Fishes, as with other animals, are subjected to a wide spectrum of diseases. Such diseases require a somewhat different approach to solving the problem than terrestrial animals.  All forms of aquaculture are susceptible to outbreaks of disease, as many pathogenic bacteria are normal inhabitants of the aquatic environment. Both in aquaculture facilities and in external aquatic environments, the occurrence of disease is a complex interaction between the host species, disease agents and the environment. 

1. A seminar on :
Use of immunological tools in fish
disease diagnosis
Submittedto, Submitted by,
Prof.T.J.Abraham Md.KaifAli
Prof.G.Dash RollNo.: F/2018/42
Asst.Prof.PrasenjitMali Reg.No.:6441 of2018-19
Dept.OfAAH BFSc.4th yr 2ndsem
FFSc.,WBUAFS
FACULTY OF FISHERY
SCIENCES
SRP-
424

2. INTRODUCTION
• Fishes, as with other animals, are subjected to a wide spectrum of diseases. Such diseases require a
somewhat different approach to solving the problem than terrestrial animals.
• All forms of aquaculture are susceptible to outbreaks of disease, as many pathogenic bacteria are normal
inhabitants of the aquatic environment. Both in aquaculture facilities and in external aquatic environments,
the occurrence of disease is a complex interaction between the host species, disease agents and the
environment.
• Traditionally, the diagnosis of infectious diseases has been accomplished by the isolation of the infecting
microorganism in pure culture. Classical methods of microbial isolation and identification have been
invaluable in the study of bacterial, viral and fungal infections. However, cultivation systems offer
disadvantages for the rapid diagnosis of infectious diseases. For example, many microorganisms, especially
viruses and slow growing bacteria, require a considerable period of time in cultivation that the results from
cultures are often not available at a time when the result can alter the course of therapy. Thus, more
sensitive means must be applied for detecting and identifying a wide range of infectious diseases of fish and
shrimps.

3. Fig. Aquaculture disease
diagram, indicating the
main factors for the
evaluation of pathogen,
and host-pathogen
interactions intervening in
fish disease outbreaks

4. Disease diagnosis
• To properly diagnose pathology in
aquaculture, we must consider
disease as a problem with multiple
levels of increasing biological
complexity, ranging from
environmental to the cell, genome
and proteome level.
• Immunological and molecular
biology-based techniques are
rapidly advancing the field of
diagnostics in fish and shrimp
diseases. Fig. Disease diagnosis concentric ring, representing layers of
disease diagnoses as environment, community, organism,
tissue, and omics as a tool to interpret cell/tissue responses.

5. IMMUNOLOGICAL
TECHNIQUE
• Immunodiagnostic tests use an antigen-antibody reaction to
detect and identify a specific antigen or antibody associated
with a disease-causing organism. The antigen-antibody
reaction itself is very specific. Consequently, if the correct
antibodies can be obtained, immunodiagnostic techniques
have the advantage of being able to identify the presence of a
specific pathogen directly in the 138 Health Management in
Aquaculture specimens and also can be used to detect the
specific antibodies produced as a result of the immune
response of the host to the organism. The primary component
in the test is the antibody. The specificity, and to some extent
the sensitivity, of the assay depends on the quality of the
antibodies used in the reagents.
• The immunodiagnostics have great prospects in fish-disease
diagnosis since they can specifically detect different microbial
diseases with great sensitivity. The advantage of these
methods over other conventional tests is that they can
differentiate between closely related strains of same species
of bacteria or virus. This is an important aspect in case of fish
pathogen, as many of the bacteria are normal inhabitant of
aquatic bodies with only some strains being pathogenic to the
fish.

6. Immunological tools used in
fish disease diagnosis
1. Agglutination test
2. Immunodiffusion test
3. Immunoelectrophoresis
4. Counter immuno electrophoresis (CIE)
5. Rocket immuno electrophoresis
6. Fluorescent Antibody Technique (FAT)
7. Enzyme-linked immunosorbent assay (ELISA)
8. Radioimmune assay
9. Western blotting

7. 1. Agglutination
test
• Agglutination reactions are among the most
easily performed of immunological tests.
• The test is based on visible
clumping (agglutination) of a
particulate antigen with antibody when
the two tests reagents are mixed together on
a glass slide.
• The test is mostly used for diagnosis of
bacterial diseases in fish.
• Agglutination has provided valuable
information on the serological relation of
bacterial fish pathogens, including species
within genera and strains of the same species.

8. i. Slide
agglutination
• In this test, 5% suspension of killed bacteria is mixed with serum samples on a clean glass slide and
kept for 5 min undisturbed. In positive case floccules or granules (agglutination) appear (Fig.).
• This is a very simple, quick and easy test and can be well adopted/accepted in field practices.
• Now attempts have been made to colour the bacterial antigen using a suitable dye such as cotton
blue or India ink, which help the proper visibility of the test and better shelf life of the antigen at
room temperature.
Fig. Agglutination of bacteria by specific antibodies where
bacterial cells have been cross-linked by the tetrameric antibodies

9. ii. Haemagglutination
test
• The test detects the antigens and their antibodies, which do not have the inherent capacity to
agglutinate red blood cells. In this test, the antigen (bacterial proteins/viral material) or their
antibodies are coated on to the RBC and used to react with the counterpart.
• In positive case, agglutination is observed and the test is performed as serial two-fold dilution of
antigen or antibody to which 1% coated RBC suspension is added. The titre is calculated as the
reciprocal of the highest dilution showing complete agglutination (Fig.).
Fig. Agglutination of antigen coated RBC with tetrameric antibodies

10. iii. Tube agglutination
test
• The test is done in tubes or ‘U’ shaped microtitre plates (Fig.). An amount of bacteria is suspended in
saline so that a standard density is obtained.
• In this test the serum is diluted serially to which a standard quantity of antigen suspension is added.
• The test is incubated for 18-24 h at 37°C. If agglutination occurs, clumps will gravitate to the bottom
of the tube and give a mat like appearance and the upper part of the liquid becomes clear.
• If the result is negative the antigens remain uniformly turbid or settle as a clump.
Fig. Agglutination of bacterial suspension by antibody solution.
Agglutination does not occur both in prozone and postzone.

11. Application in fish
diseases diagnosis
• Agglutination test has been
widely used in detecting
bacterial fish pathogens
belonging to the genera Vibrio,
Pasteurella, Aeromonas,
Yersinia, Edwardsiella and
Pseudomonas.

12. 2. Immunodiffusion
test
• The test is otherwise known as agar
gel precipitation test (AGPT), which is
very simple to perform, more specific
and less sensitive. The wells are cut in
1 % agarose gel prepared in PBS (pH
7.2-8.0), antibody (serum) and antigen
are placed in separate wells, which
diffuse towards each other and form
visible bands of precipitate. This is the
most widely applied test for diagnosis
of animal diseases and can very well
be used to diagnose the fish microbial
diseases in field conditions.
• The test can either be done as double
radial immunodiffusion (Oucterlony’s
methods) or as single radial
immunodiffusion (Mancin‘s technique)
test.

13. i. Single radial
immunodiffusion
• Using this method, it is possible to quantitate the
concentration of antibody and antigen in a
solution. The antigen diffuses in a radial direction
out from the well and a precipitin ring
develops when the reactants are close to their
optimal proportions (Figure). At the equivalence
point, when the ring is stationary, the square of
the diameter or area of the ring is directly
proportional to antigen concentration. Conversely,
though less sensitive, antigen can be mixed into
the agar and the amount of antibody in a sample
can be determined. Consequently, it is possible
to calibrate the plate using a pre-determined
constant amount of antibody (or antigen) in the
agar and placing known concentrations of antigen
(or antibody), or sample dilutions in the wells. By
calibrating the method, such radial
immunodiffusion is used to measure IgG, IgM,
complement components, and other substances
in the serum.
Fig. A representation of a single radial immunodiffusion
measurement. Typical rings obtained with positive
rabbit serum and no ring with phosphate buffered saline
(PBS) as negative control.

14. Application in fish
diseases diagnosis
• The method has been used frequently in
fish immunological studies and then only
for estimation of the
immunoglobulin concentrations in both
normal and immune serum.
• The concentration of immunoglobulins
produced in response to injection with
Salmonella bacterial and/or red blood cell
(RBC) antigens have been measured in
catfish.
• Serum IgM levels have been measured
in rainbow trout naturally infected with
VHS virus and ERM bacteria.

15. ii. Double radial immunodiffusion
• In the gel diffusion technique, gels,
usually clarified agar are used as
matrices for combining diffusion
with precipitation. Antigen and
antibody are placed in different wells
in agar and allowed to diffuse
towards each other (passive diffusion)
and precipitation results where the
optimal antibody/ antigen ratios have
been reached (Figure ). This method
(Ouchterlony) can be used either to
detect the number of major
components in an antigenic mixture
or identify the presence of
homologous and heterologous
molecules in an antigenic extract
based on the specific recognition
capacity of a prepared antiserum.
Fig. Double diffusion precipitation reactions (Ouchterlony) in gel.
As – antiserum in wells; A, B, C, D – antigens in wells; A and B –
reaction of identity; B and C reaction of nonidentity; C and D –
reaction of partial identity (cross-reaction; the “spur” is caused by
the fraction of antibody that was not precipitated by antigen D).

16. Application in fish
diseases diagnosis
• The method has been employed
frequently to determine the serological
relationships between fish bacterial
strains on the basis of lipopolysaccharide
types, between Vibrio bacteria isolated
from other marine teleosts, in the
identification of extracellular Vibrio toxins
pathogenic to eels and ayu and
for comparison of antigenicity
of Edwardsiella tarda after injection into
Eels.
• It was also used to diagnose BKD and
to determine the serological differences
of Photobacterium damsela subsp. piscici
da isolates .

17. 3.
Immunoelectrophoresis
• This technique is a widely used method in
fish immunology and combines electrophoresis
with immunoprecipitation. It has a much better
resolution than gel diffusion.
• In this system, the components of the antigen will
first be separated by electrophoresis. Separation of
the components occurs due to different
electrophoretic mobilities caused by charges on the
molecules. After the antigen has been separated into
its components, antiserum will be put into a channel
cut parallel to the direction of the electrophoresis.
From this channel, the antibodies will
diffuse toward the electrophoretically separated
antigen components, and vice versa. As the antigen
and antibody diffuse toward each other, they form a
series of arcs of precipitate (Figure). This permits the
serum proteins to be characterized in terms of their
presence, absence, or unusual pattern.
• The separated components are then visualized on
the plate by precipitin band formation due to the
diffusion of a specific antiserum into the agar parallel
to the current direction.
Fig. Schematic representation of the
immunoelectrophoresis technique.

18. Applications in fish
diseases diagnosis
• The immunoelectrophoresis
test was used to identify IPN
virus in cell culture and
determine the serological
characteristics of a typical
strains of Edwardsiella tarda
isolated from sea breams.

19. 4.
Counter immune
electrophoresis
(CIE)
• CIE is a form of crossover
electrophoresis. In alkaline pH, the
antigen is charged negative and moves
towards the anode (+) and the antibody
diffuses the way known as
electroendosmosis in the natural
medium such as agarose when electric
current is passed in the field.
• This property is utilized to enable both
reagents to move simultaneously from
their positions and form a line of
antigen-antibody reaction.
• This test is preferred to the agar gel
precipitation test because of the result is
obtained within an hour.

20. 5. Rocket
immune
electrophoresis
• In this test, the antigen is allowed to
migrate in an electric field into an
antibody- containing gel. The
antigen combines immediately with
the antibody to form immune
complexes. The complexes continue
migration at a slower rate. The
migration is stopped when the
aggregates become large enough to
be retained by the pores of the gel
and the area under precipitate takes
a rocket-like shape.
• The height of the rocket shows a
linear proportionality with the
concentration of the antigen (Fig.).
Fig. Rocket immune electrophoresis

21. 6. Fluorescent
antibody
technique (FAT)
• This technique used fluorescein dye tagged
antibody to detect specific pathogen inside
the tissue itself. The fish tissue suspected to
contain a particular pathogen is processed to
make sections for microscope. This is then
treated with antibody-fluorescein conjugate
and the slide is examined under the ultraviolet
light microscope. The parts of the tissue
(containing antigen) that bind the fluorescein-
tagged antibody fluoresce under
the fluorescence microscope. This can be used
to detect a particular microbial pathogen, as
well as, to know the part of tissue being
attacked or damaged by the pathogen.
• There are two variations in this test such as
direct and indirect methods. In direct method,
antibody is conjugated to the fluorescent dye,
and in indirect way, antiglobulin raised against
the globulin of a specific species is conjugated
with the dye.

22. Application in fish
diseases diagnosis
• Immunofluorescence is widely used for the
detection of antigen or antibody in fish,
typing and identification of a range of
microorganisms.
• FAT has been used to detect antibodies
to Aeromonas liquifaciens in fish .
• Rapid FAT diagnosis has been developed to
detect Pseudotuberculosis in
yellowtail, Renibacterium salmoninarum in
salmonids and Vibrio penaeicida in kuruma
prawn.
• FAT has also been developed for rapid
diagnosis of infectious hematopoeitic necr
osis virus (IHNV) and red sea bream
iridovirus in fish.
Fig. Indirect fluorescent antibody technique
(IFAT) staining of an impression smear prepared
from Vibrio penaeicida cell suspension

23. 7.Enzyme linked
immunosorbent assay (ELISA)
• The test is otherwise known as enzyme immunoassay (EIA). This is
one of the most sensitive and widely accepted method to detect
and quantify the antigen/antibody in the infected and suspected fish
tissues and blood sera. The test is applied when other conventional
tests fail to detect.
• Since it’s invention by Engvall and Perlman in 1971, the test is being
widely used and even is a day-to-day test for diagnosis of diseases of
human beings, animals, birds and fishes.
• In this test, the samples suspected to contain antigen are added to
wells of the Polystyrene plates. After an appropriate time, the wells
are washed and blocked with bovine serum albumin/skim milk
powder solution and treated with specific antibodies that has been
Conjugated with an enzyme. After allowing to bind, the content of
the wells are rinsed with a substrate for the enzyme and the
reaction is stopped by addition of a stopper. The assay is read by a
colour development in the plate.

24. Types of
ELISA test
• Dot-ELISA or paper ELISA: Where an antigen/antibody is adsorbed to the
nitrocellulose paper instead of plastic plates and other steps are followed
as above. The color reaction appear in the form of a dot on
the membrane.
• Indirect ELISA: This is done to quantify the antibodies in the sera of
infected and vaccinated animals. The unknown antibodies bound to the
coated known antigen are detected by addition of anti-species antibodies
conjugated with enzyme.
• Sandwich ELISA: The test is one where an antibody is coated to the plate
and treated as trapping antibody, to which suspected antigen is added
followed by addition of a second antibody raised in other species and
known as detector antibody. The detector antibody is allowed to bind
with anti-species antibody conjugated with enzyme.
• Competitive ELISA: This is done to detect and quantify the antigen or
antibody. For antigen detection in infected or suspected animal, the
standard antibody (known) bound to the polystyrene plate is allowed to
react with enzyme labelled standard antigen (known) and plain
(unknown) antigens, simultaneously. The competition between known
and unknown antigen is detected by change in color reaction.

25. Different
types of
ELISA test

26. Application in fish
diseases diagnosis
• ELISA test has been used to
detect Aeromonas salmonicida in
fish tissue, clinical cases of enteric red
mouth and furunculosis in fish
farms, Vibrio
parahaemolyticus and V. harveyi in pen
aeid shrimp.
• ELISA has also been developed for rapid
detection of
viral haemorrhagic septicaemia virus
and striped jack nervous necrosis virus
in fish.

27. 8. Radioimmunoassay
(RIA)
• In radioimmune assay, the label is a radioactive
element, commonly I125​.
• The method is exquisitely sensitive and enables to
detect the viral and bacterial antigens, fungal toxins
and protein hormones at very low concentration .
• The test is performed as ELISA except
that no substrate is added here and the binding
of radio-labelled antigen/antibody to its counterpart is
detected and measured in gamma scintillation
counter.

28. 9. Western
Blotting
• Western blotting is a rapid and sensitive assay
for the detection and characterization of
proteins. The technique allows one to identify
particular proteins by utilizing the specificity
inherent in antigen-antibody recognition.
• This technique is powerful, since it combines
electrophoretic separation of proteins,
glycoproteins and lipopolysaccharides with
immunological identification. Once such
antigens have been detected, they can be
further characterized by Western blotting.
• The technique is useful for a number of
purposes including characterization of
unknown antigens or antibody specificities,
confirmation of the presence of bacterial
antigens in sera or tissues and detection of
seropositive individuals which have been
exposed to a pathogen.

29. Process of Western Blotting
• Initially, a sample is subjected to
electrophoresis to separate antigens
according to their charge and size, or size
alone. A second electrophoretic step
transfers the antigens from the gel to an
immobilizing surface, such as nitrocellulose
paper where they are bound irreversibly
(Figure). After this transfer, the paper is
blocked with 3% gelatin in PBS to prevent
nonspecific binding of anti-body and probed
with a specific enzyme-conjugated antibody
(horseradish peroxidase-anti-
immunoglobulin conjugate). A chromogenic
substrate is then added to determine which
electrophoretic band is bound by the
antibody.
Fig. Western blotting. Organization of materials inside the
gel cassette holder for transfer of proteins from the gel to
nitrocellulose paper.

30. Advantages
and
Disadvantages
• Advantages :
 The primary advantage of Western
blotting, as opposed to other
immunoassays, is the high degree of
specificity in resolving distinct antigens.
 The technique can be used to detect as
little as 1 ng of a protein antigen.
• Disadvantages:
i. first, it is mainly a qualitative assay and
quantification of antibody or antigen is
difficult; and
ii. second, if the antigen sample must be
denatured (such as in SDS-PAGE),
antigenic activity may be reduced or
destroyed.

31. Application in fish diseases
diagnosis
• Western blotting has been useful for
characterizing the specificities of
polyclonal antisera (rabbit and salmonid)
and monoclonal antibodies to extracellular
and cell surface
antigens of Renibacterium salmoninarum.
• It has also been used in the detection of
yellowhead virus and white spot syndrome
virus in penaeid shrimp.

32. Conclusion
Nearly, all the immunodiagnostics techniques are based on either the detection of an
antigen with antibody of known specificity or detection of an antibody with a known
antigen. The antigen and antibody molecules are complementary antibody with a
known antigen. The antigen and antibody molecules are complementary to each
other when mixed, the reaction being manifested either by the precipitation or by
the agglutination etc.
The immunodiagnostics have great prospects in fish-disease diagnosis since they can
specifically detect different microbial diseases with great sensitivity. The advantage
of these methods over other conventional tests is that they can differentiate
between closely related strains of same species of bacteria or virus. This is an
important aspect in case of fish pathogen, as many of the bacteria are normal
inhabitant of aquatic bodies with only some strains being pathogenic to the fish.
Conventional diagnostic methods that involve the culture of microorganisms can take
days or weeks to complete or very tedious to perform. Immunodiagnostics offers a
rapid, very sensitive, very specific and simple alternative. Further developments in
immunodiagnostics and emerging technologies such as DNA-based tests will
revolutionize the detection and identification of infectious disease agents.

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• Class notes

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36. Thank you