542
(Antipa et al. 1980 ), which is consequently favoured. Indeed, many articles have
been published about the benefi t of immersion vaccination (Håstein et al. 1980 ;
Amend and Johnson 1981 ; Giorgetti et al. 1981 ; Horne et al. 1982 ; Johnson et al.
1982a , b ; Kawai and Kusuda 1995 ) and the longer, i.e. 2 h, ‘bath’ technique (Egidius
and Andersen 1979 ).
A further refi nement involves use of low-pressure sprays, which are easy to use,
and apparently economic in the quantity of vaccine administered (Gould et al.
1978 ). The success was illustrated by 0 % mortalities in a group of fi sh sprayvaccinated compared to 80 % mortalities among unvaccinated controls after challenge (Gould et al. 1978 ).
All of the aforementioned methods enable fi sh to develop an immune response to
the pathogen. This aspect has been discussed comprehensively, as regards chinook
salmon, by Fryer et al. ( 1972 ). It is thought that the maximum agglutination titre is
in the region of 1:8192, depending on the fi sh species used (Groberg 1982 ). The
development of immunity is clearly a function of water temperature, and generally
humoral antibodies are formed more rapidly at high rather than low temperatures.
For example, in coho salmon, humoral antibodies appeared in 25 days and 10 days
at water temperatures of 6 °C and 18 °C, respectively (Groberg 1982 ). The poor
relative performance of orally administered vaccines has been partially attributed to
an inability of the fi sh to develop humoral antibodies (Fryer et al. 1978 ; Gould et al.
1978 ; Kusuda et al. 1978b ; Groberg 1982 ). However, the role of these antibodies in
protection against disease is unclear.
Inevitably, V. anguillarum has been the target of modern approaches for vaccine
development. For example, Japanese fl ounder were protected (RPS = 85.7 %) after
4- weeks against challenge with V. anguillarum by using a DNA vaccine comprising
a mutated zinc metalloprotease gene (m- EmpA ) applied as a 50 μg dose by i.m.
injection. Protection was reduced by lowering the dose to 5 (RPS = 57.1 %) and
20 μg (RPS = 71.4 %) (Yang et al. 2009 ). Zhao et al. ( 2011 ) used the GAPDH gene
gapA to express Aer. hydrophila GAPDH in an attenuated V. anguillarum strain,
which was injected i.p. (10
6 CFU/fi sh) into turbot and challenged after 4-weeks.
Challenge of cytoplasm GAPDH expressing strain AV/pUC-gapA-vaccinated fi sh
with V. anguillarum led to an RPS value of 92 %. A plasmid-based antigen expression system has been developed and considered to have potential for future vaccine
development programmes (Xiao et al. 2011 ).
The potential of fl agella genes FlaA, FlaB, FlaD , and FlaE were evaluated in
Japanese fl ounder ( Paralichthys olivaceus ). Thus, recombinant FlaA, FlaB, FlaD,
and FlaE were expressed in Esch. coli, purifi ed and injected intraperitoneally into
Japanese fl ounder. The result was that recombinant FlaB led to a signifi cantly higher
RPS and serum antibody titre than the other three proteins (Jia et al. 2013 ).
Pseudomonas P1SW and Vibrio V3SW were recovered from seawater and
administered live separately or together by immersion and orally (the bacteria were
embedded in sodium alginate microspheres) to turbot ( Scophthalmus maximus ) at
doses equivalent to 2 × 10
8 CFU with evidence that both organisms became distributed in the internal organs, and some protection became apparent after challenge.
10 Vibrios
(Antipa et al. 1980 ), which is consequently favoured. Indeed, many articles have
been published about the benefi t of immersion vaccination (Håstein et al. 1980 ;
Amend and Johnson 1981 ; Giorgetti et al. 1981 ; Horne et al. 1982 ; Johnson et al.
1982a , b ; Kawai and Kusuda 1995 ) and the longer, i.e. 2 h, ‘bath’ technique (Egidius
and Andersen 1979 ).
A further refi nement involves use of low-pressure sprays, which are easy to use,
and apparently economic in the quantity of vaccine administered (Gould et al.
1978 ). The success was illustrated by 0 % mortalities in a group of fi sh sprayvaccinated compared to 80 % mortalities among unvaccinated controls after challenge (Gould et al. 1978 ).
All of the aforementioned methods enable fi sh to develop an immune response to
the pathogen. This aspect has been discussed comprehensively, as regards chinook
salmon, by Fryer et al. ( 1972 ). It is thought that the maximum agglutination titre is
in the region of 1:8192, depending on the fi sh species used (Groberg 1982 ). The
development of immunity is clearly a function of water temperature, and generally
humoral antibodies are formed more rapidly at high rather than low temperatures.
For example, in coho salmon, humoral antibodies appeared in 25 days and 10 days
at water temperatures of 6 °C and 18 °C, respectively (Groberg 1982 ). The poor
relative performance of orally administered vaccines has been partially attributed to
an inability of the fi sh to develop humoral antibodies (Fryer et al. 1978 ; Gould et al.
1978 ; Kusuda et al. 1978b ; Groberg 1982 ). However, the role of these antibodies in
protection against disease is unclear.
Inevitably, V. anguillarum has been the target of modern approaches for vaccine
development. For example, Japanese fl ounder were protected (RPS = 85.7 %) after
4- weeks against challenge with V. anguillarum by using a DNA vaccine comprising
a mutated zinc metalloprotease gene (m- EmpA ) applied as a 50 μg dose by i.m.
injection. Protection was reduced by lowering the dose to 5 (RPS = 57.1 %) and
20 μg (RPS = 71.4 %) (Yang et al. 2009 ). Zhao et al. ( 2011 ) used the GAPDH gene
gapA to express Aer. hydrophila GAPDH in an attenuated V. anguillarum strain,
which was injected i.p. (10
6 CFU/fi sh) into turbot and challenged after 4-weeks.
Challenge of cytoplasm GAPDH expressing strain AV/pUC-gapA-vaccinated fi sh
with V. anguillarum led to an RPS value of 92 %. A plasmid-based antigen expression system has been developed and considered to have potential for future vaccine
development programmes (Xiao et al. 2011 ).
The potential of fl agella genes FlaA, FlaB, FlaD , and FlaE were evaluated in
Japanese fl ounder ( Paralichthys olivaceus ). Thus, recombinant FlaA, FlaB, FlaD,
and FlaE were expressed in Esch. coli, purifi ed and injected intraperitoneally into
Japanese fl ounder. The result was that recombinant FlaB led to a signifi cantly higher
RPS and serum antibody titre than the other three proteins (Jia et al. 2013 ).
Pseudomonas P1SW and Vibrio V3SW were recovered from seawater and
administered live separately or together by immersion and orally (the bacteria were
embedded in sodium alginate microspheres) to turbot ( Scophthalmus maximus ) at
doses equivalent to 2 × 10
8 CFU with evidence that both organisms became distributed in the internal organs, and some protection became apparent after challenge.
10 Vibrios