Liu et al. 2014 ). A recombinant haemolysin co-regulated protein, which is an important component of T6SS, was expressed, and administered to common carp leading to improved survival after challenge (survival = 46.67 %)
compared to the controls (survival = 7.14 %) (Wang et al. 2015a , b ).
4 Aeromonadaceae Representatives (Motile Aeromonads)