This document discusses various techniques for identifying bacterial and viral pathogens. It covers microscopic, histological, microbiological, immunological, and molecular techniques. Microscopic techniques include basic staining methods to identify organisms under a microscope. Histological techniques examine stained tissue sections to detect pathogens. Microbiological techniques involve culturing samples and conducting biochemical tests on isolates. Immunological techniques like ELISA, agglutination, and immunofluorescence use antigen-antibody reactions for detection. Molecular techniques like PCR, multiplex PCR, and DNA microarrays allow sensitive detection and differentiation of pathogens through nucleic acid analysis. After detection, antibiotics are usually administered but alternative remedies like photoinactivation of pathogens using blue light and bacteri

1. Techniques for
Identification of
Bacterial and
Viral Pathogens
Ambica Bora | BBT1503

2. Classification
MICROSCOPIC HISTOLOGICAL MICROBIOLOGICAL
IMMUNOLOGICAL MOLECULAR
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3. MICROSCOPIC
• To tell whether it is a water quality or parasitic problem (basic).
• Quick method but accuracy depends on skill and equipment.
• Generally stains are used in case of bacteria, but wet mounts
of unstained samples can also be used for the detection of
fungi, parasites(helminth eggs and larvae) and motile
organisms.
• Type of stain used depends on the most likely pathogens and
no stain is 100% specific, hence not all organisms are visible
using these stains.
• Require minimum microbe concentration of 105/ml.
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4. MICROSCOPIC
• Stains generally used
1. Gram stain
2. Acid-fast stain (mycobacterium sp.),
3. Fluorescent stains (detection at lower conc.)
4. Giemsa stain (blood parasites)
5. Iron- haematoxylin stain (intestinal protozoa)
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5. HISTOLOGICAL
• Examination of thin stained tissue sections in order to study the
changes in normal structure and function by pathogens.
• Method used to detect specific pathogens in tissue culture.
• Stained tissue sections are prepared and observed under light
microscope.
• H&E (Haematoxylin and Eosin) is the most widely used histological
stain due to its ability to reveal wide range of tissue components.
Other stains used are Gram’s stain and Periodic Acid-Schiff (PAS)
stain.
• Immuno-histochemical staining methods have been developed for the
detection of viruses such as infectious pancreatic necrosis virus (IPNV),
infectious salmon anemia virus (ISAV) and nodavirus in paraffin-
embedded tissue sections.
• Viral antigen is localized by an antibody raised against the virus and
subsequent detection steps result in a colored product that can be
visualized by light microscopy
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6. 6
pictures showing how samples are made
for histological study from paraffin sections

7. MICROBIOLOGICAL
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• Sample taken aseptically from the fish are first grown in non-
selective media after which the various microbial techniques are
applied.
• Microbial techniques used:
1. Motility test ( hanging drop, semi solid agar, 3 cover slip)
2. Culturing in selective and differential media
3. Biochemical tests
http://www2.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferential.htm
http://aquafind.com/articles/FishDiseaseDiagnosis.php

8. 8

9. 3. Biochemical tests
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1. Indole 6. Coagulase 11. MRVP
2. TSI 7. Catalase 12. Casease
3. Lipase 8. Nitrate reduction 13. Gelatinase
4. Urease 9. Citrate 14. Starch hydrolysis
5. Oxidase 10.Phenol red broth 15. Decarboxylation
http://aquafind.com/articles/FishDiseaseDiagnosis.php

10. 10
UREASE TEST
TSI TEST
NITRATE TEST

11. IMMUNOLOGICAL
• Antigen- antibody interactions form the basis of this method.
• It can detect sub-clinical/latent/carrier sate of infection and can also
discriminate the antigenic differences. This technique is relatively
rapid and more specific and sensitive.
• The antibody-based test selected for the identification of pathogens
depends on a variety of factors since each method has its merits and
disadvantages.
• This method is useful for the detection of pathogens in pure culture
or/and in infected fish tissue, its sensitivity thresholds limit use in
environmental samples, especially where pathogen levels are
extremely low.
• The antibody-based tests include agglutination, latex agglutination,
immunodiffusion, fluorescent antibody tests, immunohistochemistry
(IHC) and enzyme linked immunosorbent assay (ELISA) and western
blot (WB)
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12. • Agglutination: used to identify unknown antigens. Blood with the
unknown antigen is mixed with a known antibody and whether or not
agglutination occurs helps to identify the antigen; used in tissue
matching and blood grouping and diagnose. Can be either passive or
direct. In case of latex agglutination, the process is carried out on a latex
surface, with antibodies coated on it for body fluids.
• Fluorescent antibody test (FAT): It is a laboratory test that
uses antibodies tagged with fluorescent dye that can be used to detect
the presence of microorganisms. This method offers straight-forward
detection of antigens using fluorescently labeled antigen-specific
antibodies. Because detection of the antigen in a substrate of patient
sample (cellular smear, fluid or patient- inoculated culture medium) is
the goal, this method is seldom quantitative.
• Immunodiffusion : refers to the movement of antigen or antibody or
both antigen and antibody molecules in a support medium by diffusion.
The antigen and antibody bind with each other and forms insoluble
immuno- precipitate, which is visible to naked eye as precipitin band or
line. J. Oudin described a system of single diffusion of antigen and
antibody in agar-filled tubes.
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13. • ELISA(Enzyme linked Immunosorbent Assay):In this technique the
antibody molecules are linked to enzymes either directly or indirectly. In
the direct method the enzyme is conjugated to a portion of the anitibody
molecule that does not bind to the antigen (“back end method”). In the
indirect method a carrier or secondary antibody is linked with an
enzyme, which binds to the primary antibody after it binds to the antigen.
The amount of enzyme is important in producing measurable signal
when the primary antibody binds to its target.
• One of the important uses of this technology in the field of diagnostics
involves application of antibodies to tissue sections on microscope
slides. This permits the antigen for pinpointing within a tissue using a
normal light microscope. In addition to its diagnostic applications, the
technique also helps in studying how the pathogen spreads within the
organism and causes disease.
• ELISA detects specific substances in a complex mixture by binding them
to antigen or antibody coated substances. It is also capable of detecting
viruses, bacteria, drugs, hormones, toxins, carcinogens, depending on
the nature of ELISA.
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14. Direct method
14

15. 15
INDIRECT METHOD

16. Sandwich method
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(1) Plate is coated with a capture antibody
(2) sample is added, and any antigen present binds to capture antibody
(3) detecting antibody is added, and binds to antigen
(4) enzyme-linked secondary antibody is added,
and binds to detecting antibody
5) substrate is added, and is converted by enzyme to detectable form.

17. • A working principle example for quantifying the amount of unknown antigen:
1. ELISAs are performed in 96-well plates which permits high throughput results. The bottom of
each well is coated with a protein to which will bind the antibody you want to measure.
2. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with
antibodies (called primary antibodies). The serum is incubated in a well, and each well
contains a different serum.
3. A positive control serum and a negative control serum would be included among the 96
samples being tested. After some time, the serum is removed and weakly adherent antibodies
are washed off with a series of buffer rinses.
4. To detect the bound antibodies, a secondary antibody is added to each well. The secondary
antibody would bind to all human antibodies and is typically produced in a rodent. Attached to
the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These
enzymes can metabolize colorless substrates (sometimes called chromagens) into colored
products.
5. After an incubation period, the secondary antibody solution is removed and loosely adherent
ones are washed off as before. The final step is the addition the enzyme substrate and the
production of colored product in wells with secondary antibodies bound.
6. When the enzyme reaction is complete, the entire plate is placed into a spectrophotometer
and the optical density (the amount of colored product) is determined for each well. The
amount of color produced is proportional to the amount of primary antibody bound to the
proteins on the bottom of the wells.
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18. 18
http://eprints.cmfri.org.in/8585/1/Lipton.pdf

19. • Dot-ELISA: the antigen is sandwiched between the primary immobilized
antibody and the secondary enzyme linked antibody. Both react with
different epitopes on the same antigen. The principle is same as that of
normal ELISA but the antigen concentration here is measured on the
bases of intensity of chromogenic dots.
 Applications of ELISA: this method is versatile, simple, fast, and can
quantify the target pathogen. Highly specific assays can be developed
using monoclonal antibodies(most of the time) that recognize a specific
epitope of the pathogen. However, to detect the different strains of a
given virus, for example, polyclonal antibodies that target multiple
epitopes of the pathogen are needed.
 Major limitations for the development of serological assays include that
the required antiserum for detection of a pathogen be accessible and
affordable and that the required degree of sensitivity and specificity
is often difficult to reach
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20. MOLECULAR
 Advantages
• Apart from the sensitivity and rapidity of diagnosis, principal advantage
of molecular diagnostic methods is in the detection of non-culturable
agents
• DNA amplification can assist in detecting the pathogens that are
present in low numbers and also in handling a tiny volume of specimen
• Can be used to detect latent infection and thereby identify the reservoir
hosts of infection that is significant in epizootiology
• Can be used to differentiate antigenically similar pathogens.
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21.  Disadvantages
• These methods are cost-intensive procedures
• These tests cannot detect unsuspected samples, Molecular methods will have
difficulty in detecting new pathogens as the exclusive use of these would overlook
such infections.
• Various substances such as hemoglobin, bacterial constituents, and high
concentrations of no target DNA can inhibit amplification.
• Positive results provide little quantitative assessment of the infection level, and
do not indicate whether the pathogen is replicating or causing disease in the
species tested. Thus, carrier status and viability of the pathogen are not
determined using DNA-probes.
• The extremely high specificity of these tests, coupled with the ability of many
viruses to rapidly change in genetic structure, can result in failure to detect a virus
that has altered its genetic profile.
• PCR methodologies are highly susceptible to contamination. Contamination
during processing may result in false positives, particularly in 2-step PCR
methods. PCR tests must be conducted in very well managed, clean laboratories.
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22. The various molecular methods used are
1. PCR
2. Multiplex PCR
3. Labeling and detection of nucleic acids
4. Restriction enzyme digest
a) RLFP (Restriction Fragment Length Polymorphism)
b)RAPD (Random Amplified Polymorphic DNA)
c)AFLP (Amplified Fragment Length Polymorphism)
5. In-Situ Hybridization
6. LAMP (Loop-mediated Isothermal amplification)
7. DNA Microarrays
22
http://aquafind.com/articles/FishDiseaseDiagnosis.php

23. 1. Polymerase Chain Reaction:
• Polymerase chain reaction is a technique for amplifying a specific region of
DNA, defined by a set of two "primers" at which DNA synthesis is initiated by
a thermostable DNA polymerase.
• The first cycle is characterized by a product of indeterminate length;
however, the second cycle produces the discrete "short product" which
accumulates exponentially with each successive round of amplification.
• The reaction includes template DNA that may be in various forms, from a
simple tissue lysate to purified DNA, primers, polymerase enzyme to
catalyze creation of new copies of DNA, and nucleotides to form the new
copies.
• Usually, at least a million-fold increase of a specific section of a DNA
molecule can be realized and the PCR product can be detected by gel
electrophoresis. The regions amplified are usually between 150-3,000 base
pairs (bp) in length.
23
http://www.trjfas.org/uploads/pdf_220.pdf

24. 24
Diagrams explaining
Polymerase chain reaction

25. 2. Multiplex PCR: A spinoff of PCR which allows the detection of
multiple pathogens at a time, improving cost efficiency. In multiplex
PCR more than one target sequence can be amplified by
including more than one pair of primers in the reaction. In the
field of infectious diseases, the technique has been shown to be a
valuable method for identification of viruses, bacteria, fungi and
parasites and is also applied in many areas of nucleic acid
diagnostics, including gene deletion analysis quantitative analysis
and RNA detection.
3. Labeling and detection of Nucleic acids: These include labeling
with a variety of haptens such as biotin or digoxygenin and detection
by antibody binding coupled with fluorescent, chemiluminescent or
colorimetric detection methods. The variety of labels and detection
methods now available can provide a system suitable for any
application, from dot blots to in situ hybridization.
25

26. 4. Restriction Enzyme Digest :
• Restriction enzymes (or restriction endonucleases) recognize short
sequences of DNA and cleave DNA in that very specific site. Type II
restriction enzymes, most commonly used for DNA analysis and genetic
engineering, each have a unique nucleotide sequence at which it cuts a
DNA molecule.
• A particular restriction enzyme will cleave DNA at that only recognition
sequence that is often a six base pair palindromic sequence, but others
recognize four or even eight base pair sequences.
• However all these techniques require PCR for functioning in an efficient
manner.
• It includes RFLP, AFLP-PCR and RAPD-PCR
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27. • RFLP: the DNA sample is broken into pieces (and digested)
by restriction enzymes and the resulting restriction fragments are
separated according to their lengths by gel electrophoresis. One of the
first methods of DNA profiling, redundant now due to the long time it
takes.
• AFLP-PCR: For AFLP analysis, only a small amount of purified
genomic DNA is needed; this is digested with two restriction enzymes,
one with an average cutting frequency (like EcoRI) and a second one
with a higher cutting frequency (TaqI). Ised for comparing closely
related organisms.
• RAPD-PCR: RAPD uses a single primer in low-stringency polymerase
chain reactions. Random binding of primers results in different sizes of
fragments from samples with nonidentical DNA. However problems with
reproducibility and risks of contamination render the method unsuitable
as a stand-alone method of diagnosis. RAPD can be a useful technique
as a first step in the development of specific primers or probes and has
been used in such a way in the study of bacteria.
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28. 6. LAMP- Loop Mediated Isothermal Amplification- a novel nucleic acid
amplification method that amplifies DNA with high specificity, efficiency
and rapidity under isothermal conditions. When combined with reverse
transcription, this method can also amplify RNA sequences with high
efficiency(RT-PCR).
7. DNA Microarray- (also commonly known as DNA chip or biochip) is a
collection of microscopic DNA spots attached to a solid surface.
Scientists use DNA microarrays to measure the expression levels of
large numbers of genes simultaneously or to genotype multiple
regions of a genome.
28
https://en.wikipedia.org/wiki/DNA_microarray

29. Methods of
Identification
Microscopic Histological Microbiological
Motility
Selective
culturing
Biochemical
tests
Immunological
ELISA
Agglutination
Immunodiffusion
FAT
Molecular
PCR
Multiplex
PCR
Labeling
Restriction
enzymes
DNA
Microarray
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30. REMEDIES
AFTER
DETECTION

31. After detection and diagnosis of the pathogen, antibiotics are usually
given. Continuous application of antibiotics over the years has lead to
the development of antibiotic resistance in fishes. Hence alternative
methods have been sought for the same. There are 2 novel methods that
have become prevalent over the years:
1. Photoinactivation of major bacterial pathogens
2. Bacteriophage remediation of bacterial pathogens in
aquaculture
31

32. 1. Photoinactivation of major bacterial pathogens
• It was successfully demonstrate inactivation activity of a 405/465-
nm LED on selected bacterial pathogens.
• The 405-nm light had a bactericidal effect against all seven
pathogens, indicating that blue light can be effective without the
addition of a photosensitizer.
• Photobacterium damselae, Vibrio anguillarum, and Edwardsiella
tarda were the most susceptible to the 405-nm light (36.1, 41.2, and
68.4 J cm−2, respectively, produced one log reduction in the
bacterial populations), whereas Streptococcus parauberis was the
least susceptible (153.8 J cm−2 per one log reduction).
• In general, optical density (OD) values indicated that higher
bacterial densities were associated with lower inactivating efficacy,
with the exception of P. damselae and Vibrio harveyi. In conclusion,
growth of the bacterial fish and shellfish pathogens evaluated in this
study was inactivated by exposure to either the 405- or 465-nm
light. In addition, inactivation was dependent on exposure time.
32https://fas.biomedcentral.com/articles/10.1186/s41240-016-0029-5

33. 2. Bacteriophage remediation of bacterial
pathogens in aquaculture
• A long list of bacteria can lead to opportunistic infections of fish and
shellfish. Vaccination methods have been applied in some fish species with varying
levels of success. Reductions in losses have most often been achieved with antibiotic
treatment; however, long-term antibiotic usage has led to antibiotic resistant bacterial
strains and increasing ineffectiveness of such treatments. Although antibiotics are
commonly used (overused) in many countries, there is a need to move away from
antibiotics to more natural, probiotic treatments.One such treatment involves the use of
bacteriophages (phages) to reduce morbidity and mortalities in various aquaculture
settings.
• Phages are naturally-occurring bacterial viruses which infect specific species or strains
of bacteria. There are 2 general types of phages, lytic and lysogenic. Lytic phages infect
host bacteria through a process involving attachment of the phage to the bacterium;
insertion of the phage genome into the host cell; cessation in the synthesis of host
components; host mediated replication of phage components including capsid proteins
and nucleic acids; assembly of new phage particles; lysis of the host; and release of
progeny phages. Since lytic phages replicate quickly and rapidly cause death and lysis
of the host, they are ideal for the development of phage therapies for use in treating
animal infections and in reducing pathogens in various foods and the environment.
33https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4590005/

34. Imunostimulents
a method to prevent diseases
• Immunostimulants are substances which elicit non-specific defense
mechanisms and enhance the barrier of infections against
pathogens. They are isolated from natural sources and then
synthesized chemically. Cell wall preparations from bacteria, fungi,
mushrooms and yeast are rich sources of immunostimulants. They
exist in various structural forms. The possible use of
immunostimulants in managing diseases on fish and shellfish have
been initiated recently.
• Some of the common immunostimulants include: i. Glucans, ii.
Lectins, iii. Lipopolysaccharides (LPS) from bacterial cell wall and iv.
Wheat germ agglutinins
• The immunostimulants have several advantages: i. Being natural
products, there is no environmental hazard and ii. Unlike vaccines,
which gives ptotection to a specific pathogen, immunostimulants
provide a wide range of protection against several pathogens.
34

35. CONCLUSION
There are many techniques for the detection of pathogens in fishes with
the molecular methods gaining tandem over the years. However the
necessary part is, irrespective of the technique used to detect the disease
as fast as possible so that treatment can begin, in order to reduce the
future.
With the increasing cases of antibiotic resistance, alternative methods for
the remedial action to be taken have come up over the years which
ensure that there is lesser probability of the after efects.
35