Sunday, April 28, 2019

Amoebic gill disease (AGD) has got new treatment(researchers)


Peracetic acid is an oxidising acid that is widely used as a disinfectant in recirculating aquaculture systems for rainbow trout. It is used because it disinfects without damaging beneficial bacteria in the biofilter.
Researchers are testing the efficacy of peracetic acid as a sustainable treatment for amoebic gill disease.
In recent years, amoebic gill disease (AGD) has become a serious issue for marine salmon sites. Researchers are now developing a new method of treatment that will be milder on both the fish and the environment compared to existing methods. 

Presently, farmed salmon affected by AGD are treated with fresh water or are bathed in hydrogen peroxide. Researchers are now investigating whether peracetic acid can be a more sustainable alternative to these treatments.



Treatment must be safe
The goal of the initial trials is mainly to investigate whether peracetic acid is safe to use in the treatment of salmon.

Researchers from the Norwegian research institute Nofima, the Norwegian Veterinary Institute and the Technical University of Denmark have tested various doses of peracetic acid on salmon and separately on the amoeba in three trials.

Their preliminary conclusion is that peracetic acid is not harmful to the salmon, that it has an effect on the amoeba that causes the disease and that there is low environmental risk when using peracetic acid.


“It is also very positive that we now have good interdisciplinary evidence regarding how salmon react to peracetic acid, and we have conducted preliminary investigations concerning environmental impact,” says project lead Carlo C Lazado, a fish health researcher at Nofima.

Infectious Salmon Anaemia (ISA) well explained

What Is It?

Infectious salmon anaemia (ISA) is an infectious viral disease of Atlantic salmon (Salmo salar L.). The disease was first reported in Norway in 1984, but has since been reported in Canada, the USA, the Faroe Islands, Ireland and Scotland. The outbreak of ISA in Scotland in 1998-99 was successfully eradicated. Atlantic salmon is the only susceptible species known to develop clinical disease, but ISA virus can replicate in rainbow trout (Oncorhynchus mykiss) and sea trout (Salmo trutta L.).

Where and When Might it Occur?

In Norway, cases of ISA have occasionally been reported in fresh water farms but generally in hatcheries which use part sea water. The overwhelming majority of cases occur in farmed fish in sea water. The virus has been detected in wild fish but cases of clinical disease have only been reported in farmed fish.
The disease has also been detected in the UK, Canada and Chile.

Diagnosis

Cases of clinical disease may be characterised by severe anaemia, ascites (accumulation of fluid in the body cavity), haemorrhage in internal organs and darkening of the liver. Fish also tend to appear lethargic with pale gills and blood spots in the eyes.

Indirect fluorescent antibody test Indirect fluorescent antibody test (IFAT) showing the presence of ISA virus in the kidney cells of an Atlantic salmon



The causative agent of ISA is an orthomyxovirus. The presence of the virus is confirmed by isolation and identification techniques, indirect fluorescent antibody test (IFAT) and reverse transcriptase polymerase chain reaction (RT-PCR).

Control

The virus can be transmitted through water, but the highest risk factors for spread of disease are movement of live fish, discharge of untreated blood and contact with infected vehicles and equipment.



ISA is exotic to the European Union. It is a notifiable disease under UK legislation and a List I disease under European Directive 91/67/EEC. Under EU legislation action must be taken to contain any outbreak, to eradicate sources of infection and to protect other fish farms by:
  • Compulsory slaughter and disinfection of infected farms
  • Strict movement controls on suspect farms
  • Placing farms in the vicinity of an outbreak under surveillance

Treatment

There are no treatments for ISA and no licensed vaccines in the EU. Vaccine trials in Canada have yielded equivocal results.

Friday, April 26, 2019

RadioImmunoAssay | The principle and Procedure of RIA


Radioimmune assay (RIA): As the name indicates, it is an immunological assay to analyze any antigen or antibody in the patient’s serum to diagnose the disease.
This is one of the most sensitive & specific methods of immune assays available.
It involves competitive binding of radio-labeled antigen and unlabeled antigen to a high-affinity antibody.
The sensitivity range is 0.0006–0.006 µg antibody/ml.
The technique was developed by S. A. Berson, and Rosalyn Yalow and Rosalyn R. Yalow received the Nobel Prize for it in 1977.
It involves three principles which make it most specific & sensitive than other immune assays.
  1. An immune reaction, i.e., antigen, antibody binding.
  2. A competitive binding or competitive displacement reaction. (It gives specificity)
  3. Measurement of radio emission. (It provides sensitivity)
IMMUNE REACTION:
When a foreign biological substance enters into body bloodstream through non-oral route, the body recognizes the specific chemistry on the surface of the foreign material as antigen and produces specific antibodies against the antigen so as nullify the effects and keep the body safe. Body immune system produces the antibodies so; it is an immune reaction. Here the antibodies or antigens bind move due to chemical influence. This is different from the Principle of electrophoresis where proteins are separated due to charge.
A competitive binding or competitive displacement reaction:
This is a phenomenon wherein when there are two antigens which can bind to the same antibody, the antigen with more concentration binds extensively with the limited antibody displacing other. So here in the experiment, the radiolabelled antigen is allowed to bind to high-affinity antibody. Then when patient serum is added unlabelled antigens in it start binding to the antibody displacing the labeled antigen.
Measurement of radio emission:
Once the incubation is over, then washings are done to remove any unbound antigens. Then radio emission of the antigen-antibody complex is taken, the gamma rays from radiolabeled antigen are measured.
The approach for RIA:
The steps of radioimmunoassay experiment {Image Credit: Georgia state university}
The first step to set up an RIA is to determine the amount of antibody needed to bind 50–70% of a fixed quantity of radioactive antigen (Ag*) in the assay mixture. This ratio of antibody to Ag* is chosen to ensure that the number of epitopes presented by the labeled antigen always exceeds the total number of antibody binding sites. Consequently, unlabeled antigen (from patient serum) added to the sample mixture will compete with a radiolabeled antigen to bind to the limited number of antibody.
Even a small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a decrease in the amount of radioactive antigen bound, and this decrease will be proportional to the amount of unlabeled antigen added. To determine the amount of labeled bound antigen, the Ag-Ab complex is precipitated to separate it from free antigen (antigen not bound to Ab), and the radioactivity in the precipitate is measured. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample), and from this plot, the amount of antigen in the test mixture may be precisely determined.
Procedure of RIA 
  1. The labeled antigen is mixed with an antibody at a concentration that saturates the antigen-binding sites of the antibody.
  2. Then test samples of an unlabeled antigen of unknown concentration are added in progressively more substantial amounts.
  3. The antibody does not distinguish labeled from the unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. As the concentration of unlabeled antigen increases, the more labeled antigen will be displaced from the binding sites.
  4. The decrease in the amount of radiolabeled antigen bound to the specific antibody in the presence of the test sample is measured to determine the amount of antigen present in the test sample.
  5. The antigen is generally labeled with a gamma-emitting isotope such as I125, but beta-emitting isotopes such as tritium (3H) are also routinely used as labels.
  6. The radiolabeled antigen is part of the assay mixture; the test sample may be a complex mixture, such as serum or other body fluids, that contains the unlabeled antigen.
The procedure requires small amounts of sample and can be conducted in small 96-well microtiter plates; hence this procedure is suitable to determine the concentration of a particular antigen in large numbers of samples. For example, a microtiter RIA can be used to screen for the presence of the hepatitis B virus. RIA screening of donor blood has sharply reduced the incidence of hepatitis B infections in recipients of blood transfusions
Radioimmunoassay is not widely used as that of ELISA tests in health care. Because RIA is a time-consuming method and also very expensive.

Thursday, April 25, 2019

Testosterone Levels Test(layman's terms)

What is a testosterone levels test?

Testosterone is the main sex hormone in males. During a boy's puberty, testosterone causes the growth of body hair, muscle development, and deepening of the voice. In adult men, it controls sex drive, maintains muscle mass, and helps make sperm. Women also have testosterone in their bodies, but in much smaller amounts.

This test measures the levels of testosterone in your blood. Most of the testosterone in the blood is attached to proteins. Testosterone that is not attached to a protein is called free testosterone. There are two main types of testosterone tests:

Total testosterone, which measures both attached and free testosterone.
Free testosterone, which measures just free testosterone. Free testosterone can give more information about certain medical conditions.
Testosterone levels that are too low (low T) or too high (high T) can cause health problems in both men and women.

Other names: serum testosterone, total testosterone, free testosterone, bioavailable testosterone

What is it used for?

A testosterone levels test may be used to diagnose several conditions, including:

Decreased sex drive in men and women
Infertility in men and women
Erectile dysfunction in men
Tumors of testicles in men
Early or delayed puberty in boys
Excess body hair growth and development of masculine features in women
Irregular menstrual periods in women

Why do I need a testosterone levels test?

You may need this test if you have symptoms of abnormal testosterone levels. For adult men, it's mostly ordered if there are symptoms of low T levels. For women, it's mostly ordered if there are symptoms of high T levels.


Symptoms of low T levels in men include:


Low sex drive
Difficulty getting an erection
Development of breast tissue
Fertility problems
Hair loss
Weak bones
Loss of muscle mass

Symptoms of high T levels in women include:


Excess body and facial hair growth
Deepening of voice
Menstrual irregularities
Acne
Weight gain
Boys may also need a testosterone levels test. In boys, delayed puberty can be a symptom of low T , while early puberty may be a symptom of high T.


What happens during a testosterone levels test?

A health care professional will take a blood sample from a vein in your arm, using a small needle. After the needle is inserted, a small amount of blood will be collected into a test tube or vial. You may feel a little sting when the needle goes in or out. This usually takes less than five minutes.


Will I need to do anything to prepare for the test?

You don't need any special preparations for a testosterone levels test.


Are there any risks to the test?

There is very little risk to having a blood test. You may have slight pain or bruising at the spot where the needle was put in, but most symptoms go away quickly.


What do the results mean?

Results mean different things depending on whether you are a man, woman, or boy.

For men:


High T levels may mean a tumor in the testicles or adrenal glands. Adrenal glands are located above the kidneys and help control heart rate, blood pressure, and other bodily functions.
Low T levels may mean a genetic or chronic disease, or a problem with the pituitary gland. The pituitary gland is a small organ in the brain that controls many functions, including growth and fertility.

For women:


High T levels may indicate a condition called polycystic ovary syndrome (PCOS). PCOS is a common hormone disorder affecting women of childbearing age. It is one of the leading causes of female infertility.
It may also mean cancer of the ovaries or adrenal glands.
Low T levels are normal, but extremely low levels may indicate Addison disease, a disorder of the pituitary gland.
For boys:

High T levels may mean cancer in the testicles or adrenal glands.
Low T levels in boys may mean there is some other problem with the testicles, including an injury.
If your results are not normal, it doesn't necessarily mean you have a medical condition needing treatment. Certain medicines, as well as alcoholism, can affect your results. If you have questions about your results, talk to your health care provider.

Is there anything else I need to know about a testosterone levels test?

Men who are diagnosed with low T levels may benefit from testosterone supplements, as prescribed by their health care provider. Testosterone supplements are not recommended for men with normal T levels. There is no proof they provide any benefits, and in fact they may be harmful to healthy men.

Semen analysis(layman's terms)

What is a semen analysis?

A semen analysis, also called a sperm count, measures the quantity and quality of a man's semen and sperm. Semen is the thick, white fluid released from the penis during a man's sexual climax (orgasm). This release is called ejaculation. Semen contains sperm, the cells in a man that carry genetic material. When a sperm cell unites with an egg from a woman, it forms an embryo (the first stage of an unborn baby's development).

A low sperm count or abnormal sperm shape or movement can make it difficult for a man to make a woman pregnant. The inability to conceive a baby is called infertility. Infertility can affect men and women. For about one-third of couples unable to have children, male infertility is the reason. A semen analysis can help figure out the cause of male infertility.

Other names: sperm count, sperm analysis, semen testing, male fertility test

What is it used for?

A semen analysis is used to find out if a problem with semen or sperm may be causing a man's infertility. The test may also be used to see if a vasectomy has been successful. A vasectomy is a surgical procedure that is used to prevent pregnancy by blocking the release of sperm during sex.

Why do I need a semen analysis?

You may need a semen analysis if you and your partner have been trying to have a baby for at least 12 months without success.

If you've recently had a vasectomy, you may need this test to make sure the procedure has worked.

What happens during a semen analysis?

You will need to provide a semen sample. The most common way to provide your sample is to go to a private area in your health care provider's office and masturbate into a sterile container. You should not use any lubricants. If masturbation is against your religious or other beliefs, you may be able to collect your sample during intercourse using a special type of condom. Talk to your health care provider if you have questions or concerns about providing your sample.

You will need to provide two or more additional samples within a week or two. That's because sperm count and semen quality can vary from day to day.

Will I need to do anything to prepare for the test?

You will need to avoid sexual activity, including masturbation, for 2–5 days before the sample is collected. This will help make sure your sperm count is at its highest level.

Are there any risks to the test?

There is no known risk to a semen analysis.

What do the results mean?

The results of a semen analysis include measurements of quantity and quality of semen and sperm. These include:

Volume: the amount of semen
Sperm count: the number of sperm per milliliter
Sperm movement, also known as motility
Sperm shape, also known as morphology
White blood cells, which may be a sign of an infection
If any of these results are not normal, it may mean there is problem with your fertility. But other factors, including the use of alcohol, tobacco, and some herbal medicines, can affect your results. If you have questions about your results or other concerns about your fertility, talk to your health care provider.

If your semen analysis was done to check the success of your vasectomy, your provider will look for the presence of any sperm. If no sperm is found, you and your partner should be able to stop using other forms of birth control. If sperm is found, you may need repeat testing until your sample is clear of sperm. In the meantime, you and your partner will have to take precautions in order to prevent pregnancy.

Is there anything else I need to know about a semen analysis?

Many male fertility problems can be treated. If your semen analysis results were not normal, your health care provider may order more tests to help figure out the best approach to treatment.

Tuesday, April 23, 2019

Gel electrophoresis well explained

Key points:

  • Gel electrophoresis is a technique used to separate DNA fragments according to their size.
  • DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel.
  • DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.
  • When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.

Introduction

Suppose you have just done a PCR reaction, making many copies of a target DNA region. Or perhaps you’ve done some DNA cloning, trying to "paste" a gene into a circular DNA plasmid.
Now, you want to check and see whether your PCR worked, or whether your plasmid has the right gene in it. What technique can you use to visualize (directly observe) the fragments of DNA?

Gel electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another.
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All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only. Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another. We can also determine the absolute size of a piece of DNA by examining it next to a standard "yardstick" made up of DNA fragments of known sizes.

What is a gel?

As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.
At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed:
Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is hooked to a positive electrode, while the other end is hooked to a negative electrode. The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current. Although you may not be able to see in the image above (thanks to my amazing artistic skills), the buffer fills the gel box to a level where it just barely covers the gel.
The end of the gel with the wells is positioned towards the negative electrode. The end without wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode.

How do DNA fragments move through the gel?

Once the gel is in the box, each of the DNA samples we want to examine (for instance, each PCR reaction or each restriction-digested plasmid) is carefully transferred into one of the wells. One well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so we would want to pick one with good "coverage" of the size range of our expected fragments.
Next, the power to the gel box is turned on, and current begins to flow through the gel. The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone, so they start moving through the matrix of the gel towards the positive pole. When the power is turned on and current is passing through the gel, the gel is said to be running.
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DNA samples are loaded into wells at negative electrode end of gel.
Power is turned on and DNA fragments migrate through gel (towards the positive electrode).
After the gel has run, the fragments are separated by size. The largest fragments are near the top of the gel (negative electrode, where they began), and the smallest fragments are near the bottom (positive electrode).
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As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones. After the gel has run for awhile, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells. Very short pieces of DNA may have run right off the end of the gel if we left it on for too long (something I've most definitely been guilty of!).

Visualizing the DNA fragments

Once the fragments have been separated, we can examine the gel and see what sizes of bands are found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel.
The bp next to each number in the ladder indicates how many base pairslong the DNA fragment is.

A well-defined “line” of DNA on a gel is called a band. Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position. A single DNA fragment (or even a small group of DNA fragments) would not be visible by itself on a gel.
By comparing the bands in a sample to the DNA ladder, we can determine their approximate sizes. For instance, the bright band on the gel above is roughly 700 base pairs (bp) in size.

Check your understanding


Leftmost lane: ladder with 3000 bp, 1500 bp, and 500 bp bands marked on it.
Lane 1: 5000 bp band.
Lane 2: 100 bp band.
Lane 3: 1500 bp and 2000 bp bands.
Lane 4: 500 bp band.
Four lanes are numbered on the gel above. (A lane is a corridor through which DNA passes as it leaves a well.)