671
• Dot blotting
Monospecifi c antibodies have been in used in dot blotting techniques to record
the presence of specifi c pathogens. Dot blotting involved spotting 1 μl quantities of
bacterial samples onto nitrocellulose membranes, baking at 60 °C for 10 min, incubating in a 1:200 solution of 5 % blotto (5 % non fat dry milk, 0.1 % Triton X-100 in
phosphate buffered saline [PBS]) for 5 h, washing in blotto, incubating in a 1:500
dilution of horseradish peroxidase goat anti-mouse gamma immunoglobulin for 3 h,
re-washing in 0.5 % blotto, and incubation for 5 min in substrate (0.03 % diaminobenzidine, 0.006 % hydrogen peroxide, and 0.05 % cobalt chloride in PBS)
(Longyant et al. 2010 ).
Immunohistochemistry
Immunohistochemistry, based on avidin-biotin complexes, have identifi ed V. salmonicida in fi xed tissues (Evensen et al. 1991 ). Also, a peroxidase-antiperoxidase
immunohistochemical technique appears to be useful for differentiating Y. ruckeri
(Jansson et al. 1991 ). Complications with the interpretation of slides by interference
with melanin and/or melanomacrophages resulted in the adoption of melaninbleaching with 3.0 g/l potassium permanganate for 20 min followed by 1 % (w/v)
oxalic acid for 1.5 min before immunostaining, which removed some of the problems of interpreting the presence/absence of Ren. salmoninarum (Morris et al.
2002 ).
• Immunomagnetic separation of antigens
A comparatively novel approach concerns the recovery of Aer. salmonicida cells
with immunomagnetic beads coated with monoclonal antibodies to LPS coupled
with culturing (Nese and Enger 1993 ). Thus, the cells are recovered by serological
procedures for culturing on routine bacteriological media.
Molecular Techniques
Molecular methods have become the norm in many diagnostic laboratories, and
have gained the reputation for sensitivity and specifi city. Thus, PCRs have been
used with increasing regularity for the detection of bacterial pathogens both to
determine the presence of single organisms and to recognize and discriminate
between multiple taxa within the same genus, and across different genera. An example of the recognition of three species from within the same genus is the triplex
loop-mediated isothermal amplifi cation method that detected V. alginolyticus, V.
anguillarum and V. harveyi specifi cally and sensitively in the tissues of
Immunohistochemistry
• Dot blotting
Monospecifi c antibodies have been in used in dot blotting techniques to record
the presence of specifi c pathogens. Dot blotting involved spotting 1 μl quantities of
bacterial samples onto nitrocellulose membranes, baking at 60 °C for 10 min, incubating in a 1:200 solution of 5 % blotto (5 % non fat dry milk, 0.1 % Triton X-100 in
phosphate buffered saline [PBS]) for 5 h, washing in blotto, incubating in a 1:500
dilution of horseradish peroxidase goat anti-mouse gamma immunoglobulin for 3 h,
re-washing in 0.5 % blotto, and incubation for 5 min in substrate (0.03 % diaminobenzidine, 0.006 % hydrogen peroxide, and 0.05 % cobalt chloride in PBS)
(Longyant et al. 2010 ).
Immunohistochemistry
Immunohistochemistry, based on avidin-biotin complexes, have identifi ed V. salmonicida in fi xed tissues (Evensen et al. 1991 ). Also, a peroxidase-antiperoxidase
immunohistochemical technique appears to be useful for differentiating Y. ruckeri
(Jansson et al. 1991 ). Complications with the interpretation of slides by interference
with melanin and/or melanomacrophages resulted in the adoption of melaninbleaching with 3.0 g/l potassium permanganate for 20 min followed by 1 % (w/v)
oxalic acid for 1.5 min before immunostaining, which removed some of the problems of interpreting the presence/absence of Ren. salmoninarum (Morris et al.
2002 ).
• Immunomagnetic separation of antigens
A comparatively novel approach concerns the recovery of Aer. salmonicida cells
with immunomagnetic beads coated with monoclonal antibodies to LPS coupled
with culturing (Nese and Enger 1993 ). Thus, the cells are recovered by serological
procedures for culturing on routine bacteriological media.
Molecular Techniques
Molecular methods have become the norm in many diagnostic laboratories, and
have gained the reputation for sensitivity and specifi city. Thus, PCRs have been
used with increasing regularity for the detection of bacterial pathogens both to
determine the presence of single organisms and to recognize and discriminate
between multiple taxa within the same genus, and across different genera. An example of the recognition of three species from within the same genus is the triplex
loop-mediated isothermal amplifi cation method that detected V. alginolyticus, V.
anguillarum and V. harveyi specifi cally and sensitively in the tissues of
Immunohistochemistry