2.9. Plasma total immunoglobulin assay
This assay was similar to the previous one. 50 µl plasma and 50 µl
polyethylene glycol (PEG) was added to each well of a 96-well microtiter plate.
After 2 h of incubation at room temperature, plates were centrifuged at 1000 G
for 15 min. The protein content of the supernatant was determined by the assay
described above. This value was subtracted from the total protein level, and the
result was equal to the total immunoglobulin concentration of the plasma.
2.10. Challenge tests with A. hydrophila
At the end of the experiment, a challenge test was performed on each
experimental group with A. hydrophila. B2/12 strain of A. hydrophila, which
had originally been isolated in Bangladesh, was used for the test. This strain was
chosen because in our preliminary experiments it proved to be the most virulent
of our strains (data not shown). A. hydrophila strains were obtained from
Bacteriology Unit, Institute of Aquaculture, University of Stirling, Stirling,