670
ratio of 10:1 and incubated at 25 °C for 3 h. An equal volume of a boiled bacterial
suspension and the sensitised staphylococci are mixed on a glass slide. Following
incubation in a moist chamber at room temperature for up to 2 h, a positive response
is indicated by clumping of the cells. The advantages of this technique concern its
simplicity and reliability. Moreover, it was considered suitable for deployment in
fi eld conditions.
• Passive agglutination
For rough colonies of Aer. salmonicida , which were unsuitable for use with
whole cell agglutination [because of auto-agglutination], McCarthy and Rawle
( 1975 ) recommended the mini-passive agglutination test. This technique involves
the use of sheep erythrocytes sensitised with Aer. salmonicida O-antigen (extracted
with hot physiological saline). This reacts with dilute anti- Aer. salmonicida immune
serum, assuming that the antigen is present. The obvious advantages of this method
concern its application to the detection of both rough and smooth colonies. However,
McCarthy and Rawle ( 1975 ) cautioned that false negative results may sometimes be
obtained with cultures that have been maintained in laboratory conditions for prolonged periods. Hence, old cultures may not be suitable for use in serological studies (or, for that matter, vaccine production!).
• Immuno-India Ink technique (Geck)
Another rapid technique, which allows diagnosis within 15 min, is the India ink
immunostaining reaction as developed initially by Geck ( 1971 ). This is a microscopic technique, in which the precise mode of action is unknown, although Geck
suggested that it could be regarded as an immuno-adsorption method. The technique has been described only for use with Aer. salmonicida (McCarthy and
Whitehead 1977 ). A drop of bacterial suspension is smeared into a clean (de-fatted)
microscope slide, air-dried, and heat-fi xed. The smear is covered with a 1:1 mixture
of India ink and antiserum, before incubation in a moist chamber for 10 min at room
temperature. Subsequently, the mixture is removed by washing with ferric chloride
(0.00001 % w/v), and the slide air-dried prior to microscopic examination. A positive result is indicated by the presence of cells, clearly outlined with India ink.
• Enzyme-linked immunosorbent assay (ELISA)
This is a technique which is becoming widely adopted for the detection and diagnosis of bacterial fi sh pathogens, some commercial kits having been developed.
This is a useful technique, which has already gained widespread use in human and
veterinary medicine. Essentially, there is a requirement for a specifi c antiserum, an
enzyme, e.g. alkaline phosphatase or horseradish peroxidase, and a substrate, e.g.
o -phenylenediamine (for use with alkaline phosphatase) (See Austin et al. 1986 ). A
positive result is indicated by a colour change, which may be recorded quantitatively with a specially designed reader. Variations of the technique have been published, and include indirect ELISA, indirect blocking ELISA and competitive
ELISA (e.g. Swain and Nayak 2003 ).
13 Diagnosis
ratio of 10:1 and incubated at 25 °C for 3 h. An equal volume of a boiled bacterial
suspension and the sensitised staphylococci are mixed on a glass slide. Following
incubation in a moist chamber at room temperature for up to 2 h, a positive response
is indicated by clumping of the cells. The advantages of this technique concern its
simplicity and reliability. Moreover, it was considered suitable for deployment in
fi eld conditions.
• Passive agglutination
For rough colonies of Aer. salmonicida , which were unsuitable for use with
whole cell agglutination [because of auto-agglutination], McCarthy and Rawle
( 1975 ) recommended the mini-passive agglutination test. This technique involves
the use of sheep erythrocytes sensitised with Aer. salmonicida O-antigen (extracted
with hot physiological saline). This reacts with dilute anti- Aer. salmonicida immune
serum, assuming that the antigen is present. The obvious advantages of this method
concern its application to the detection of both rough and smooth colonies. However,
McCarthy and Rawle ( 1975 ) cautioned that false negative results may sometimes be
obtained with cultures that have been maintained in laboratory conditions for prolonged periods. Hence, old cultures may not be suitable for use in serological studies (or, for that matter, vaccine production!).
• Immuno-India Ink technique (Geck)
Another rapid technique, which allows diagnosis within 15 min, is the India ink
immunostaining reaction as developed initially by Geck ( 1971 ). This is a microscopic technique, in which the precise mode of action is unknown, although Geck
suggested that it could be regarded as an immuno-adsorption method. The technique has been described only for use with Aer. salmonicida (McCarthy and
Whitehead 1977 ). A drop of bacterial suspension is smeared into a clean (de-fatted)
microscope slide, air-dried, and heat-fi xed. The smear is covered with a 1:1 mixture
of India ink and antiserum, before incubation in a moist chamber for 10 min at room
temperature. Subsequently, the mixture is removed by washing with ferric chloride
(0.00001 % w/v), and the slide air-dried prior to microscopic examination. A positive result is indicated by the presence of cells, clearly outlined with India ink.
• Enzyme-linked immunosorbent assay (ELISA)
This is a technique which is becoming widely adopted for the detection and diagnosis of bacterial fi sh pathogens, some commercial kits having been developed.
This is a useful technique, which has already gained widespread use in human and
veterinary medicine. Essentially, there is a requirement for a specifi c antiserum, an
enzyme, e.g. alkaline phosphatase or horseradish peroxidase, and a substrate, e.g.
o -phenylenediamine (for use with alkaline phosphatase) (See Austin et al. 1986 ). A
positive result is indicated by a colour change, which may be recorded quantitatively with a specially designed reader. Variations of the technique have been published, and include indirect ELISA, indirect blocking ELISA and competitive
ELISA (e.g. Swain and Nayak 2003 ).
13 Diagnosis