Diagnostic Tests Used for Viruses

Haemagglutination Assay


A simple and rapid method of quantifying the amount of virus in a sample. Haemagglutination is the agglutination of red blood cells. Viruses with envelops or surface proteins are able to bind to the sialic acid, N-Acetylneuraminic acid found in the cell membrane of red blood cells. Because each agglutinating molecule (in this case each viral molecule) can bind to multiple red blood cells, a clump of cells begins to form, this is agglutination. This agglutination forms a lattice structure.

Haemagglutination Inhibition

Haemagglutination inhibition is the addition of an inhibitor of the virus. Antisera (a blood serum which contains antibodies) is used in this case. The antibodies will bind to the virus and thus prevent haemagglutination.

By creating multiple, increasing levels of dilution e.g. 1:2, 1:4, 1:8, etc. of virus to antiserum we can determine a haemagglutination titre, this is the highest value of dilution (i.e. smallest amount of virus) which inhibits agglutination.

A typical haemagglutination inhibition assay looks like this:

You can see that point of agglutination, where the contents of the well go from a homogenous cloudy red, to containing a red spot which is the agglutinated red blood cells. In the above example, sample E (the highlighted row) has a haemagglutination titre of 1:512 as this is the largest dilution which prevents haemagglutination.

By monitoring the haemagglutination titre, we can determine how the virus is progressing over time, for example:

  • 1st assay – an endpoint is reached at a dilution of 1:4
  • 2nd assay – an endpoint is reached at a dilution of 1:16
  • 3rd assay – an endpoint is reached at a dilution of 1:8

From this would we be able to see initially the virus count is low as it is inhibited at a low dilution, however at the next assay the haemagglutination titre is higher (greater dilution of virus reached before it is inhibited) this must mean the virus is replicating as the initial count of virus is higher (i.e. takes greater amount of dilution to reach inhibition). WIth the final assay, we can see that because the haemagglutination titre has decreased, the virus must be reducing in numbers, indicating recovery.



ELISA is an acronym for Enzyme-linked immunosorbent assay and is used to detect whether or not a certain antibody or antigen is present in a sample. To indicate the presence of an antibody or antigen, reporter molecules are used which are identifiable by a change in colour. Therefore, if an antigen or antibody is present, a colour change will be observed.


A typical ELISA requires the use of a 96-well microtitre plate (as used in the haemagglutination assay), but the wells of the plate are coated with a known or unknown antigen. When diagnosing viruses, the antigen will be unknown as it is the sample which we are testing. To this an antiserum is added, if the virus antigen is present in the sample, then the antibodies will bind. If not, no binding will occur.

To determine whether or not an antibody-antigen complex has formed (i.e. a positive diagnosis of the virus), an antiglobulin is added. This is an antibody which binds to the antibodies used in the initial serum. For example, Goat anti-rabbit IgG is a rabbit antibody (Immunoglobulin G [IgG]) which will bind to goat antibodies. There are many possible variations of antiglobulins.

The antiglobulin will be ‘labelled’ with an enzyme (an enzyme is attached to the antibody which has a negligible effect on its binding capabilities).

Finally, the substrate of the enzyme using to label the antibody is added. This substrate will be broken down if the secondary antibody (antiglobulin) bound to the primary antibody. The break down of the substrate will be coupled with a colour change, for easy identification that this has occurred. The greater the intensity of the colour change, the higher the concentration of the initial antigen.

Sometimes a spectrophotometer (a device used to detect light intensity) will be used to determine the degree of change in colour, as this can be used to calculate reasonably accurate values for initial antigen concentration and thus virus concentration in the host.

Below is an example of the variations in colour which occur during an ELISA test. The coloured wells indicate positive samples:

This type of ELISA is known as an indirect ELISA test, however there are other forms of this test, such as sandwich ELISA, which is where the well is coated in antibody, to which an antigen binds and to this another antibody is added. The antiglobulin tests for the secondary antibody.

There are also competition ELISA tests, where the added sample is an antibody-antigen complex, this is added to antigen coated wells. If the concentration of initial antigen in the sample is high then there will be fewer available antibodies to bind with the antigen in the wells. The wells are washed to remove unbound antibodies and as with the indirect assay, enzyme-coupled antiglobulins are added with substrate that elicit a colour change. However in this case a high initial concentration of antigen in the sample will yield a low change in colour.



Leptospirosis is a zoonotic disease caused by the bacterial genus Leptospira. Leptospires are spirochetes, a group of Gram-negative bacteria with long, thin, spiral structures and an internal flagella used for movement. The size of a typical leptospire is around 0.1μm wide and 6-20μm long. This narrow, helical structure enables them to burrow in to tissues, within tissues they may adopt a more spherical or granular appearance.


The primary pathogenic strain of Leptospira is Leptospira interrogans, however there are also non-pathogenic strains such as Leptospira biflexa which is an environmental saprophyte (i.e. consumes dead organic matter).There are currently around >16 species of identified Leptospira.

Serovars & Serogroups

Serovars are groups of organisms, categorised depending on the antigens they present on their surface. Therefore if a number of organisms within a Leptospira species share the same antigens on their surface, they will be grouped together into one serovar. Differences in surface antigens occur within the same species for example, within the pathogenic L. interrogans species, around >250 serovars have been identified worldwide.

Serogroups are clusters of serovars, as there may be hundred of serovars within a species, it is useful to group those together which share similar properties. Important serovars of L. interrogans include:

  • Canicola (Primary reservoir host – Dog)
  • Icterohaemorrhagiae – (Rat, mouse)
  • Bratislava – (Rat, pig, horse)
  • Pomona – (Cattle, pig, skunk)
  • Grippotyphosa – (Rodents)
  • Hardjo – (Cattle)

Each serovar is not limited to its primary reservoir host however. They be transmitted to incidental hosts fairly easily. Incidental hosts include humans, dogs and cats as well as other domesticated animals.

Different serovars are also responsible for different clinical conditions which can range from abortion to haemorrhagic disease. An individual serovar may also cause different clinical conditions in different species, for example L.interrogans serovar hardjo causes abortion and still births in cattle, but in humans it can cause an influenza-like illness or liver/kidney diseases.


Transmission Cycle

The transmission cycle of a typically Leptospira species is as follows:

  • Rodents shed Leptospira in their urine
  • Direct transmission of Leptospira to humans may occur at this stage
  • The urine contaminates the environment (e.g. soil, water) with Leptospira
  • Indirect transmission to humans may occur at this stage
  • Leptospira may be transmitted to other domestic animals via the environment
  • These animals may become infected and can shed Leptospira in their urine which can lead to direct transmission to humans or contaminate the environment as before

Rodents can acquire Leptospira from the urine contaminated environment, thus creating a cycle of transmission

Transmission via direct contact usually occurs by urine which contains the Leptospira organisms. However direct transmission may also occur via veneral or placental transfer as well as bite wounds or the ingestion of infected tissue material. Crowding of animals (such as in kennels or intensive farming) will enhance transmission of Leptospira. Animals which recover from the disease, may still be infected, thus making them carriers of Leptospira which can still be excreted chronically in their urine. This continues the spread of infection

Transmission via indirect contact can also occur. Methods of indirect transmission generally requires exposure to contaminated sources such as; soil, food, bedding or water sources. The bacteria enters susceptible hosts from the contaminated source via damaged skin or exposed mucous membranes such as in the nose, mouth, eyes etc. Leptospira remains viable in the environment (still able to cause infection) for months, this further enhances transmission.

Environmental Factors

The optimal habitat for Leptospira depends on their environment, if aquatic, optimal conditions are stagnant or slow moving waters. If terrestrial, a neutral or slightly alkaline soil pH is preferred. However, organisms may survive transiently in undiluted acidic urine. A typical temperature range of 0-25C is preferred, this often leads to seasonal fluctuations in the incidence of Leptospirosis.


Leptospira enters the host by penetrating mucous membranes via vunerable areas such as damaged skin, eyes, nose or the mouth. Their helical shape and flagella aids in tissue penetration. Upon entering the blood system, they begin to multiply rapidly. The presence of bacteria in the blood is called bacteraemia. They are then distributed around the body via the blood stream.

Once distributed around the body, they then further replicate in target organs and tissues (including the kidney, liver, spleen, central nervous system, eyes and genital tract). The incubation period is around 7 days, this factor depends on the species and the strength of the host immune system however.

The initial immune response will usually remove all Leptospira organisms from the blood and tissues but some will persist in the kidney tubules where they can continue to replicate. The Leptospira organisms in the kidney tubules manage to evade the host immune response by avoiding phagocytosis.

The damage done to the host’s organs and tissues is variable and depends on the virulence of the Leptospira serovar and how susceptible the host immune system is. The most serious of diseases occur in the incidental hosts, i.e. not the primary reservoir host.

Overview of Pathogenesis


At present there are three different methods of leptospirosis diagnosis:

  • Detect leptospire antigens – Leptospire antigens will induce agglutination of antibodies. This can be tested using a microscopic agglutination test (MAT).
  • Isolation of Leptospires – Leptospires are isolated from the urine or infected tissues. However this can be very labour and time intensive as Leptospira species are slow to culture using growth medium, meaning it can take weeks before a positive/negative result is returned. Despite this, this method of diagnosis is probably the most reliable.
  • Polymerase Chain Reaction (PCR) – Molecular methods of diagnosis (such as PCR) are gaining popularity for diagnosing Leptospirosis, however PCR is unable to distinguish between serovars.


For dogs there are currently two forms of vaccine available:

  • In the UK, a bivalent vaccine is used which protects against two serovars – canicola and icterohaemorrhagiae.
  • In the USA however, a quadrivalent vaccine is used, this protects against four serovars – grippotyphosa and pomona as well the canicola and icterohaemorrhagiae which the bivalent vaccine covers.

Some preventative measures are also being taken in cattle to protect against Leptospira borgpetersenii serovar hardjo.

The widespread use of these bivalent vaccines may be responsible for the observed decline in classic canine Leptospirosis infections, however this vaccine does not provide cover for other serovars.

In the USA canine leptospirosis has been classified as a re-emerging disease due to the increasing amounts of newly diagnosed cases. This may be due to the prevalence of grippotyphosa, pomona and bratislava in wild reservoir species which are spreading Leptospira through the domesticated animal population. This is good reasoning behind the introduction of the quadrivalent vaccine as it protects against these serovars (not bratislava, however cases of bratislava are low).

In the UK, rural cases of canine leptospirosis are greater than urban cases, possibly hinting at a greater transmission via wildlife. This is a breakdown of serovar cases diagnosed in the UK:

  • 60% – L. icterohaemorrhagiae
  • 20% – L. canicola
  • 6% – L. icterohaemorrhagiae copenhageni
  • 1.3% – L. bratislava

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2010 in review

I would like to thank everyone who visited VetSci in 2010! This year, the site proved very popular and it could not have achieved it without you! Averaging at around 5,000 views per month now (and continuing to grow) this blog has become more popular than I ever thought. I can only hope that it has helped you all. It is the high level of activity that ensures my motivation to keep adding to the blog – if you want to be a part of this then contact me by clicking my ‘Gravatar’ in the sidebar to the right. I thought you may like to see how the blog did in 2010, which is why am I sharing these stats provided to me by the wordpress team:

The stats helper monkeys at WordPress.com mulled over how this blog did in 2010, and here’s a high level summary of its overall blog health:

Healthy blog!

The Blog-Health-o-Meter™ reads This blog is on fire!.

Crunchy numbers

Featured image

The average container ship can carry about 4,500 containers. This blog was viewed about 21,000 times in 2010. If each view were a shipping container, your blog would have filled about 5 fully loaded ships.

In 2010, there were 34 new posts, growing the total archive of this blog to 54 posts. There were 27 pictures uploaded, taking up a total of 6mb. That’s about 2 pictures per month.

The busiest day of the year was December 6th with 308 views. The most popular post that day was Arterial Blood Supply to the Brain .

Where did they come from?

The top referring sites in 2010 were facebook.com, jameswatts.co.uk, kmle.co.kr, en.wordpress.com, and search.conduit.com.

Some visitors came searching, mostly for circle of willis, blood supply to the brain, meninges, brain blood supply, and intrasexual selection.

Attractions in 2010

These are the posts and pages that got the most views in 2010.


Arterial Blood Supply to the Brain February 2010
2 comments and 1 Like on WordPress.com,


Cellular Anatomy of the Nervous System March 2010


Meninges, CSF & Venous Drainage February 2010
1 comment


Special Senses – Hearing May 2010
1 comment


Appetite Control & Maintaining Constant Body Weight May 2010

Strategies of Diving Mammals


Diving to depth is a typical behaviour observed in many aquatic mammals such as; whales, otters, seals, walrus and many others. Such mammals are well adapted at diving. The Weddell seal for example, is able to dive for periods of 80 minutes and more. Compare this to the average 1-3 minutes in untrained humans and you can see that diving mammals have developed great abilities for diving to depths.

The two major problems associated with diving are; limited oxygen stores (limiting the length of the dive) and hydrostatic pressure (limiting the depth of the dive). Oxygen is an important factor during diving, an absence of oxygen (anoxia) means anaerobic respiration becomes the only source of energy. Anaerobic respiration consists only of glycolysis, which produces around 5% of the energy (ATP) produced during aerobic respiration. Such a great deficit in ATP production can damage brain cells and have fatal consequences. In anaerobic respiration, lactate is produced as a waste product. Normally (in aerobic conditions) the lactate is metabolised by the presence of oxygen, in its absence however, the lactate builds up and dissociates into lactic acid. This causes fatigue, which decreases swimming ability.

The dangers of hydrostatic pressure come include; physical damage as a result of the pressure (barotrauma). Especially gas filled spaces of the body such as the lungs and ears. As well as decompression sickness (referred to as ‘the bends’ in humans), caused by rapid changes in pressure.

Oxygen Stores

The main stores or oxygen in mammals are; the lungs, blood and muscle. Oxygen is also present dissolved in body tissue. In adept diving mammals such as the Weddell seal the primary store of oxygen is the blood (in haemoglobin), followed by the muscle (in myoglobin) and then the lungs. The Weddell seal actually exhales before performing a dive.

Having a large store of oxygen in the lungs causes some problems. A large lung volume can make the dive more energetically costly due to the extra energy spent negating the effect of increased buoyancy. Exhaling pre-dive decreases energy required to dive. Another factor to consider is that, being a gas filled space, the lungs are susceptible to hydrostatic pressure. At large enough depths, the pressure can cause the lungs to collapse. So, a large lung volume is not necessary for accomplished diving mammals and some will exhale before diving to reduce the lung volume further.

The blood has an excellent oxygen carrying capacity, oxygen binds with haemoglobin in the blood and this essentially dissolves the gas, thus reducing the volume and associated problems. Adept divers will have a greater volume of blood (and haemoglobin) and therefore are able to store more oxygen. Humans are typically able to store 15ml of oxygen per Kg of body weight, whereas accomplished divers can store anything between 40-70+ ml Kg-1.

Myoglobin is similar to haemoglobin, it is found in the muscles and transports oxygen, but it has a much higher affinity for oxygen. Large concentrations of myoglobin mean diving mammals are able to dive for much longer, therefore high levels of myoglobin are associated with accomplished divers.

No matter how great the ability to store oxygen, there will always be a time when oxygen stores are exhausted. This is known as the aerobic diving limit. It is the length of time an organism is able to dive and respire aerobically from stored oxygen. Dives longer than the aerobic diving limit are possible, but are often rare even in capable diving mammals. Taking the Weddell seal as an example, 80% of dives occur within the aerobic diving limit (about 18-20 minutes) but some still dive for periods of 80 minutes or more. This shows they have developed systems to help combat the buildup of lactate and the lack of oxygen which occurs during anaerobic dives of 20 minutes or more.

Extending Dives beyond the Aerobic Diving Limit

The body is still able to produce energy (ATP) without oxygen via glycolysis during anaerobic respiration but this is at an effective efficiency of 5%. Therefore glucose and glycogen stores (the body’s fuel) would be depleted at 20x the normal rate if glycolysis increased to match aerobic ATP production. Such an increase is known as the ‘Pasteur Effect’, but if the diving mammal is able to decrease metabolism, this might not be necessary.

Diving mammals are able to undergo metabolic depression, which reduces the energy demand of certain body systems by reducing their output. The only problem is that the mammalian brain is very energy hungry and requires a lot of ATP to remain functional.

The ‘Diving Reflex’ is observed when diving past the aerobic diving limit. This reflex includes the slowing of the heart rate (bradycardia) and regional vasoconstriction to produce metabolic depression.

The Diving Reflex

The bradycardia seen in the diving reflex, reduces the heart rate from around 60-70 to 5-10 beats per minute. This is combined with regional vasoconstriction to increase the length of dives. The regional vasoconstriction is seen across the body except the brain as it would be unable to survive any reduction in blood flow. The amount by which blood flow is reduced across the rest of the body varies from 80-95%. This essential sets up a near closed blood flow from the lungs, to the heart, to the brain. Blood flow from the heart to the rest of the body is minimal.

Bradycardia prevents a change in stroke volume, blood pressure and blood flow, which if altered may damage the body. So even though the dimensions of the cardiovascular system have been reduced, the blood flows around the ‘closed circuit’ of the heart, lungs and brain as it would flow round the complete cardiovascular system under aerobic conditions.

The ‘closed circuit’ is able to remain aerobic from oxygen stores in the blood, muscles and lungs. The rest of the body remains aerobic only for as long as the oxygen bound to the myoglobin of the muscles lasts, barely any oxygen is received from the blood or lungs due to the extreme vasoconstriction. This means the rest of the body becomes anaerobic much faster than the ‘closed circuit’, the anaerobic respiration results in production of lactate.

Because of this, high levels of lactate are observed in the muscles, whereas levels are low in heart, lung and brain. Lactate produced by anaerobic respiration of the rest of the body is stored in the muscles until resurfacing. Lactate remains in the muscles due to the regional vasoconstriction, which essentially has disconnected them from the blood flow.

Upon resurfacing, regional vasoconstriction and bradycardia cease. This results in a ‘Wash out’ of lactate. All the lactate stored in the muscles during the dive is released into the bloodstream, thus increasing blood lactate levels. The lactate is metabolised however, in the presence of oxygen. The lactate produced during the dive must be metabolised before another anaerobic dive may occur. Therefore the longer the dive, the longer it takes before another dive may occur. The period of time between dives spent metabolising lactate, is known as the recovery period. Because of this, long dives are not desirable (unless necessary, such as escaping a predator) as it means less time can be spent beneath the water.

Energy Saving Behaviour

To further preserve energy, diving mammals will perform diving locomotion which reduces muscle energy requirements as much as possible. Diving mammals will use a combination of actively stroking for propulsion and gliding to preserve energy. Typically the descent of a dive requires stroking (combined with brief glides) to reach large depths due to buoyancy of the animal. The ascent however consists almost completely of long gliding behaviour in an attempt to expend as little energy as possible. Stroking it is not needed due to buoyancy, which causes the animal to rise to the surface.

Another energy saving behaviour is the induction of voluntary hypothermia. Whilst diving, mammals actively seek cooler waters, as a decrease in temperature causes a decrease in metabolic rate. If the animal is able to decrease their metabolic rate, the length of the dive can be extended.

Hydrostatic Pressure

Pressure can be measured in terms of atmospheres. At sea level (0m), pressure is the equivalent of 1 atmosphere or 1atm.Underwater, the pressure increases by 1atm per 10 meters, therefore at 10 meters below sea level, the pressure is 2atms. At 100 meters below sea level, the pressure rises to 11atms.

Boyle’s law states that pressure multiplied by volume equals a constant value (at a constant temperature) or PV= Constant. This means, if pressure increases, volume decreases (and vice versa). This is important because the body contains many gas filled spaces such as the lungs. Gas is easy to compress, and so as pressure increases (i.e. As we travel deeper into the ocean) the volume of these gas filled spaces within the body decrease in volume.

In order to understand the changes of the body with pressure, there are two other important pressure laws, these are:

Dalton’s Law – The total pressure of a gas constitutes all of the partial pressures within the gas. For example, the total pressure of air at sea level (or Ptot) = 1atm. This means all partial pressures of gases within air must total to 1atm. PN2 (or the partial pressure of nitrogen) = 0.78atm and PO2 = 0.21atm therefore the partial pressures of all the other gases in air must equal 0.01atm.

Henry’s Law – The solubility of a gas in a liquid is proportional to the partial pressure of the gas (above the liquid). This means that if the PN2 in the lungs increases, the amount of nitrogen which dissolves into the blood also increases.

Problems Associated with Hydrostatic Pressure

The main concerns of hydrostatic pressure are; the toxicity of the gases, decompression sickness and barotrauma. The potential of damage being caused by these factors increases as hydrostatic pressure increases.

At certain depths, nitrogen and oxygen can become toxic. At around 20m depth nitrogen can begin to have affect consciousness – altering perception. At around 50m oxygen can cause damage to the central nervous system, lungs and eyes.

Barotrauma is physical damage as a result of pressure. Gas filled spaces are prone to such damage. Major targets of barotrauma are the thorax and the trachea, if the trachea is not strengthened at great depths, the high pressure can cause the trachea to collapse. This is similar with the thorax, as pressure increases, the size of the lungs decreases. At a depth of 40m the lungs are 1/5 of their size at sea level. Such a large decrease in size could cause damage to thoracic muscles. By having a strengthened trachea, elastic diaphragm and strong sternum, it is possible to prevent such damage.

Decompression Sickness

Decompression sickness occurs due to resurfacing too quickly, this is a result of how pressure of nitrogen changes within the body at depth:


PN2 (0.8atm) is equilibrated between the lungs blood and tissues.

Dive to 30 meters (Compression)

Ptot has increased to 4atms. Due to Dalton’s law, PN2=3.2atms. Due to Boyle’s law, the volume of the lungs has decreased.

PN2 in the lungs quickly reaches 3.2atms, due to their compression. PN2 in the blood slowly equilibrates followed by the tissues. Eventually lungs, blood and tissues have all equilibrated to PN2=3.2atms.

Resurfacing (Decompression)

Resurfacing too quickly means PN2 in the blood and tissues remains at a value near 3.2atms as there was not a chance to equilibrate with the lungs. At the surface, PN2=0.8atms in the lungs, whilst blood and tissues PN2 still equals 3.2atms. This results in a rapid release in pressure resulting in the formation of bubbles in the blood. The bubbles consist of nitrogen at a partial pressure of 0.8atms and so it is energetically favourable for any remaining dissolved nitrogen to diffuse into these bubbles. Because of this, the bubbles can quickly grow in size.

Decompression sickness arises due to any rapid change in pressure (not just changes in pressure when diving). It is also known as either ‘Caisson’s Disease’ or the ‘Bends’. The only treatment available is to contain the sufferer in a hyperbaric chamber – a chamber where pressure can be controlled and allowed to rise steadily, preventing further formation of nitrogenous bubbles in the blood.

Decompression sickness can arise due to the rapid resurfacing from large depths underwater, this results in a large gradient of partial pressures between the blood, lungs and tissues. The offloading of nitrogen from the blood into the lungs is a relatively slow process and therefore takes time. By resurfacing too fast, the pressure is quickly released causing gases (nitrogen) to go out of solution. This results in the formation of nitrogen bubbles in the blood. The bubbles can join together to become larger and more dangerous, whilst nitrogen is able to diffuse in, further increasing the size. It is energetically favourable for nitrogen to diffuse into the bubbles as they are at normal atmospheric pressure. If decompression sickness reaches the spinal cord, it can result in paralysis.

Avoiding Decompression Sickness

There are basically two ways to combat decompression sickness; limit the load of nitrogen into the body and prevent its distribution. To limit the load of nitrogen, diving mammals have evolved specialised alveoli.

An alveolus consists of a sac-like, bulbous area (where gas exchange takes place) and a terminal bronchiole (a tube connecting to the alveolus where no gas exchange takes place). In terrestrial mammals, pressure (such as that experienced whilst diving) causes the bronchiole to collapse, trapping nitrogen within the bulbous end of the alveoli. With nowhere to go, the nitrogen moves out into the tissues and blood (increasing nitrogen load and the possibility of decompression sickness).

The bronchioles of diving mammals on the other hand consist of strengthened cartilage, preventing them from collapse. Under pressure it is the alveoli which compresses first. Nitrogen in the alveolus moves out into the bronchiole. As no gas exchange occurs in the bronchiole, nitrogen is not able to move into the surrounding blood and tissues. Nitrogen load is therefore decreased as is the possibility of suffering from decompression sickness upon resurfacing. Such alveolar collapse occurs at a depth of 30m in Weddell seals, meaning the partial pressure of nitrogen in the tissues does not increase further than 3.2atms.

The other method of avoiding decompression sickness is to limit the distribution of nitrogen. This is done by peripheral vasoconstriction. As with oxygen limited dives that extend past the aerobic diving limit, regional or peripheral vasoconstriction result in decreased blood flow to the body. Blood flow is only preserved to the lungs, heart and brain. The benefit of this is that fatty tissue (i.e. the areas to where blood flow is restricted) has high nitrogen solubility, so reducing the blood flow to these areas also reduces the amount of nitrogen able to dissolve into the tissue. The brain on the other hand consists mainly of watery tissue which has much lower nitrogen solubility; it also offloads nitrogen much faster and therefore has a reduced risk of bubble growth.

Surviving Anoxia


Oxygen comprises a large proportion of our atmosphere (21%) and has become a vital gas for many organisms on Earth. Without it our ability to produce energy (as ATP) via aerobic respiration fails. Oxidative phosphorylation, the primary method of aerobic ATP production, produces large amounts of ATP (from our glycogen stores) just as long as we are able to continually supply O2. Without oxygen we resort to anaerobic respiration, where the primary method of ATP production is glycolysis. In comparison to oxidative phosphorylation, the amount of ATP produced is minimal.

Whilst we take oxygen for granted in our environment, there is a large variety of situations where oxygen levels are limited, or where oxygen is completely absent. Typically, such conditions arise in aquatic environments where the ability of oxygen to diffuse is greatly reduced and oxygen levels depend heavily on the ability of organisms to photosynthesise. Yet many respiring organisms are able to live in these environments due to adaptations to low oxygen/ zero oxygen situations.

When referring to the content of oxygen in an environment, we usually state its partial pressure i.e. the proportion of the total gas which comprises oxygen. At sea level, the total pressure of the gases in the environment is 101kPa (or 1 atmosphere), as oxygen makes up 21% of these gases, the partial pressure of oxygen at sea level is 21kPa.

From this we can define four terms:

Hyperoxia (>21kPa)  – When oxygen levels are greater than normal

Normaxia (21kPa)      – When oxygen levels are normal

Hypoxia (<21kPa)      – When oxygen levels are below normal

Anoxia (0kPa)              – When oxygen is completely absent

Why We Require Oxygen

Aerobic Respiration

The major role of oxygen is to provide the body with metabolic energy in the form of ATP. This is done during aerobic respiration via a process known as oxidative phosphorylation which occurs within the inner mitochondrial membranes. The role of oxygen in this process is to act as a terminal electron acceptor. The transfer of electrons to oxygen provides enough energy to phosphorylate ADP to ATP.

However, for oxygen to form ATP in this manner, there are a number of prerequisites. Glucose (a 6-carbon molecule) must be readily available, if it is not it must be mobilised from the body’s glycogen stores. There are 3 major steps which occur before oxidative phosphorylation:

Glycolysis – Glucose is metabolised into 2 molecules of pyruvate (a 3-carbon molecule). This provides a small amount of ATP as well (2 per glucose molecule).

Pyruvate is metabolised into 2 molecules of Acetyl CoA (a 2-carbon molecule). One molecule of carbon is lost as CO2.

The Krebs cycle – Each molecule of acetyl CoA undergoes a number of enzyme-catalysed reactions producing a number of highly energised electrons. These electrons are used in the electron transport chain to produce vast amounts of ATP.

Anaerobic respiration

However, oxygen isn’t always available. In this situation, respiration may continue but it can go no further than glycolysis. As a result, the ATP yield per molecule of glucose alters dramatically:

Glycolysis Krebs Cycle Oxidative Phosphorylation
Aerobic 2 2 34
Anaerobic 2 0 0

Anaerobic Glycolysis

The efficiency of ATP production drops to around 5% during anaerobic reproduction. One way the body can cope with this is to increase the glycolytic rate (the rate of glycolysis). Increasing the glycolytic rate by about 20x returns the level of ATP production to that seen in aerobic respiration. This is known as the Pasteur Effect.

There are of course negative effects as a result of this:

Increasing the glycolytic rate causes a rapid drop in the body’s store of glycogen. Without glycogen, the body will be unable to perform any form or respiration – anaerobic or not, which will inevitably lead to death.

The Krebs cycle stops soon after oxygen becomes absent. This means there is no demand for acetyl CoA. The result of this is excess production of pyruvate. Lactate dehydrogenase converts the excess pyruvate to lactate (and also recycles NADH + H+ to NAD+ allowing glycolysis to continue). A build up of lactate is also undesirable however as it immediately dissociates into lactic acid. The low pH of lactic acid will begin to reduce cellular pH and denature sensitive enzymes.

Decreasing Cellular pH

As a result of decreased pH and ATP, maintaining the membrane potential becomes very difficult. The Na/K ATPase pump which actively removes N+ from the cell and uptakes K+ in order to maintain the correct (-70mV) membrane gradient, stops functioning when ATP production diminishes. The maintained -70mV gradient begins to equilibrate at 0mV, causing a breakdown of the transmembrane gradient within a matter of minutes.

This has dire consequences on the mammalian brain in particular; the change in the membrane potential allows calcium ions to enter the cell. Intracellular calcium causes cell damage and hastens enzyme breakdown. The breakdown of these important brain cells causes permanent brain damage and if not supplied with oxygen quickly enough, will lead to death.

Strategies to Survive Anoxia

To survive anoxic conditions, an organism must be able to deal with the problems associated with anaerobic respiration; keeping up ATP levels (particularly in the brain) and avoiding acidification and associated cellular damage. Two organisms which are highly adapted to surviving anoxic conditions are; the crucian carp and the painted turtle. Both have different strategies for maintaining the body without oxygen, but both do so by combating the associated problems with anaerobic respiration.

Glycogen Stores in the Crucian Carp

One of the problems with anaerobic respiration is that, after a short time in anoxia, glycogen levels decrease rapidly. The Pasteur effect (where glycolytic rate increases for a short period to accommodate the lack of ATP production from oxidative phosphorylation) is only temporary. All body stores of glycogen are quickly depleted and the body is no longer able to survive.

The crucian carp (and to a smaller extent the painted turtle) has an unusually large glycogen reserve in the liver (30% of the total liver mass). Such large stores of glycogen allow the crucian carp to survive in anoxia by glycolysis alone for a much longer period of time.

The crucian carp will typically only face anoxic conditions during the winter months, when a thick layer of ice forms over the water sources they inhabit. Because of this, the crucian carp has adapted to build up glycogen stores throughout the year so that come winter, the glycogen stores are as great as possible. To do this, during the warmer summer months, instead of using carbohydrates for energy (i.e. Glucose / glycogen) lipids are used, allowing the crucian carp to build glycogen stores.

Metabolic Depression in the Crucian Carp

Although increased glycogen stores length the period of time the crucian carp is able to survive in anoxic conditions, it is not the only factor which prolongs this survival period. Metabolic depression means slowing down the rate at which the body consumes ATP, so combining reduced ATP requirements with greater stores of glycogen to produce ATP ensures a near maximum survival time in anoxia.

To reduce metabolic rate, locomotion is reduced during anoxia this means muscles use as little energy as possible. Also, some sensory functions are suppressed, these include vision and hearing. During the winter months when anoxic conditions arise, the thick layer of ice above reduces light levels to a minimum so vision is not a necessity.

Crucian carp will also induce voluntary hypothermia during anoxia. This is actually beneficial to the carp because at lower temperatures, metabolic rate decreases. Therefore when anoxic conditions arise, the crucian carp will swim to cooler waters to reduce their metabolism.

Avoiding Acidification in the Crucian Carp

The problem with glycolysis is the production of lactate and hence lactic acid, causing a decrease in cellular pH and thus acidification. If the crucian carp is producing ATP from glycolysis alone then cellular concentrations of lactic acid would normally quickly rise and pose a major threat for the carp. However, instead of producing lactate as the end product of glycolysis, the crucian carp has adapted to produce ethanol.

By producing ethanol instead of lactate, the crucial carp benefits in a number of ways. Primarily ethanol is non-acidic and thus will not decrease intracellular pH (therefore avoiding the associated damage to the cell as a result of acidification). Ethanol is also lipid soluble meaning it will not build up in the body as it is easily excreted across the gills.

Normally, during glycolysis, pyruvate is metabolised to acetyl CoA by pyruvate dehydrogenase – producing lactate as the end product. Yet a mutation in the pyruvate dehydrogenase enzyme of the crucian carp means that instead of acetyl CoA, acetaldehyde is produced. Acetaldehyde is then metabolised by alcohol dehydrogenase to produce ethanol.

However, the brain is unable to produce ethanol in this manner, only skeletal muscle can. Any glycolysis occurring in the brain will therefore produce lactate as the end product. This lactate however, is transported via the blood, to the skeletal muscle. Here, the lactate is turned into pyruvate where glycolysis will metabolise it into ethanol, it is subsequently excreted across the gills.

Avoiding Acidification in the Painted Turtle

The painted turtle deals with anoxia in a different way to the crucian carp. Whereas the crucian carp has very large glycogen stores and avoids acidification by producing ethanol, the painted turtle favours metabolic depression. Although the crucian carp also depresses its metabolism, it is on a much smaller scale. The crucian carp is still aware and the brain functions as normal. The painted turtle on the other hand becomes almost ‘comatose’ as many more body systems are shut down.

The painted turtle must too deal with the excess of lactate produced during glycolysis to avoid acidification. The painted turtle produced lactate as the end product of glycolysis and does not have the ability to form ethanol from pyruvate. To avoid acidification, it must therefore buffer the low pH of the lactic acid.

To buffer the lactic acid, the painted turtle uses bicarbonate and carbonate. The turtle has large stores of these in its shell (which has a rich blood and nerve supply). The carbonates are able to ‘mop up’ excess H ions (which make the blood acidic) and through a number of reactions, the carbonate and H ions and turned into carbon dioxide and water.

The shell is also a rich store of calcium ions. These calcium ions are able to bind with lactate and the resulting molecule becomes stored in the shell until it is possible to excrete the lactate during aerobic respiration.

This method of avoiding acidification is arguably not as efficient as the crucian carp (producing non-acidic ethanol) but it does help the turtle to avoid acidification for a much longer period of time than would normally be possible.

Metabolic Depression in the Painted Turtle

The painted turtle is able to survive anoxia for such extended periods of time due to the amazing amount at which it is able to decrease its metabolic rate by (90-95%). All non-essential systems are shut down which causes the turtle to become ‘comatose’ as the brain becomes quiescent. Even essential systems such as protein synthesis cut their ATP demand drastically. However on system reduces its ATP demand as little as possible -the Na/ K ATPase pump.

As we know, if the Na/ K ATPase pump stops functioning, then the transmembrane gradient breaks down due to the leakage of Na & K ions across the membrane. This allows calcium ions to enter the cell and cause fatal destruction if normoxic conditions are not resumed. However, the painted turtle is not able to maintain 100% efficiency of this pump during anoxia due to the large demand of ATP it requires. Its efficiency is essential cut down to 25%.

To combat the reduction in the ATPase pump’s efficiency a phenomenon known as ‘Channel Arrest’ occurs. This allows the retention of the transmembrane gradient. Normally Na & K are able to leak into and out of a cell via transmembrane ion channels, down their respective concentration gradients. However during channel arrest these ion channels are forced to close greatly decreasing the amount of ion leakage into and out of the cell. This essentially counteracts the 4-fold reduction in the ATPase pump’s efficiency and allows for the preservation of the transmembrane gradient – thus preventing cell damage.

Another phenomenon of the painted turtle which allows for further depression of the metabolic rate is ‘Spike arrest’. This inactivates Na & K ion channels normally associated with producing action potentials. However as these channels are inactivated the action potential threshold becomes greatly elevated – thus forcing the brain into a quiescent state and preventing responses to stimuli.

The Role of Neurotransmitters and Metabolites

Certain molecules are involved with mediating the transition to an anoxic state. The main ones are the neurotransmitters; glutamate and GABA as well as the metabolite adenosine.

Adenosine release is associated with low levels of oxygen. When low blood oxygen is detected, adenosine is released shortly after. The immediate result is an increase of cerebral blood flow. This decreases soon after in the painted turtle as the metabolic rate is lowered – however as the crucian carp remains ‘active’ during anoxia, cerebral blood flow does not reduce.

Adenosine is basically responsible for ensuring ATP production is able to meet demand as it is responsible for a number of factors contributing to the metabolic depression associated with anoxia. It blocks the production of glutamate (an excitatory neurotransmitter) and has been found to suppress protein synthesis and down-regulate NDMA receptor activity.

The NDMA receptor is highly permeable to Ca2+ ions, which are responsible for cell damage in anoxic conditions of unspecialized organisms. Normally in anoxic conditions excessive glutamate over-stimulates this receptor and causes a massive influx of calcium ions into the cell. Therefore the ability of adenosine to down regulate this receptor is highly beneficial during anoxia.

GABA and glutamate are highly related neurotransmitters; GABA is an inhibitory neurotransmitter and glutamate an excitatory neurotransmitter. During anoxia, we see a steady rise in GABA and a steady decline in glutamate. An initial steep rise of GABA in the painted turtle is due to increased levels of adenosine (released at low blood oxygen levels). Because GABA is an inhibitory neurotransmitter it is able to effectively reduce the amount of ATP the body requires, as the painted turtle reduces its metabolism by 90-95% we see very large amounts of GABA released. Release of GABA is seen in the crucian carp too, but as the carp remains active, GABA levels observed are much more modest.


Effectively we see two different strategies for combating anoxic conditions. The painted turtle, although it has larger glycogen stores than other animals, chooses to reduce its metabolism by such a large amount that it becomes almost comatose. This is achieved by Spike arrest, channel arrest, nearly a total suppression of nervous activity and a huge increase in extracellular inhibitory neurotransmitter, GABA. The limiting factor of how long the painted turtle can remain in anoxia is therefore the build-up of lactate. Although it is steadily removed and stored in the shell, it eventually builds up enough to reduce cellular pH to fatal levels if normoxic conditions are not returned.

The crucian carp on the other hand has glycogen stores so large it is able to survive on glycolysis alone at a metabolic rate not too far below normal. In terms of metabolic depression the crucian carp only moderately suppresses nervous activity, coupled with moderate release of GABA and suppression of locomotor activity (as well as voluntary hypothermia to decrease metabolic rate further). The key strategy for survival is the production of the non-acidic glycolytic end product – ethanol, thus preventing cellular pH from falling to fatal levels. Because of this, the limiting factor of anoxia survival in crucian carp is therefore the amount of glycogen stored in the liver.


Crucian Carp Painted Turtle
Activity level during anoxia Slightly reduced Comatose, quiescent brain
Production of ATP from glycolysis Great Above average
Glycogen storage Great Above average
End product of glycolysis Ethanol Lactate (Lactic acid)
Blood pH and cellular pH Stable (ethanol is non-acidic) Slowly rises when uptake of lactate decreases
Overall metabolic depression 70% 90-95%
ATP levels and membrane potential of the brain Maintained at normoxic levels – Brain function is preserved Maintained at normoxic levels – Brain function is preserved
Suppression of neuronal activity Above average Great
Channel & Spike Arrest No Yes