Transcript Diagnosis of fish diseases

Diagnosis of Fish Diseases
Principles of disease diagnosis
• Diagnosis of the aetiological agent is the most
important aspect of the health management
of any animal against any of the infectious
diseases.
• Identification of causative agent of infection
as well to identify subclinical infections if any.
Investigation of the possible disease
problems
• Level I - changes in feeding or swimming
behaviour followed by appropriate sampling
and analysis
• Level II - traditional diagnostic techniques like
histopathology, bacteriology, virology and
water analysis
• Level III - Advanced techniques like PCR using
molecular biological methods for fast and
confirmatory diagnosis of infections
Case history and clinical sign in diagnosis
Case history would give a presumptive idea about the nature
of problem that caused the infection.
Look for clinical signs indicative of diseased condition
Clinical signs not giving direct indication of aetiology go for
confirmatory diagnosis using various diagnostic tests.
Behavioural changes in
fish
Reduced or stop feeding
Viral, bacterial,
parasitic and
environmental
Lethargic swimming
Viral, bacterial,
parasitic, fungal and
water quality
Spinning, erratic
swimming
Viral, parasitic or
toxicants.
Normally found external signs in fish
and causes
• Gills necrotic – Bacterial, parasitic, fungal
• Gills with excess mucus – Bacterial, parasitic,
environmental or nutritional
• Gills pale – Viral, bacterial, nutritional
• Skin with excess mucus – Parasitic, environmental
• Red pigmented areas in skin – Bacterial or parasitic
• Dark skin pigmentation – Viral, bacterial, nutritional,
eye parasite
• Haemorrhage, erythraemia – viral, bacterial,
parasitic, toxicants
Contd…..
• Frayed, eroded, erythraemia in fins – Bacterial,
Parasite, mechanical/Physiological disorder
• Exophthalmia, haemorrhaged opaque eyes –
Viral, Bacterial, parasitic, gas super saturation
• Ulceration, necrotisation – Bacterial, parasitic
• Hydropsy – Bacterial, viral, metazoan parasite.
• Enlarged abdomen (fluid accumulation) – Viral,
bacterial, parasitic
• Growth, nodules, raised ativite spot on skin –
Viral, parasitic neoplasmic, fungal.
Clinical signs observed in shrimps
• Major behavioural changes that can be observed
in shrimp are:
• reduced escape reflexes
• abnormal swimming near pond edges or at the
surfaces associated with lethargy
• unusual aggregations
• reduced preening activity and
• increased feed consumption followed by
anorexia.
Morphological abnormalities observed
in shrimps
• surface fouling of appendages and cuticle for
filamentous bacteria and protozoans.
• oral region for bacterial "plaques"
(presumptive for Vibrio spp.).
• larval mycosis - diagnosed by demonstration
of hyphae, discharge tube, and motile
zoospores.
Contd…..
• muscle atrophy in abdomen, especially in 6th
abdominal segment (muscle should fill half of
space available).
• hepatopancreas atrophy, vacuolisation, lipid
droplets and BP inclusion bodies.
• cuticular deformities: bent or broken setae or
spines
• melanized appendages tips or foci.
Histological techniques
• Histology:- Microscopic examination of thin,
stained tissue sections in order to study their
structure and function.
• determine changes that occur in the tissues due
to pathogens and disease
• Histopathology can be used as diagnostic tool for
identifying a variety of pathological conditions
associated with many of the disease conditions.
• Immunohistochemical methods are also used to
detect specific pathogens in tissue sections.
Direct microscopical observation of
the pathogen in the tissue sample
• Fresh preparations are made from different
fish tissues and observed microscopically
• Many of the parasites are detected primarily
by their movement.
• The preparations of skin scrapings and gill are
examined under the microscope and
examined for the presence of for parasites.
Direct culture of the infectious
agents and identification
• Aseptically collected samples of tissues are
used for the isolation and culture of
aetiological agents such as bacteria, fungi or
viruses using appropriate culture techniques.
• Using standard biophysical characteristics,
biochemical tests, immunological or nucleic
acid based diagnostic techniques these
pathogen can be identified.
Immunodiagnostic techniques
• Make use of the basic principle of the
specificity of antigen and Antibody
• Simplest form –agglutination test – for
particulate antigens- bacterial identification
• Improved techniques like ELISA are now
available
NATURE OF ANTIGEN-ANTIBODY
REACTIONS
• A. Lock and Key Concept
Fab portion - constructed from the hypervariable regions of
the heavy and light chains
• B. Non-covalent Bonds
The bonds are non-covalent - hydrogen bonds, electrostatic
bonds, Van der Waals forces and hydrophobic bonds.
• C. Reversibility
Since antigen-antibody reactions occur via non-covalent
bonds, they are reversible.
Factors affecting measurement of
antigen-antibody reactions
• 1. Affinity
• 2. Avidity
• 3. Antigen to antibody
ratio
• 4. Physical form of the
antigen
Slide Agglutination
• Simple form of Ag-Ab reaction
• Microscopic slides
• rapid and convenient method for
determining the presence of
agglutinating antibodies.
• Positive - granulation or flocculation
Precipitation
• when the antigen is soluble
• when antigen and antibody are mixed under correct
conditions
• quantitative precipitation is done in a series of test
tubes.
• tubes where most precipitate appears contain
optimal proportions of antigen and antibody.
Immunofluorescence
• For localisation of antigen at tissue level
• Fluorescent compounds covalently attached to antibody
molecules without impairing the biological activity
• fluorescein isothyocyanate (FITC) forms covalent bonds with
free amino groups on antibody molecules
• antibody molecules highly fluorescent - can be seen with
fluorescent microscope
• used to identify antigens.
• direct and the indirect methods
Direct and indirect methods
• direct method - fluorescein tagged antibodies contact with
antigens in a smear on a slide
• Ag bind the tagged antibody
• slide is examined with a fluorescent microscope.
• indirect method - for detecting specific antibodies or
identifying antigens
• A secondary labeled antibody for detecting primary
antibodies
• Examine through microscope
Immunoperoxidase test
Glucagon α cells
Insulin β cells
Haemagglutination assays
• Erythrocytes- microparticulate-agglutinate with antibodies
against the antigenic determinants on them.
• Passive haemagglutination- when erythrocytes are carrier
particles for antigens.
• Commonly used sheep erythrocytes (SRBC)-treat the SRBC
with tannic acid.
• Antigen coating on SRBC.
• Antigen coated SRBC will be made to react with the antibody
which will lead to agglutination.
Haemagglutination
Passive haemagglutination
Detection of bacterial toxins
Latex agglutination test
• Polystyrene latex particles sensitized with antibody
(antiserum) raised against the toxin
• Agglutination done with the test sample and the latex
particles sensitized with antiserum
• Positive reaction –clumps of agglutinated latex particles settle
giving irregular appearance
Negative reaction- latex particles settle as a button
• Best example – Test kits for cholera toxin
– Heat labile enterotoxin of E. coli
Gel diffusion techniques
• Ouchterlony(1949) for soluble antigens
• Soluble antigen and antibody are allowed to diffuse
passively in a solid agar gel medium
• When the reactants meet at the optimal
concentration, visible insoluble complex is formed
due to precipitation
• Simple rapid and needs only small volumes of
reactants
Radial immunodiffusion
• The antibody or mono/poly-specific antiserum
incorporated into the agarose, and the antigen is applied
into the well made inside the agarose (Mancini et
al,1965)
• Antigen diffuses into the gel radially around the well and
a precipitin ring is formed around the well when the
concentration is optimum
• Reaction can be done with antigen in the gel and
antibody in the well- but less sensitive.
Immunoelectrophoresis
• Combines the principle of electrophoresis and
immunoprecipitation
• Partial separation of mixed antigens
• Negatively charged molecules migrate rapidly in an antibody
gel.
• Can be used for serum immunoglobulin identification
• Bottleneck:- Large volume of soluble antigen is required
Immunoelectrophoresis
Counter current electrophoresis
• Active migration of antibody and antigen in an agar
medium under the influence of an electrical field.
• the antigens with higher negative charge move towards
the anode and meet the antibodies moving towards the
cathode
• Facilitate precipitation
Counter current electrophoresis
Enzyme linked immunosorbent Assay
(ELISA)
• Detection of antigen or antibody
• Chemical conjugation of an enzyme to the primary or
secondary antibody
• Allows the detection of immune complex formed on a solid
surface
• Enzyme will react with its substrate and give rise to a colored
product
• Visualized and measured by optical density
Sandwich Elisa protocol
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Coating with capture antibody
Blocking with BSA
Addition of antigen
Addition of labeled antibody
Addition of substrate
Conversion of chromogen
Addition of stop solution
Reading in ELISA reader at 450nm
Nucleic acid based rapid
diagnostics
• Recent advances in molecular biology has
revolutionised the diagnostic capabilities
against many infectious agents.
• The most efficiently used nucleic acid based
techniques at present include PCR
(Polymerase Chain Reaction) and DNA probes.
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