text:
Tools Used in Fish Disease Diagnosis
Arun Sudhagar S, Ezhil Nilavan S, Linga Prabu D,
Rathi Bhuvaneswari G, Chandrasekar S and Rajesh kumar R.
Central Institute of Fisheries Education,
Email: firstname.lastname@example.com
is a label given for a medical condition or disease identified by its
signs, symptoms, and from the results of various diagnostic
procedures. The term "diagnostic criteria" designates the
combination of signs, symptoms, and test results that allows the
clinician to ascertain the diagnosis of the respective disease. It
can be definition as "the recognition of a disease or condition
by its outward signs and symptoms," or "the analysis of the
underlying physiological/biochemical cause(s) of a disease or
condition." Disease diagnosis is refers to the various
procedures and techniques used to identify the nature of disease and
to precisely pinpoint the primary and secondary pathogens involved.
Proper diagnosis leads to accurate therapies and avoid indiscriminate
use of chemotherapeutics. Disease diagnosis is an integral part of
aquatic health management. Proper diagnosis helps to adopt accurate
therapy and avoid indiscriminate use of chemotherapeutics.
Information on case history and clinical signs should be carefully
used while examining samples for diagnosis.
of case history
history information is very vital for proper disease diagnosis and
for taking proper remedial steps. During a disease outbreak, careful
scrutiny of the case history information will help to precisely
pinpoint the circumstances under which the disease has developed.
Some of the case history that are taken in to
account are,
of last Algal bloom
of seed, stocking details
of disease diagnostic methods
disease diagnostic methods can be classified in to following types
Without a microscope it
is simply impossible to tell the difference between a water quality
and a parasite problem. The microscope should be considered the most
basic of tools in fish disease diagnosis. Microscopy can be done
quickly, but accuracy depends on the experience of the microscopist
and quality of equipment. Most specimens are treated with stains that
color pathogens, causing them to stand out from the background,
although wet mounts of unstained samples can be used to detect fungi,
parasites (including helminth eggs and larvae) and motile organisms.
Visibility of fungi can be increased by applying 10% potassium
hydroxide (KOH) to dissolve surrounding tissues and nonfungal
stain is based on the likely pathogens, but no stain is 100%
specific. Most samples are treated with Gram stain and, if
mycobacteria are suspected, an acid-fast stain. However, some
pathogens are not easily visible using these stains; if these
pathogens are suspected, different stains or other identification
methods are required because microscopic detection usually requires a
microbe concentration of about 1 x
The microscopes that
are commonly used in fish disease diagnosis are
Light microscope
Electron microscope
Light microscope
The light microscope is
the most commonly used microscope in disease diagnosis. In this
microscope the scrapings from the fish can be directly visualized or
a squash of organ can be prepared for the examination. It can also be
used for interpreting histological slides and stained microorganisms.
Electron microscope
They are broadly
classified in to two types
Electron Microscopy
They are used for
observing the changes in the tissue at higher magnification.
Scanning Electron
They are used for
observing the surface level changes in the tissues at higher
is a branch of biology that involves the microscopic examination of
thin, stained tissue sections in order to study their structure and
function and, in the case of histopathology, to determine changes
which may be due to pathogens and disease. Histology and have a
central role in disease diagnosis.Stained fish tissue sections are
prepared and examined by light microscopy. Tissue changes resulting
from infectious or non-infectious disease are identified and
described. Immunohistochemical methods are also used to detect
specific pathogens in tissue sections.
sampling, fish tissues are placed in an aqueous fixative. This
fixative preserves the morphology (structure and chemical
constituents) of tissues and cells, so that they are capable of
withstanding further preparatory steps without change. It is
essential that tissues are fixed within a very short time after death
to avoid disintegration of tissues or cells by the action of their
own enzymes. Following fixation, tissues are gradually dehydrated to
remove any tissue water, using a series of graded alcohols. The
tissues are then 'cleared', which involves treatment with a
substance that mixes completely with both the dehydrating fluid and
the embedding agent. Next the tissues are embedded in molten paraffin
wax and cooled to harden the wax so that thin sections can be cut
using a microtome and then mounted onto glass microscope slides. The
wax is removed from the sections before staining.
clearing and rehydration, the tissue sections can be stained using
biological stains or dyes. Haematoxylin and eosin (H&E) is the
most widely used histological stain because of its ability to reveal
a wide range of different tissue components. Gram's stain is a
staining method for differentiating microorganisms. The technique is
based on the capability of bacteria cell walls to retain the crystal
violet dye in the Gram stain during solvent treatment. The cell walls
for Gram positive micro-organisms retain the primary violet as they
have a higher peptidoglycan (sugars) and a lower lipid content than
Gram negative bacteria. The Periodic Acid-Schiff (PAS) reaction is
used to demonstrate certain carbohydrates that are present in some
tissues, and provide identification of infecting fungus in fish
tissues. The PAS positive sites stain pink/red.
staining methods have been developed for the detection of viruses
such as infectious pancreatic necrosis virus (IPNV), infectious
salmon anaemia virus (ISAV) and nodavirus in paraffin-embedded tissue
sections. Viral antigen is localised by an antibody raised against
the virus and subsequent detection steps result in a coloured product
that can be visualised by light microscopy
techniques enable the description of tissue pathology and highlight
the sequence of cellular changes and their progression caused by
infectious and noninfectious diseases. By examining stained sections,
viruses, bacteria, fungi and parasites can be identified, and using
immunohistochemical techniques, certain infectious agents can be
detected in tissue sections. The increased use of image analysis
tools by FRS allows qualitative data to be generated to enhance
disease diagnosis.
Bacterial diseases can
be identified by various microbiology methods. The samples are taken
from the fish under aseptic condition and they are first grown in
non-selective media, then the various microbial diagnostic methods
are used. The different microbiological diagnostic methods are
Staining methods
in selective medium
Biochemical test
Staining method
Various staining
methods are used to identify various bacterial pathogens among them
gram staining is the most important and most commonly used method.
stain: The
Gram stain classifies bacteria according to whether they retain
crystal violet stain (gram-positive — blue) or not
(gram-negative — red) and highlights cell morphology (eg, bacilli,
cocci) and cell arrangement (eg, clumps, chains, diploids). Such
characteristics can direct antibiotic therapy pending definitive
identification. To do a Gram stain, technicians heat-fix specimen
material to a slide and stain it by sequential exposure to Gram's
crystal violet, iodine, decolorizer, and counterstain (typically
and moderate (modified) acid-fast stains:
These stains are used to
identify acid-fast organisms (Mycobacterium
sp) and moderately acid-fast organisms (primarily Nocardia
sp). These stains are also useful for staining Rhodococcus
and related genera, as well as oocysts of some parasites (eg,
stains: These
stains allow detection at lower concentrations (1
cells/mL). Examples are acridine orange (bacteria and fungi),
auramine-rhodamine and auramine O (mycobacteria), and calcofluor
white (fungi, especially dermatophytes). Coupling a fluorescent dye
to an antibody directed at a pathogen (direct or indirect
immunofluorescence) should theoretically increase sensitivity and
specificity. However, these tests are difficult to read and
interpret, and few (eg, Pneumocystis
and Legionella
direct fluorescent antibody tests) are commercially available and
commonly used.
ink (colloidal carbon) stain:
This stain is used to detect
mainly encapsulated fungi in a cell suspension. The background
field, rather than the organism itself, is stained, which makes any
capsule around the organism visible as a halo. Leukocytes may appear
stain and Giemsa stain:
These stains are used for
detection of parasites in blood, phagocytes and tissue cells,
intracellular inclusions formed by viruses and some intracellular
stain (Gomori-Wheatley stain) and iron hematoxylin stain: These
stains are used to detect intestinal protozoa. The Gomori-Wheatley
stain is used to detect microsporidia. It may miss helminth eggs and
larvae. The iron hematoxylin stain differentially stains cells, cell
inclusions, and nuclei. Helminth eggs may stain too dark to permit
Motility test
are three methods to see the motility of the bacteria and they are
Hanging Drop Method
Solid Agar Method (Craigie)
Coverslip Method
Drop Method
Place a small drop of liquid bacterial culture in the
center of a coverslip
Place a small drop of water at each corner of the
Invert a slide with a central depression over the
The coverslip will stick to the slide and when the
slide is inverted the drop of bacterial culture will be suspended in
the well
Examine microscopically (x400) for motile organisms
Note: If well slides are not available, a ring of
Vaseline or plasticine may instead be made on an ordinary microscope
Positive result: A darting, zigzag, tumbling or other
organised movement
Negative result: No movement or Brownian motion only.
Semi Solid Agar Method
Inoculate the test organism - the central glass tube
Incubate at the relevant temperature for 18-24 hours
Subculture from the outer section of the medium
Positive result: Organism can be recovered from the
outer section of the mediumNegative result: Organism remains in
the inner tube
Three coverslip method
Set microscope slide according to Figure 1
Place a small drop of bacterial culture in center of third
Place a small drop of water at each corner of the coverslip.
Invert the prepared microscope slide over the third coverslip.
The coverslip should stick to the other coverslips on the slide and
when inverted the drop of bacterial culture should be suspended in
the gap between the microscope slide and third coverslip.
Examine microscopically (x400) for motile organism.
Positive result: A darting, zigzag, tumbling or other organised
Negative result: No movement or only Brownian motion.
bacteria pathogen in selective medium
bacteria have the capacity to grow in selective media. This property
of bacteria is used to identify the bacterial pathogen. Only
particular kind of bacteria can grow in selective media. Example for
selective medium is TCBS for Vibrios.
biochemical test are used for the identification of bacteria based on
the character of a particular bacteria to give its positive and
negative result for a particular biochemical test.
Indole Test
The test organism is inoculated into tryptone broth, a
rich source of the amino acid tryptophan. Indole positive bacteria
such as Escherichia coli produce tryptophanase, an enzyme that
cleaves tryptophan, producing indole and other products. When Kovac's
reagent (p-dimethylaminobenzaldehyde) is added to a broth with indole
in it, a dark pink color develops. The indole test must be read by 48
hours of incubation because the indole can be further degraded if
prolonged incubation occurs. The acidic pH produced by Escherichia
coli limits its growth.
Methyl Red and Voges-Proskauer Tests
methyl red (MR) and Voges-Proskauer (VP) tests are read from a single
inoculated tube of MR-VP broth. After 24-48 hours of incubation the
MR-VP broth is split into two tubes. One tube is used for the MR
test; the other is used for the VP test. Media contains glucose and
peptone. All enterics oxidize glucose for energy; however the end
products vary depending on bacterial enzymes. Both the MR and VP
tests are used to determine what end products result when the test
organism degrades glucose. E. coli is one of the bacteria that
produces acids, causing the pH to drop below 4.4. When the pH
indicator methyl red is added to this acidic broth it will be cherry
red (a positive MR test). Klebsiella and Enterobacter produce more
neutral products from glucose (e.g. ethyl alcohol, acetyl methyl
carbinol). In this neutral pH the growth of the bacteria is not
inhibited. The bacteria thus begin to attack the peptone in the
broth, causing the pH to rise above 6.2. At this pH, methyl red
indicator is a yellow color (a negative MR test).
reagents used for the VP test are Barritt's A (alpha-napthol) and
Barritt's B (potassium hydroxide). When these reagents are added to a
broth in which acetyl methyl carbinol is present, they turn a
pink-burgundy color (a positive VP test). This color may take 20 to
30 minutes to develop.
is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2.
Hydrogen peroxide is often used as a topical disinfectant in wounds,
and the bubbling that is seen is due to the evolution of O2 gas. H2O2
is a potent oxidizing agent that can wreak havoc in a cell; because
of this, any cell that uses O2 or can live in the presence of O2 must
have a way to get rid of the peroxide. One of those ways is to make
Citrate Test
citrate test utilizes Simmon's citrate media to determine if a
bacterium can grow utilizing citrate as its sole carbon and energy
source. Simmon's media contains bromthymol blue, a pH indicator with
a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's
(around 6), and gradually changes to blue at more alkaline pH's
(around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so
it is an intermediate green color. Growth of bacteria in the media
leads to development of a Prussian blue color (positive citrate).
Enterobacter and Klebsiella are citrate positive while E.coli is
Oxidase Test
The oxidase test identifies organisms that produce the
enzyme cytochrome oxidase. Cytochrome oxidase participates in
the electron transport chain by transferring electrons from a donor
molecule to oxygen. The oxidase reagent contains a chromogenic
reducing agent, which is a compound that changes color when it
becomes oxidized. If the test organism produces cytochrome
oxidase, the oxidase reagent will turn blue or purple within 15
Nitrate Reduction Test
Nitrate broth is used to determine the ability of an
organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme
nitrate reductase. It also tests the ability of organisms to perform
nitrification on nitrate and nitrite to produce molecular nitrogen.
Nitrate broth contains nutrients and potassium nitrate as a source of
nitrate. After incubating the nitrate broth, add a dropperful of
sulfanilic acid and a-naphthylamine. If the organism has reduced
nitrate to nitrite, the nitrites in the medium will form nitrous
acid. When sulfanilic acid is added, it will react with the nitrous
acid to produce diazotized sulfanilic acid. This reacts with the
a-naphthylamine to form a red-colored compound. Therefore, if the
medium turns red after the addition of the nitrate reagents, it is
considered a positive result for nitrate reduction.If the medium does
not turn red after the addition of the reagents, it can mean that the
organism was unable to reduce the nitrate, or it could mean that the
organism was able to denitrify the nitrate or nitrite to produce
ammonia or molecular nitrogen. Therefore, another step is needed in
the test. If the medium does not turn red after the addition of the
nitrate reagents, add a small amount of powdered zinc. Be careful,
as powdered zinc is hazardous! If the tube turns red after the
addition of the zinc, it means that unreduced nitrate was present.
Therefore, a red color on the second step is a negative result. The
addition of the zinc reduced the nitrate to nitrite, and the nitrite
in the medium formed nitrous acid, which reacted with sulfanilic
acid. The diazotized sulfanilic acid that was thereby produced
reacted with the a-naphthylamine to create the red complex. If the
medium does not turn red after the addition of the zinc powder, then
the result is called a positive complete. If no red color forms,
there was no nitrate to reduce. Since there was no nitrite present in
the medium, either, that means that denitrification took place and
ammonia or molecular nitrogen were formed.
Urease Test
Urease broth is a differential medium that tests the
ability of an organism to produce an exoenzyme, called urease that
hydrolyzes urea to ammonia and carbon dioxide. The broth
contains two pH buffers, urea, a very small amount of nutrients for
the bacteria, and the pH indicator phenol red. Phenol red turns
yellow in an acidic environment and fuchsia in an alkaline
environment. If the urea in the broth is degraded and ammonia
is produced, an alkaline environment is created, and the media turns
pink. Many enterics can hydrolyze urea; however, only a few can
degrade urea rapidly. These are known as "rapid
urease-positive" organisms. Members of the genus Proteus
are included among these organisms. Urea broth is formulated to test
for rapid urease-positive organisms. The restrictive amount of
nutrients coupled with the use of pH buffers prevent all but rapid
urease-positive organisms from producing enough ammonia to turn the
phenol red pink.
Phenol Red Broth
Phenol Red Broth is a general-purpose differential test
medium typically used to differentiate gram negative enteric
bacteria. It contains peptone, phenol red (a pH indicator), a
Durham tube, and one carbohydrate. We use three different kinds
of phenol red broths. One contains glucose; one contains lactose, and
the last contains sucrose. The objective of the exercise is to
determine which organisms can utilize each sugar. Phenol red is a pH
indicator which turns yellow below a pH of 6.8 and fuchsia above a pH
of 7.4. If the organism is able to utilize the carbohydrate, an acid
by-product is created, which turns the media yellow. If the
organism is unable to utilize the carbohydrate but does use the
peptone, the by-product is ammonia, which raises the pH of the media
and turns it fuchsia. When the organism is able to use the
carbohydrate, a gas by-product may be produced. If it is, an air
bubble will be trapped inside the Durham tube. If the organism
is unable to utilize the carbohydrate, gas will not be produced, and
no air bubble will be formed.
Casease Test
Skim milk agar is a differential medium that tests the
ability of an organism to produce an exoenzyme, called casease, that
hydrolyzes casein. Casein forms an opaque suspension in milk that
makes the milk appear white. Casease allows the organisms that
produce it to break down casein into smaller polypeptides,
peptides, and amino acids that can cross the cell membrane and be
utilized by the organism. When casein is broken down into these
component molecules, it is no longer white. If an organism can
break down casein, a clear halo will appear around the areas where
the organism has grown.
Gelatinase Test
Nutrient gelatin is a differential medium that tests the
ability of an organism to produce an exoenzyme, called gelatinase,
that hydrolyzes gelatin. Gelatin is commonly known as a component of
gelled salads and some desserts, but it's actually a protein derived
from connective tissue. When gelatin is at a temperature below 32°C
(or within a few degrees thereof), it is a semisolid material.
At temperatures above 32°C, it is a viscous liquid. Gelatinase
allows the organisms that produce it to break down gelatin into
smaller polypeptides, peptides, and amino acids that can cross the
cell membrane and be utilized by the organism. When gelatin is broken
down, it can no longer solidify. If an organism can break down
gelatin, the areas where the organism has grown will remain liquid
even if the gelatin is refrigerated.
Lipase Test
Tributyrin agar is a differential medium that tests the
ability of an organism to produce an exoenzyme, called lipase, that
hydrolyzes tributyrin oil. Lipases break down lipids (fats).
Tributyrin oil is a type of lipid called a triglyceride. Other lipase
tests use different fat sources such as corn oil, olive oil, peanut
oil, egg yolk, and soybean oil. Lipase allows the organisms that
produce it to break down lipids into smaller fragments. Triglycerides
are composed of glycerol and three fatty acids. These get
broken apart and may be converted into a variety of end-products that
can be used by the cell in energy production or other processes.
Tributyrin oil forms an opaque suspension in the agar. When an
organism produces lipase and breaks down the tributyrin, a clear halo
surrounds the areas where the lipase-producing organism has grown.
Starch Hydrolysis
Starch agar is a differential medium that tests the
ability of an organism to produce certain exoenzymes, including
a-amylase and oligo-1,6-glucosidase, that hydrolyze starch.
Starch molecules are too large to enter the bacterial cell, so some
bacteria secrete exoenzymes to degrade starch into subunits that can
then be utilized by the organism. Starch agar is a simple
nutritive medium with starch added. Since no color change
occurs in the medium when organisms hydrolyze starch, we add iodine
to the plate after incubation. Iodine turns blue, purple, or black
(depending on the concentration of iodine) in the presence of starch.
A clearing around the bacterial growth indicates that the organism
has hydrolyzed starch.
Triple Sugar Iron Agar
Triple sugar iron agar (TSI) is a differential medium
that contains lactose, sucrose, a small amount of glucose (dextrose),
ferrous sulfate, and the pH indicator phenol red. It is used to
differentiate enterics based on the ability to reduce sulfur and
ferment carbohydrates. As with the phenol red fermentation
broths, if an organism can ferment any of the three sugars present in
the medium, the medium will turn yellow. If an organism can
only ferment dextrose, the small amount of dextrose in the medium is
used by the organism within the first ten hours of incubation. After
that time, the reaction that produced acid reverts in the aerobic
areas of the slant, and the medium in those areas turns red,
indicating alkaline conditions. The anaerobic areas of the slant,
such as the butt, will not revert to an alkaline state, and they will
remain yellow. This happens with Salmonella
and Shigella
Decarboxylation Test
Decarboxylase broth tests for the production of the
enzyme decarboxylase, which removes the carboxyl group from an amino
acid. Decarboxylase broth contains nutrients, dextrose (a fermentable
carbohydrate), pyridoxal (an enzyme cofactor for decarboxylase), and
the pH indicators bromcresol purple and cresol red. Bromcresol purple
turns purple at an alkaline pH and turns yellow at an acidic pH. We
also add a single amino acid to each batch of decarboxylase broth.
The three amino acids we test in our decarboxylase media are
arginine, lysine, and ornithine. The decarboxylase test is useful for
differentiating the Enterobacteriaceae.Each
decarboxylase enzyme produced by an organism is specific to the amino
acid on which it acts. Therefore, we test the ability of organisms to
produce arginine decarboxylase, lysine decarboxylase, and ornithine
decarboxylase using three different but very similar media. If the
organism is unable to ferment dextrose, there will be no color change
in the medium. If an organism is able to ferment the dextrose,
acidic byproducts are formed, and the media turns yellow. As
the organisms ferment the dextrose, the media initially turns yellow,
even when it has been inoculated with a decarboxylase-positive
organism. The low pH and the presence of the amino acid will
cause the organism to begin decarboxylation. If an organism is able
to decarboxylate the amino acid present in the medium, alkaline
byproducts are then produced. Arginine is hydrolyzed to
ornithine and is then decarboxylated. Ornithine decarboxylation
yields putrescine. Lysine decarboxylation results in
cadaverine. These byproducts are sufficient to raise the pH of the
media so that the broth turns purple. If the inoculated medium
is yellow, or if there is no color change, the organism is
decarboxylase-negative for that amino acid. If the medium turns
purple, the organism is decarboxylase-positive for that amino acid.
Coagulase Test
The coagulase test identifies whether an organism
produces the exoenzyme coagulase, which causes the fibrin of blood
plasma to clot. Organisms that produce catalase can form protective
barriers of fibrin around themselves, making themselves highly
resistant to phagocytosis, other immune responses, and some other
antimicrobial agents. The coagulase slide test is used to identify
the presence of bound coagulase or clumping factor, which is attached
to the cell walls of the bacteria. Bound coagulase reacts with the
fibrinogen in plasma, causing the fibrinogen to precipitate. This
causes the cells to agglutinate, or clump together, which creates the
"lumpy" look of a positive coagulase slide test. You may
need to place the slide over a light box to observe the clumping of
cells in the plasma.The coagulase tube test has been set up as a demo
for you to observe in class. This version of the coagulase test is
used to identify the presence of either bound coagulase or free
coagulase, which is an extracellular enzyme. Free coagulase reacts
with a component of plasma called coagulase-reacting factor. The
result is to cause the plasma to coagulate. In the demo, the
coagulase plasma has been inoculated with Staphylococcus
aureus and Staphylococcus
epidermidis and allowed to incubate at 37°C
for 24 hours. Staphylococcus aureus
produces free coagulase; Staphylococcus
epidermidis does not. The coagulase test is
useful for differentiating potentially pathogenic Staphylococci
such as Staphylococcus aureus
from other Gram positive, catalase-positive cocci.
Immunological Diagnosis
Antigen — Antibody reactions are highly specific and
sensitive. This forms the basis of immunodiagnostics. These tools are
used for the qualitative and quantitative estimation of the pathogens
and/or the protective antibody. These tests can be used at the farm
level without the aid of the instruments. It is apparent that
conventional diagnostic methods rely solely on the microscopic
examination and its visual recognition, which requires a great deal
of experience with the organisms that often change so dramatically in
their morphology during the course of their development. Therefore,
in the evolution of disease diagnostic procedures in aquaculture,
antibody-based (protein-based) immunodiagnosis plays a crucial role.
This method has the advantage over other traditional method in that
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. Further refinement
of conventional immunodiagnostic techniques has resulted in the
development of monoclonal antibody-based techniques and this has
increased the accuracy of detection and has allowed studying the
pathogenesis of diseases. Nevertheless, the specificity of
antibodies also limits their usefulness because major antigens are
not conserved among life stages of certain pathogens (Bartholomew et
al., 1995). Although there is an array of polyclonal and monoclonal
antibody-based diagnostics available for various aquatic animal
Gel Precipitation Test,
Agglutination Test and
Antibody Test.
Gel Precipitation Test
In this test, antibody and possible antigens are placed
in wells in agar plates and allowed to diffuse toward one another.
The antibody is placed in a center well and antigens (specific or
nonspecific) are placed in surrounding wells. When an antibody and
its specific antigen meet one another and are at the proper
concentrations, the precipitate will form a visible white line
between the two wells. This line is called a precipitin line.
test is 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. In the direct agglutination
test, serum is added to a suspension of cells that have the
surface self-Ag to be tested. If the individual's serum
contains the specific auto-Abs, Ig will bind and, at the appropriate
Ab concentration, the cells will become cross linked. This will cause
agglutination, and the cells will form a mat at the bottom of the
test well. Auto-Abs attached to a patient's cells can be detected by
the addition of a second Ab and observed for agglutination. Selective
soluble self-Ags can also be used to assay auto-Abs by attaching them
to the surface of red blood cells. This latter type of agglutination
test is called passive or indirect hemagglutination.
purpose of an ELISA is to determine if a particular protein is
present in a sample and if so, how much. There are two main
variations on this method: you can determine how much antibody is in
a sample, or you can determine how much protein is bound by an
antibody. The distinction is whether you are trying to quantify an
antibody or some other protein. In this example, we will use an ELISA
to determine how much of a particular antibody is present in an
individual's blood. 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.
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. 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. 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. 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.When the enzyme reaction is
complete, the entire plate is placed into a plate reader and the
optical density (i.e. 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
(Enzyme Linked Immunosorbent Assay) is an extensively used
immunological tool in research as well as analytical/diagnostic
laboratories. In sandwich Dot-ELISA, the antigen is sandwiched
directly between two antibodies which react with two different
epitopes on the same antigen. Here one of the antibodies is
immobilized onto a solid support and the second antibody is linked to
an enzyme. Antigen in the test sample first reacts with the
immobilized antibody and then with the second enzyme-linked antibody.
The amount of enzyme linked antibody bound is assayed by incubating
the strip with an appropriate chromogenic substrate, which is
converted to a coloured, insoluble product. The latter precipitates
onto the strip in the area of enzyme activity, hence the name
Dot-ELISA. The
enzyme activity is indicated by intensity of the spot, which is
directly proportional to the antigen concentration.
Agglutination Test
latex agglutination test is a laboratory method to check for certain
antibodies or antigens in a variety of bodily fluids including
saliva, urine, cerebrospinal fluid or blood. In latex agglutination,
an antibody (or antigen) is coated on the surface of latex particles
which is known as sensitized latex. When a sample containing the
specific antigen (or antibody) is mixed with the milky-appearing
sensitized latex, it causes visible agglutination
Antibody Test
is a laboratory test that uses antibodies
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, DFA is
seldom quantitative.
techniques are potentially faster and more sensitive than culture,
serology, and histology methods that are traditionally used to
identify fish pathogens. During the last 15 years or so, molecular
techniques have been increasingly employed to diagnose fish diseases.
These techniques include polymerase chain reaction (PCR), restriction
enzyme digestion, probe hybridization, in situ hybridization, and
microarray. Pathogens can be detected from asymptomatic fish by
molecular diagnostic techniques so disease outbreak could be
prevented. Thus antibiotic treatment can be reduced so that creation
of antibiotic resistant bacteria may be eliminated. In this paper
molecular techniques for detection of fish pathogens are reviewed and
the potential for their application are discussed. The application of
new techniques as a routine tool in a diagnostic laboratory is an
area where relevant literature is scarce and this may contribute to
the reticence of some to adopt these methods.
Chain Reaction
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. 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. Primer design is important to obtain
greatest possible sensitivity and specificity. Therefore, the primers
should be sufficiently long to allow a high annealing temperature and
reduce the opportunity for nonspecific primer annealing, but primers
that are too long may facilitate nonspecific annealing even to
regions of DNA that are not perfectly complementary to the primer
sequence. 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. During each round of the
thermocycling reaction, the template DNA is denatured, primers anneal
to their complementary regions and polymerase enzyme catalyses the
addition of nucleotides to the end of each primer, thus creating new
copies of the target region in each round. Theoretically, the
increase in amount of product after each round will be geometric.
Reverse transcriptase polymerase chain reaction (RT-PCR) is used to
detect specific mRNA and determine levels of gene expression.
Compared to the two other commonly used techniques for quantifying
mRNA levels, Northern blot analysis and RNase protection assay,
RT-PCR can be used to quantify mRNA levels from much smaller samples.
In fact, this technique is sensitive enough to enable quantitation of
RNA from a single cell. Over the last several years, the development
of novel chemistries and instrumentation platforms enabling detection
of PCR products on a real-time basis has led to widespread adoption
of real-time RTPCR as the method of choice for quantitative changes
in gene expression. Furthermore, real-time RT-PCR has become
preferred method for validating results obtained from array analyses
and other techniques that evaluate gene expression changes on a
global scale. The sensitivity and specificity achieved in a
well-designed RT-PCR make it an ideal tool for use in the
surveillance and monitoring of covert infections. As in the
eukaryotes, the prokaryotic rRNA genes contain highly conserved
sequences. The potential utility of conserved regions to identify or
amplify the rRNA genes, followed by exploitation of more variable
regions of the genes or spacers to detect or identify bacteria that
may be difficult or even impossible to culture has long been
developments such as design of PCR conditions that can detect several
pathogens at one time in a multiplex reaction will improve time and
cost-efficiency of this methodology, countering one of the major
arguments against the adoption of these techniques as routine. In
multiplex PCR more than one target sequence can be amplified by
including more than one pair of primers in the reaction. Multiplex
PCR has the potential to produce considerable savings of time and
effort within the laboratory without compromising test utility. Since
its introduction,multiplex PCR has been successfully applied in many
areas of nucleic acid diagnostics, including gene deletion analysis,
quantitative and RNA detection. 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 detection of nucleic acids
variety of labeling and detection systems exist for nucleic acid
probes. Radioisotopes were once the norm but, in the interests of
researcher safety, other methods are becoming increasingly popular.
The variety of labels and detection methods now available can provide
a system suitable for any application, from dot blots to in situ
hybridization. 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
enzyme digestion
enzymes (or restriction endonucleases)
cleave DNA in a very specific fashion.
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. A common
use for restriction enzymes is to generate a
"fingerprint" of a particular DNA
molecule. Because of
the sequence specificity of restriction enzymes,
these enzymes can cut DNA into discrete
fragments which can be
resolved by gel electrophoresis. This pattern
of DNA fragments generates a "DNA
fingerprint" and each DNA molecule has
its own fingerprint.
Other restriction enzymes can be used to
further characterization of a particular DNA
molecule. The location
of these restriction enzyme cleavage sites
on the DNA molecule can be compiled to create
a restriction enzyme map. These
maps are very useful for identifying and
characterizing a particular DNA plasmid or
region. Restriction enzymes recognize specific short sequences of DNA
and cleave the DNA at that site. Single nucleotide changes can result
in the gain or loss of a restriction site, thus altering the number
of fragments produced following digestion of DNA. These RFLP can be
visualized following gel electrophoresis of the digested DNA to
separate the fragments according to size. Differences in the RFLP
profiles have revolutionized criminal investigations and have become
powerful tools in the identification of individuals in paternity and
maternity cases, population genetics, and in the diagnosis of a
variety of diseases.
Fragment Length Polymorphism (RFLP)
is a technique in which organisms may be
differentiated by analysis of patterns
derived from cleavage
of their DNA. If two organisms differ in the
distance between sites of cleavage of a
particular restriction
endonuclease, the length of the fragments
produced will differ when the DNA is digested
with a restriction
enzyme. The similarity of the patterns
generated can be used to differentiate
species (and even
strains) from one another. Isolation
of sufficient DNA for RFLP analysis is
time-consuming and labor intensive. However,
PCR can be used to
amplify very small amounts of DNA, usually
in 2-3 hours, to the levels required for RFLP
analysis. Therefore, more samples can be
analyzed in a shorter
Amplified Fragment
Length Polymorphism (AFLP)
rapid PCR-based technique, AFLP can be used for typing prokaryotes
and eukaryotes. The method is based on the selective PCR
amplification of genomic restriction fragments of the whole genome
and has been shown to be rapid, reproducible, and highly
discriminatory. Selected markers are amplified in a PCR, which makes
AFLP an easy and fast tool for strain identification in agriculture,
botany, microbiology, and animal breeding. The AFLP method used was
essentially that described by Valsangiacomo et
al., (1995). AFLP analysis belongs to the
category of selective restriction fragment amplification techniques,
which are based on the ligation of adapters to genomic restriction
fragments followed by a PCR-based amplification with adapter-specific
primers. 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 (like MseI
or TaqI).
Double-stranded oligonucleotide adapters are designed in such a way
that the initial restriction site is not restored after ligation,
which allows simultaneous restriction and ligation, while religated
fragments are cleaved again. An aliquot is then subjected to two
subsequent PCR amplifications under highly stringent conditions with
adapter specific primers that have at their 3' ends an extension of
one to three nucleotides running into the unknown chromosomal
restriction fragment. Alternative AFLP typing procedures are based on
one enzyme with a single adapter and analysis by agarose gel
electrophoresis. A major improvement has been obtained by switching
from radioactive to fluorescently labeled primers for detection of
fragments in an automatic sequence. In addition, it has been shown
that for small bacterial and fungal genomes a single PCR
amplification with one and two selective nucleotides, respectively,
on both primers are sufficient.
Amplified Polymorphic DNA (RAPD)
technically demanding method of RAPD has been applied to the study of
crayfish plague fungus, Astacus astaci .
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. Application of the RAPD
technique grouped different isolates of the fungus and provides the
means to carry out epidemiological investigations (Lilley et
al., 1997; Oidtmann et
al., 1999). The method has also been used to
examine another Aphanomyces species
that has resulted in serious losses in both farmed and wild fish in
Asia (Lilley et al.,
1997). Other fish pathogens have been studied using RAPD, but
problems with reproducibility and risks of contamination render the
method unsuitable as a stand-alone method of diagnosis. However, 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.
Situ Hybridization
situ PCR has become a powerful molecular tool in research as well as
clinical practice. This technique has resulted in an increased
understanding of infectious and neoplastic diseases and improvements
in diagnosis of disease. In situ RT-PCR gives more detailed
information by allowing for highly sensitive detection of low
abundance gene expression in a given cell while providing anatomical
information. The usefulness of these techniques has been hampered by
low detection sensitivity, poor reproducibility, and high
backgrounds. Moreover, many of the methods used to visualize the
results of PCR amplification within cells and tissues employ
radioactive tracers, making performance of the techniques cumbersome
and costly. Researchers have developed a method for specific
fluorescent detection of gene expression using in situ RT-PCR. This
method enables the researcher or clinician to detect low levels of
gene expression within tissues with very low background interference
while addressing many of the other existing drawbacks to using in
situ RT-PCR. Potential Areas of Application are detection and
diagnosis of viruses and other infectious agents in specific cell
types within tissues, detection, and characterization of tumor cells
within a tissue, detection, and diagnosis of genetic mutations in
inherited diseases, and detection of genes and gene expression in
tissue. Fluorescence in situ hybridization,
or FISH, is a method used to label cells or chromosomes according to
the sequences of nucleic acids contained within them. In
microbiology, the nucleic acid that is labelled as RNA or DNA of the
ribosomes and the target is usually whole cells. The process works by
taking fluorescently labelled pieces of DNA or RNA called probes that
are around 20 nucleotides in length. The probes are incubated in the
presence of cells munder appropriate conditions to permit specific
hybridization of probe to target nucleic acid. Cells types that
contain ribosomes with complementary RNA sequences become labeled by
the binding of the fluorescent probe in situ. These labelled cells
can then be visualized by flow cytometric or fluorescence microscopy.
are a number of ways of using DNA microarrays for the detection of
unique DNA (or RNA) sequences. One method is to fluorescently label
all the DNA sequences in the test sample. The sample DNA that
hybridizes to a specific location on the microarray can be detected
by fluorescent array detection and the data analyzed by computer
programs. Often more practical is to use competitive hybridization in
which the test sample competes for hybridization to the tethered
oligonucleotide, on the chip, with a fluorescent labeled competitor
oligonucleotide. When the test DNA is perfectly complimentary to the
tethered oligonucleotide, it will hybridize to the chip. When the
test DNA is not perfectly complementary to the tethered
oligonucleotide, the fluorescent labeled competitor oligonucleotide
will bind to the tethered oligonucleotide on the chip and displace
the test DNA. A fluorescent microarray detector and computer program
can then analyze the fluorescent array for the presence or absence of
the species/strain specific DNA sequence. Compared with traditional
nucleic acid hybridization with membranes, microarrays offer the
additional advantages of high density, high sensitivity, rapid
detection, lower cost, automation, and low background levels.
Microarrays may provide a better option for largescale diagnostic
testing and can survey a sample for a multitude of sequences
simultaneously. Since most of the pathogens genetic sequences are
available in the GenBank, oligonucleotide probes complementary to all
pathogens can be made and inserted into microarray so that variety of
microbes could be detected by a single microarray chip. As a result,
microarray-based technology is potentially well suited for
identifying fish pathogens in fish populations. The microarray
techniques does not require such sequence conservation, however,
because all of the diverse gene sequences from different populations
of the same functional group can be fabricated on arrays and used as
probes to monitor contagious fish disease especially during the
asymptomatic period of the diseases. Microarrays are already proving
valuable for assaying gene expression. A large number of probes on an
array can reveal which genes are expressed or are present in the
sample. This type of array would be particularly useful in studies of
pathogens, where the presence of certain genes or gene products
indicate whether the organism is pathogenic or not. Set up cost for
the use of DNA microarrays is high. However, once the equipment is
available and microarrays have been prepared, cost per unit of sample
analyzed will be low. Furthermore, analysis time is extremely short.
DNA microarray technology will be used in the future for fish
diseases diagnosis especially during the asymptomatic period of
Loop Mediated
Isothermal Amplification (LAMP)
isothermal amplification (LAMP) is a novel nucleic acid amplification
method that amplifies DNA with high specificity, efficiency and
rapidity under isothermal conditions (Natomi et al, 2000). When
combined with reverse transcription, this method can also amplify RNA
sequences with high efficiency. The method relies on auto-cycling
strand displacement DNA synthesis using a DNA polymerase with a high
strand displacement activity and a set of four specially designed
primers. These four primers, termed as inner and outer primers,
recognize six distinct sequences of the target DNA, which improves
the specificity of the reaction. The reaction is carried out at
isothermal condition, as the denaturation of strands takes place by
strand displacement.
LAMP Reaction:
In the initial stages of LAMP reaction all four primers are
involved, however, in the later cycling reaction only the inner
primers are used for strand displacement DNA synthesis. The LAMP
reaction is initiated by an inner primer containing sequences of
sense and anti-sense strands of the target DNA. This is followed by
the release of a single-stranded DNA through the priming by an outer
primer. This single-stranded DNA will serve as a template for DNA
synthesis primed by the second inner and outer primers that can
hybridize at the other end of the target. This process will result
in the formation of a stem-loop DNA structure. In the subsequent
step of LAMP cycling, one inner primer will hybridize to the loop on
the product and initiate strand displacement DNA synthesis which
will result in the original stem-loop DNA and a new stem-loop.
Cycling continues for a period of approximately 1 h and results in
the accumulation of 109 copies the target. The final
products of the reaction are stem-loop DNA with several inverted
repeats of the target and cauliflower-like structures with multiple
of Amplified Products: Several methods can be employed to
visualize the end products of LAMP reaction. The most common method
of visualization is by agarose gel electrophoresis. The agarose gel
is stained with intercalating dyes such as ethidium bromide or SYBR
Green I. Since the end products of LAMP consist of stem-loop DNA
and cauliflower-like structures with multiple loops of various
lengths, the agarose gel electrophoresis will reveal the products
from the minimum length of the target DNA to the loading well, which
appears as smear at the top and bands at the base of the gel.
Since, one of the characteristics of the LAMP reaction is its
ability to synthesize extremely large amount of DNA, addition of
intercalating dye, SYBR Green I, into the reaction tube itself would
help in visualizing the product under a UV- transilluminator. This
method is useful in the field-level application, where gel
electrohoresis will be a limiting factor. Another method is also
based on the accumulation large amount of byproduct of the reaction.
In the LAMP reaction large amount of byproduct, pyrophosphate ion is
produced, which will yield white precipitate of magnesium
pyrophosphate in the reaction mixture. Hence, detection of presence
or absence of white precipitate will provide an easy distinction of
whether the target DNA is amplified during the reaction. Further,
since increase in the turbidity of the reaction mixture according to
the production of the precipitate correlates to the amount of target
DNA synthesized, a colorimetric estimation of the turbidity in
real-time is also being used as an efficient method of visualizing
the amplified product.
Advantages of
LAMP: LAMP amplifies the target DNA under isothermal
amplification with high efficiency; The detection limit of LAMP is a
few copies and comparable to PCR; No significant influence of the
co-presence of non-target DNA; LAMP allows simple, easy and
selective detection; Lamp is highly specific for the target
sequence, as it employs four primers targeting multiple sequences;
LAMP is simple and easy to perform, as it requires (after
appropriate primers are prepared) only a regular laboratory water
bath or heat block for the reaction; BY incorporating reverse
transcription, LAMP can be used for amplifying RNA as well.
Because amplification of the target DNA is so high at the final
stage it is vulnerable to contamination in subsequent
amplifications; Multiplexing is not possible with LAMP.
of Molecular Methods
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 sued to detect latent infection and thereby identify
the reservoir hosts of infection that is significant in
epizootiology; Can be used to differentiate antigenically similar
of Molecular methods
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.
methods for aquatic animal pathogens have advanced from microscopic
characterization and morphological descriptions to molecular
characterization and probe-based diagnosis. Molecular tools are
increasingly relevant to fish diseases. The sequencing of the
complete genomes of pathogens is allowing great advances in studying
the biology, and improving diagnosis and control of pathogens. Using
nucleic acid as targets, and new methods of analyzing polymorphism in
this nucleic acid, can improve specificity, sensitivity, and speed of
diagnosis and offer means of examining the relationships between
genotype and phenotype of various pathogens. Progress in techniques
aids epidemiological studies as well as identifying causes of disease
outbreaks or the presence of pathogens. Therefore, molecular biology
can be a routine tool in the search for improved methods of diagnosis
and control of fish pathogens and the epidemiology of infectious fish
Riji John & Rosalind George, 2004, Fin Fish & Shell Fish
Diseases (Practical Manual). Fisheries College & Research
Institute, Thoothukudi.
Felix, Riji John, Prince Jeyaseelan & V Sundararaj, 2001,
National Traning Course On Fish Disease Diagnosis & Health
Management, Fisheries College & Research Institute, Thoothukudi.
Shankar & C V Mohan, 2002, Fish And Shellfish Health Management,
College Of Fisheries, Mangalore.
Altinok & Ilknur
Kurt, 2004, Molecular Diagnosis Of Fish Diseases: A Review,
Turkish Journal Of Fisheries And Aquatic
Sciences 3: 131-138
Standard Methods, Center For Infection, Wales.
H.A. PCR Technology Principles And Applications For DNA
Amplification. Stockton Press, New York, 1989.
Technology: Current Innovations, 2nd Edition. Edited By Thomas
Weissensteiner Et Al.
K., Logan J., Saunders N. (Eds.). 2004. Real-Time PCR: An Essential
Guide. Horizon Bioscience, Norfolk, UK, P. 346.
S.V., Lightner D.V. 2002. Quantitative Real Time PCR For The
Measurement Of White Spot Syndrome Virus In Shrimp. J. Fish Dis. 25,
Fauce K.A., Layton R., Owens L. 2007. Taqman
Real-Time PCR For Detection Of Hepatopancreatic Parvovirus From
Australia. J. Virol. Methods,
140, (1-2), 10-16.
K.P., Klimpel K.R., Dhar A.K. 2003. Improvement In The Specificity
And Sensitivity Of Detection For The Taura Syndrome Virus And Yellow
Head Virus Of Penaeid Shrimp By Increasing The Amplicon Size In SYBR
Green Real-Time RT-PCR. J. Virol. Methods, 111 (2), 121-127.
K.V., Jeff A. Cowley, Russell J. Mcculloch, Peter J. Walker. 2006. A
Taqman Real-Time RT-PCR For Quantifying Mourilyan Virus Infection
Levels In Penaeid Shrimp Tissues. J. Virol. Methods 137, 265-271.
K.F., Wang J., Lightner D.V. 2004. Quantification
Of Taura Syndrome Virus By Real-Time RT-PCR With A Taqman Assay. J.
Viro. Methods 11, 109-114.
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The Climate Equity Reference Calculator is a general equity reference tool, designed to allow the user to specify their own preferred interpretation of national responsibility and capacity for climate action, which in turn is used to determine each country's fair share of the global climate effort.
The Calculator was developed by Tom Athanasiou & Paul Baer of EcoEquity and Sivan Kartha & Eric Kemp-Benedict of the Stockholm Environment Institute, who also developed a specific effort-sharing approach, the Greenhouse Development Rights framework (which has since been generalized into the Climate Equity Reference framework. Most of the initial Calculator programming was also done by Eric, with the assistance of Douglas Wang. Tyler Kemp-Benedict did most of the user-interface design and implementation. More recently, Christian Holz has joined the CERP team, and among much else, has become a key team programmer. The core funding for these effort has been provided by the Stockholm Environment Institute, the Rockefeller Brothers Fund and Christian Aid.
The core of the Climate Equity Reference framework is still the dynamic Responsibility and Capacity Index approach. Or, as we sometimes call it, the "Responsibility, Capacity, and Need approach." However, the CERP approach has been substantively generalized, and much more generalization and extension is planned for the future.
We would also like to thank Kirk Smith, who as far as we can tell invented the Responsibility and Capacity Index, or at least the approach that it embodies. (We thought that we had done so, but in fact Kirk beat us by over a decade!) See Kirk R. Smith, Joel Swisher, and Dilip R. Ahuja, "Who pays (to solve the problem and how much)?," in Peter Hayes and Kirk Smith (eds.), The global greenhouse regime: Who pays?, United Nations University Press/Earthscan, London, 1993.
Adaptation Need
In these calculations, a country or region's share of the Global Mitigation Requirement is proportional to its Responsibility and Capacity Index. The Responsibility and Capacity Index can also quite properly be used to estimate national fair shares in a global adaptation effort. Not that it can help us to estimate the global adaptation need, or the adaptation need of any given country, but it does offer a way to think about national fair shares of any monetized, global, climate-related effort.
(Global adaptation need is properly estimated as a function of projected temperature change and national vulnerability. At the moment, in the Calculator, all we offer is a user-defined parameter that is specified as a percentage of projected Gross World Product.)
Business as Usual
Any effort-sharing framework (as opposed to resource-sharing frameworks that divides up a fixed emissions budget) requires baselines. This is because "effort" must be measured against a baseline, and – by definition – it should be a "no effort" or "no policies" baseline.
The construction of such baselines is challenging, but plausible projections can be made.
In this system, we have approached this problem in a straightforward manner. Our baselines are derived from an existing set of standard sources: UN Population Division, IMF, US EPA, McKinsey and Company. We update and calibrate these to be consistent with the most current available annual emissions data, based on fossil fuel CO2 from numerous other sources.
For a much more detailed discussion, see Definition, sourcing, and updating of the emissions baselines.
This calculator defines capacity in income terms. It allows the user use a progressive definition, in that it is possible to define a level of income (a development threshold) below which income does not count toward capacity, similar to the way typical income tax schedules do not tax income below a certain exemption level. In this calculator, national capacity is calculated as the sum of all individual incomes, excluding income below the development threshold.
Capacity is measured in market exchange rate (MER) terms, while the development threshold is expressed in Purchasing power parity (PPP) terms and then converted to country-specific values using the ratio of MER and PPP exchange rates appropriate to each country.
This capacity is combined with responsibility to calculate a Responsibility and Capacity Index, which is taken as a measure overall obligation to act.
Development Threshold
The development threshold defines an income threshold below which an individual's income, whatever country they may reside in, is taken to be exempt from the calculation of national fair shares. Which is to say that income below the development threshold is not taken to contribute to national capacity, nor are emissions corresponding to consumption below this threshold taken to contribute to national responsibility.
This threshold can be set between $0 and $20,000 per person per year (in Purchasing Power Parity terms). For reference, a development threshold of $7,500 PPP per person per year, which is a bit above a reasonably-defined global poverty line based on empirical observations, is the standard setting presented to the user.
Emissions Intensity
Emissions Intensity is a measure of the efficiency of a country's economic activity, in terms of greenhouse gas emitted per unit of economic output. It is defined as greenhouse-gas emissions divided by Gross Domestic Product, and can be calculated in CO2-only terms or it can include non-CO2 gases.
Equity Settings - what they are and how to use them
The Climate Equity Reference Calculator does not produce one, single, "correct" result. Rather, it offers a set of controls, and when you click past this page you'll have the opportunity to change them at will. This Equity Settings page, on the other hand, contains only the key equity parameters, the ones where you specify how ambitious a climate transition you'd like to model, and how you want to interpret the core parameters defining responsibility and capacity.
Choose your preferences. You can always change them later.
Level of Global Ambition
First, chose a mitigation pathway. You have three options, which are carefully defined to represent three politically distinct storylines. You can read more about them here.
Common but Differentiated Responsibilities and Capacities
The core of the Equity Reference Calculator is the Responsibility and Capacity Index. The controls in the center of the Equity Settings page determine how national RCIs are calculated.
- If you choose a box from the top row of the matrix (or the first bullet under "Responsibility vs. Capacity, relative weight") you instruct the calculator to consider only responsibility. Similarly, if you select a box from the bottom row, you instruct it to consider only capacity. If you choose a box in the middle row, responsibility and capacity are weighted equally -- a 50/50 weighting. Note that, once you've clicked past this Equity Settings page to the Calculator itself, you can choose any weighting of capacity and responsibility.
- The columns relate a different aspect of effort-sharing problem -- progressivity
- If you choose a box from the center column (or the first bullet under "Progressivity, between and within countries") you're selecting the "standard" development threshold. That is, you're instructing the system to exclude all income below $7,500 (PPP) from the calculation of capacity, and all emissions corresponding to consumption below that same threshold from the calculation of responsibility.
- If you choose a box from the left column, you're instructing the system to use a much lower development threshold, one just a bit higher than the average per-capita income in the Least Developed Countries. That is, you're reducing the overall progressivity of the effort-sharing calculations.
- If you select a box from the right column, you're choosing settings that are more progressive than those in the central column. The development threshold stays $7,500 (PPP), but you also activate a luxury threshold that is defaulted to a per-capita income of $100,000 (MER dollars) per person per year, which is just about the point that defines the "global one percent." In this case, as income rises above the development threshold, a linearly increasing percentage of that income (and the associated emissions) are counted towards national capacity (and responsibility), until, when the luxury threshold is reached, it is all counted toward national capacity. The effect is to shift obligation (if you will) up the global income scale. This is a simple analog to a progressive income tax, with a zero tax bracket up to the Development Threshold, and a maximum tax bracket starting at the Luxury Threshold. Again, note that the Calculator itself provides a wider set of controls.
Historical Responsibility Start Date
Since responsibility is defined as cumulative emissions (arising from consumption above the user-specified development threshold), it is necessary to specify an initial year for accounting for emissions. While the Equity Reference Calculator is not a budget-sharing system, if you select 100% Responsibility and a development threshold of $0, it becomes a effort-sharing system in which national fair shares are entirely proportional to national historical emissions.
Fair Share
A nation's overall fair share of the global climate effort is calculated as a percentage of that global effort -- whether mitigation or adapation related -- and is based on the country's share of the global responsibility for causing climate change and capacity for addressing it. These are expressed in a Responsibility and Capacity Index, which is calculated based on user's own preferred interpretation of national responsibility and capacity. These preferences are set in the panel to the left labeled Calculator Settings, and a core subset of them are also accessible in the Equity Settings panel.
Critically, a nation's fair share is not seen in domestic terms, though its mitigation potential of course is. The CERP views climate as a global commons problem that can only be solved within a high-cooperation international regime. Such a regime can only be established if each Party sees others to be doing their fair shares in the face of the common challenge. In practice, of course, each country will decide, on the basis of its own specific considerations (e.g. it's own view of costs, co-benefits and political-economic tradeoffs) what fraction of its fair share of the global effort it will attempt domestically, and what fraction it will make off shore" by supporting action in other countries.
Global Mitigation Pathways / Level of Global Ambition
A mitigation pathway is a global emissions trajectory that is designed to, over time, keep the climate system within a given carbon budget, or to keep temperature increases below a certain limit. The more stringent the budgetary or temperature limit, the higher the level of global ambition.
The Climate Equity Reference Calculator supports three mitigation pathways. They range from the Strong 2ºC pathway, which is an emergency mitigation pathway by any definition, to the G8 pathway, which is based on the (politically influential) targets specified in the G8's 2009 Declaration in the Italian town of L'Aquila.
- The Strong 2ºC pathway is defined to conform to an extremely challenging carbon budget - the most challenging we could defend as being techno-economically achievable. While we cannot give a quantified estimate of its likelihood of staying below 1.5ºC, we can say that, by comparison with the emission budgets given in the IPCC's Fifth Assessment Report, it has a greater than 66% likelihood of keeping total warming below 2ºC. In this pathway, global emissions peak in 2014 and annual fossil CO2 reductions reach a maximum rate of 6% annually. Non-CO2 emissions have a "floor" of 5 GtCO2 annually, to account for the potentially irreducible requirements of agriculture. Importantly, we do not assume net-negative emissions in either the fossil-fuel or land-use sectors.
- The Weak 2°C pathway is deliberately similar to well-known emissions pathways that have generally been considered to have a "likely" (roughly 67%, in the IPCC's terminology) chance of maintaining less than 2ºC of warming. However, such pathways now carry an important caveat – the IPCC's Fifth Assessment Report suggests that these pathways and their associated CO2 budgets carry substantially higher risks of exceeding 2ºC than was previously estimated. In any case, this pathway, like our Strong 2ºC pathway, peaks immediately (2014); its rate of decline then increases gradually, reaching a maximum of 4.4% in 2050. Like the Climate Action Tracker's 2ºC pathway, its emissions in 2020 are 44 GtCO2equivalent.
- The G8 pathway, for its part, is not calibrated to a specific temperature threshold, but is based on the politically influential targets specified in the G8's 2009 Declaration in the Italian town of L'Aquila. The L'Aquila text specifies only that the peak must be "as soon as possible" and omits the reference year against which goal the 50% global reductions by 2050 is to be calculated. In our rendering, its peak year is 2020, and the maximum annual rate of post-peak emissions decline is 6.0%. (The late peak requires very rapid reductions in order to reach its 50% in 2050 specification, and this despite its relatively larger budget.) The pathway's 2012 to 2100 CO2 budget is ~1600 GtCO2, which is well above the largest of the IPCC's budgets, the one that is given a 33% chance of staying below 2ºC. One can safely say that it is "very likely" to exceed 2ºC by 2100.
For much more information on all of this, including a comparison of these pathways to the IPCC's emissions budgets, see the Mitigation Pathway Overview.
Global Mitigation Requirement
The global mitigation requirement is the "mitigation gap" between emissions under a global Business-as-Usual pathway and emissions under the specified global mitigation pathway. In any given year, for any given BAU and mitigation pathway pair, this gap can be expressed as a number of Gigatonnes of CO2 or CO2equivalent. This is the number of tons to be mitigated in that year. This global mitigation requirement can be allocated to individual countries, in proportion to their Responsibility and Capacity Index, to determine their fair share of the global mitigation effort.
Greenhouse Development Rights (GDRs)
Greenhouse Development Rights was a ground-breaking equity reference framework based upon national responsibility and capacity for climate action, and national (development and adaptation) need. It has been generalized into and superceded by the Climate Equity Reference framework. The core of the Climate Equity Reference framework is still the dynamic Responsibility and Capacity Index approach. However, the CERP approach has been substantively generalized, and much more generalization and extension is planned for the future.
Historical Responsibility Start Date
Here the user specifies the initial year in the calculation of cumulative emissions for the purposes of assessing a country's Responsibility, which is defined as cumulative emissions (arising from consumption above the user-specified development threshold).
Intro to Climate Equity Pledge Scorecard
The Climate Equity Pledge Scorecard is is intended as an easy-to-use tool to compare countries' "pledges" – their promises of mitigation action – to their fair share of the global effort. The Scorecard is a general equity reference tool designed to allow users to specify their own preferred interpretation of national responsibility and capacity for climate action, which in turn is used to determine each country's fair share of the global climate effort.
In principle, the Scorecard views national fair shares as the sum of domestic mitigation and financial support for international mitigation. Unfortunately, explicit pledges tend to be announced only for domestic mitigation alone, and not for financial support for international mitigation. Some wealthy (high capacity) countries do provide some support for international action of various kinds, but generally not in a manner that is explicitly reported and accounted in a coherent manner.
Because of this lack of explicit and credible information, the Scorecard does not include the financial support for international mitigation in the assessment of a country's pledge. If accountable financial pledges are ultimately made, these will be included, and many scores will improve.
Note that pledges score better when evaluated against the G8 marker pathway than they do when scored against the Stong 2°C pathway. Unfortunately, the G8 marker pathway provides only a poor chance of keeping warming below 2°C. (See the Mitigation Pathway Overview for details).
Kyoto Obligation
The Kyoto Protocol is an agreement under which the world's industrialized countries commited to limits on greenhouse gas emissions. Some countries complied with the Kyoto protocol emission limits, but some did not ratify, and some withdrew from the agreement. Views differ on how this should affect fair shares going forward, and thus, the Equity Reference Calculator allows the user to select whether "Kyoto obligations" are to be included or excluded when calculating national fair shares and evaluating national pledges.
If they are included, then countries like Canada which not met their Kyoto target must reduce their emissions from their actual level in 2012 to their Kyoto target before any further reductions are counted towards their global (Responsibility and Capacity Index-based) fair shares. The effect is that Kyoto non-compliers have fair shares going forward that are larger by an amount equal to the amount by which they exceeded their targets. All other countries have somewhat smaller fair shares, because the shared global climate-related need (which is divided among countries on the basis of responsibility and capacity) becomes smaller by an amount equal to the aggregate amount by which Kyoto non-compliers exceed their Kyoto targets.
There are three different ways in which Kyoto obligations can be treated.
- They can be included for all countries. Thus, the US and Canada are treated as if they were still parties to the Kyoto protocol. This allows the calculator to take account of the moral, if not legal, obligations that were implied by the original signature (the US) or ratification (Canada) of the Kyoto Protocol. More generally, it highlights the significance of non-compliance as a form a "free riding" – an issue that goes far beyond Kyoto.
- They can be excluded for all countries, which means that Kyoto non-compliance is ignored. The Kyoto Protocol thus has no bearing on future fair share. This option, importantly, is the default.
- They can be included for all countries except the US and Canada, which are not or are no longer parties to the Kyoto protocol, regardless of their previous intent to comply.
Mitigation cost as % of GWP
Estimates of the cost of a mitigation vary widely, and depend on a wide range of assumptions. The Equity Reference Calculator does not assume a particular cost, but rather permits the user to specify an estimate of the cost. The cost specified by the user is an annual cost, as a % of GWP, that is assumed for simplicity to be constant over the entire time period. The specified cost also determines a global average (not marginal) per-ton carbon-dioxide mitigation cost.
This hypothetical cost estimate -- added to the adaptation cost estimate -- is used (in the Country Report and the "Fair share" table view) to estimate fair shares (national and per capita) of total costs. (The specified cost is not used in calculating fair shares of the mitigation effort, which are expressed in terms of tons.)
Mitigation Fair Share
A country's mitigation fair share is its fair share of the global mitigation requirement. It is proportional to both the stringency of the mitigation pathway and the country's Responsibility and Capacity Index.
A developed country, with relatively large fractions of total global responsibility and capacity, typically has a mitigation fair share that is larger than it's domestic mitigation potential. That is, a mitigation fair share that is larger than any that it can efficiently discharge within its own borders.
A developing country, with relatively small fractions of total global responsibility and capacity, typically has a mitigation fair share that is smaller than its domestic mitigation potential. That is, it contains mitigation potential that must be used in order to realize a high-ambition global transition. In practice, this means that it must be "matched" with support provided by developed countries that need it in order to discharge their mitigation fair shares.
More Detailed Calculations
This link takes you to the Climate Equity Reference Calculator, where you can see and experiment with an analysis that is much more detailed than the one available in the Climate Equity Pledge Scorecard system.
Pledged Effort
The Equity Reference Calculator assesses the national pledges that countries have submitted to UNFCCC Secretariat. (The official Annex 1 submissions and non-Annex 1 submissions are available on the UNFCCC website, and helpful compilations are available from the Climate Action Tracker and Climate Interactive websites. We rely upon these roll-ups and do not attempt to collect additional information about pledges. In a few cases, we adjust our pledge database to take account of important, but not yet widely reported, changes in national pledges.
To this point, almost all mitigation pledges are given for the year 2020. Some countries have submitted both an "unconditional" pledge, which they intend to meet unilaterally, and a more ambitious "conditional" pledge, which is contingent on other countries raising their own mitigation ambition and/or providing financial and technological support.
While this Equity Reference Calculator views national fair shares as the sum of domestic mitigation and financial support for international mitigation, only the mitigation pledges can currently be assessed. This is because explicit pledges tend to be announced only for domestic mitigation alone, and not for financial support for international mitigation (not to mention adaptation). Some wealthy (high capacity) countries do provide some support for international action of various kinds, but they do not generally do so in a manner that is explicitly reported and accounted in a coherent manner. Because of this lack of explicit and credible information, it is not possible to consider financial support for international mitigation in the assessment of a country's pledge.
In the calculations here, responsibility – contribution to the climate problem – is defined as the sum of all emissions corresponding to consumption above the user-specified development threshold (as is the case with capacity). Emissions corresponding to consumption below that threshold are not included in the calculation of responsibility, as they are exempted on the grounds of, say, being associated with basic or survival consumption.
Responsibility is measured cumulatively since some user-specified start date. (To the extent that emissions before the start date contributed to current capacity, they are accounted for implicitly in the capacity measure.) In calculating national fair shares, responsibility and capacity (a simple average, in the default case, though the weighting can be changed) are combined to compute a Responsibility and Capacity Index.
Responsibility and Capacity Index (RCI)
The Responsibility and Capacity Index (RCI) combines measures of responsibility and capacity (using a user-specified weighting) into a combined indicator of national obligation.
The RCI is then used to straightforwardly calculate each country's fair share of the global climate effort -- a country's fair share of the global effort (say, in total tons of mitigation required) is proportional to its RCI.
A country's RCI is affected by its income distribution, because both responsibility and capacity are calculated in terms of a user-specified development threshold. For any non-zero development threshold, the resulting effort-sharing allocation, if interpreted as a climate tax, is mildly progressive. in other words, a dollar of income that is just above the development threshold is "taxed" at the same rate as a dollar earned by a billionaire.
Scores reflect the amount by which a country's pledged reductions fall short of (or, in rare cases, surpass) their fair share of the global mitigation requirement.
On the scorebar, a country's fair share of 2020 global emissions reductions is shown as the distance from its 2020 business-as-usual (BAU) emissions to the zero point. Its pledge is shown as the distance from its BAU to its score.
Most country's pledges fall far short of their fair share of the effort. This leaves a red gap to the left of the zero point, and yields a negative score.
A few country's pledges surpass their fair share of the effort, by an amount shown as a green bar to the right of the zero point, and yields a positive score.
Type of Pledge
Many countries have expressed their national emissions mitigation pledges in the form of an unconditional pledge and a conditional pledge. The unconditional pledge is weaker (lower in ambition), but will be met unilaterally. The conditional pledge is stronger (higher in ambition) but becomes active only if other countries satisfy certain requirements relating to increasing their own ambition with respect to mitigation pledges and/or the provision of finance and technology.
Note that some countries have offered only a single pledge, and sometimes an ambiguous definition of it as well. In rare cases -- to illustrate the implications of high- and low-ambition interpretations of such pledges -- we have denoted the weaker interpretation to be 'unconditional' and the stronger interpretation to be 'conditional'.


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Nalbari District
Nalbari is an administrative district in India and is located in the state of Assam.
District name: Nalbari
State: Assam
Total population: 771,639
House Holds: 155,248
Population by Sex
Males: 396,006.
Females: 375,633.
Google Map of Nalbari
View Larger Map of NalbariView satellite images/ street maps of villages in Nalbari, Assam, India. The Map data on this website is provided by Google Maps, a free online map service one can access and view in a web browser.


text:
To me the stories seem to flow together- they had to be concieved together. However, I think there are instances that could have been simply added later. Jack and Illusio are mentioned as saints, yes, but beyond a brief outline you know almost nothing about them. I'd forgotten in the seven odd years it between reading the Saga and trilogy for the first time. The Rebellion is hinted at, but not really explained. The CE-rigs were almost a complete mystery, and I wonder why d-jumping hasn't been studied in the Milieu (they did say it wasn't an unknown phemonenon, but you'd have thought that people would have studied it. It could be very useful, at least for GM+ heads).
Any thoughts on the matter?
(Apologies if I'm posting too much)


text:
Joining our Tennis Summer Camp is a great way to meet new friends and learn the game of tennis! The goal of our camp is to provide students the opportunity to learn and enjoy the lifetime sport of
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How many people died in the waters from each year?
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Air pollution is killing as many as 2.4 million people each year. Written by Gwynn ... air pollution kills up to 2.4 million people a year around the ...
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36 %
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17 %
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10 %
Between 350,000 and 500,000 Chinese die prematurely each year ... 000 and 500000 people die prematurely each year as a ... died in 2016 ...
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