Cure closer for sweet itch in horses

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The pony in the picture shows typical signs of sweet itch. In severe cases such as this, the scabs and sores extend all the way down the mane along the back to the base of the tail and are also seen on the flanks, belly and legs. Inset is a photograph of a typical response that occurs following an injection of Midge proteins. There is a small swelling in the area of skin that has been tested for sensitivity to midge extract in salt solution (square 2) compared to an area of skin injected with the salt solution alone (square 1).
The pony in the picture shows typical signs of sweet itch. In severe cases such as this, the scabs and sores extend all the way down the mane along the back to the base of the tail and are also seen on the flanks, belly and legs. Inset is a photograph of a typical response that occurs following an injection of Midge proteins. There is a small swelling in the area of skin that has been tested for sensitivity to midge extract in salt solution (square 2) compared to an area of skin injected with the salt solution alone (square 1).

“Why can’t you vets do something about this terrible sweet itch?”, asks a horse owner.

This question was put to me one afternoon by a lady at pony club when I arrived to pick up my children. The story was a familiar one of a young pony that had been purchased in the winter months then suddenly developed the characteristic rubbing and weeping sores of sweet itch the following summer.

Of course the vendor was certain that the pony had shown no signs of the disease the previous year, which may well be true as the peak age of onset for sweet itch is about four years and it can first appear in much older horses.

Initially I was rather taken aback by this abrupt demand to “do something”, yet on reflection I could only agree that it was time something was done about this distressing condition which affects about 3% of horses and ponies in Britain.

Furthermore, as a vet with an interest in immunology I was in an undeniably good position to take on this task and any chance of backing out was completely lost when my children joined in with a chorus of “Yeah dad, you can fix it”. Fine words; but before one can fix anything it is essential to understand the exact nature of the problem and this is also largely true of treating diseases.

Midges are to blame

Sweet itch is most often an allergic reaction of horses to the bites of midges, which occur in almost every country in the world where horses are kept. Midges are small, only a couple of millimetres in length and like many insects that feed on blood, only the females do so as they need the high protein meal to enable them to lay their eggs.

Although the disease has been known for centuries, the first scientific description of the link with midge bites was made in Australia in the 1950s by a vet called Riek. Riek first noticed that the disease occurred only in areas where midges were abundant, then showed that when an extract of midges was injected into the skin of the affected horses, a small swelling appeared within a few minutes.

A female Culicoides nubeculosus looking for a meal on someone's arm.
A female Culicoides nubeculosus looking for a meal on someone’s arm.

This type of “acute” reaction is typically seen in certain types of immune “hypersensitivity” or allergy and so Reik concluded that sweet itch was an allergy to midge bites.

Why do midges spit?

A few days after the pony club incident, I was out in the field checking our ponies. It was a calm sunny evening and as I watched I could see large numbers of midges, which are most active around dawn and dusk or on overcast days when there is less risk of them drying out in the hot sun.

After alighting they crawl down the hair shafts to the skin surface. Their mouthparts are too short to probe for a blood vessel like their larger cousins the mosquitoes, so they have to chew their way through the tough outer layers of skin. To assist their efforts they secrete saliva containing a mixture of enzymes that digest and soften the skin tissue as well as “vasodilators” to encourage extra blood to flow to the site of the bite and several factors that will prevent the blood clotting.

A small pool of blood forms just under the skin surface and is then sucked up by the midges.

The picture at left is a microscope section through a midge that has been stained pink. The antibodies in the horse's blood have bound to the midge's salivary gland in the area next to the arrow. At right is a similar section treated with serum from a horse living in Iceland where midges do not occur. The saliva glands in this case are not stained pink, because horses never exposed to midge bites do not make antibodies to midge saliva proteins.
The picture at left is a microscope section through a midge that has been stained pink. The antibodies in the horse’s blood have bound to the midge’s salivary gland in the area next to the arrow.
At right is a similar section treated with serum from a horse living in Iceland where midges do not occur. The saliva glands in this case are not stained pink, because horses never exposed to midge bites do not make antibodies to midge saliva proteins.

Once full they make their way back to the ends of the hairs from where, laden with blood and weighing twice as much as when they arrived, they launch themselves into the air. The whole process takes about 15-20 minutes and over the course of an evening a horse may be bitten by hundreds or even thousands of midges, each one injecting a small amount of saliva containing foreign proteins into the horse.

The response of the immune system to midge saliva

When an animal is injected with something foreign like midge saliva, its immune system responds by making antibodies that can bind on to the foreign proteins. The action of antibodies is often compared to that of a key for a lock, in that one end is like the key’s handle, while the other end has a unique shape that fits the lock or in the case of antibodies allows them to bind their target.

Antibodies are made by specialised cells called B-cells. Each individual B-cell is programmed to make a unique antibody. An animal like a horse (or human) has millions of B-cells which between them can make millions of different antibodies. But one B-cell on its own will not make very much antibody, so when a B-cell encounters a foreign substance that binds its own unique antibody it is stimulated to grow and divide so that in a few days there are thousands of them and lots of antibodies can be made.

This is how a flu vaccine works; your horse is injected with a small amount of the influenza virus and those B-cells which make antibodies that can bind to the virus increase in number. Then when a real flu virus turns up there are lots of B-cells primed and ready to immediately make antibodies that bind onto the virus and “neutralise it”, preventing the infection spreading, without your horse having to go to the trouble of getting ill and waiting several days for its immune system to catch up.

So, if the immune system of horses with sweet itch is reacting to the saliva of midges, the horses should have antibodies that will bind specifically to the proteins in midge saliva. The first stage in our research was to identify which midge proteins are important.

There was no problem getting some midges – all that was needed was to catch them in a suction trap as they landed on the horse to feed. The first approach we used to look for the antibodies that bind to midge saliva proteins, was a technique called immuno-histochemistry. First the midges are sliced into very thin sections then placed on a glass slide. The slides are exposed to serum from horses so that any antibodies in the serum will bind to their target. We then detect the bound horse antibody using a label that produces a red colour.

However, we found that all British horses had antibodies that bound midge saliva; perhaps this is not surprising as all horses living in Britain will be bitten by midges. Fortunately we were able to get some serum from horses living in Iceland, one of the few places in the world where there are no midges. The serum from Icelandic horses that had never encountered a midge did not stain the midge saliva glands, confirming that their serum contained no antibodies to midge saliva.

A special kind of antibody

But if all British horses have antibodies to midges, why do only some develop sweet itch? The answer lies in understanding more about the immune system and about antibodies. Think again of the antibody being like a key, with one end specially shaped to bind a foreign substance and a handle at the other end.

Mast cells are found just under the skin. They are identified by their granules which store several chemicals that can be stained blue. In sweet itch the mast cells become coated with Immunoglobulin E (IgE) antibodies (blue Y shapes) that recognise midge saliva proteins (red crosses). When the midge bites, their saliva triggers the release of chemicals like histamine from the mast cell storage granules (blue circles) that inflame the skin, causing the severe itching and giving rise to swellings like the one seen in the picture of the pony with sweet itch.
Mast cells are found just under the skin. They are identified by their granules which store several chemicals that can be stained blue. In sweet itch the mast cells become coated with Immunoglobulin E (IgE) antibodies (blue Y shapes) that recognise midge saliva proteins (red crosses). When the midge bites, their saliva triggers the release of chemicals like histamine from the mast cell storage granules (blue circles) that inflame the skin, causing the severe itching and giving rise to swellings like the one seen in the picture of the pony with sweet itch.

The B-cell can attach a different handle to its antibody for different purposes, for example, one type of antibody handle is best for binding to an influenza virus and preventing it from infecting its host. A different type of antibody handle is needed to deal with a bacteria like the one that causes strangles, in this case the antibodies bind to the bacteria’s surface and their handle enables white blood cells to catch hold of the bacteria then ingest and destroy them.

The kind of antibody that is important in allergies like sweet itch is called Immunoglobulin E or IgE for short. Its role is in immune responses to parasites such as worms. Adult worms of course are very common in the horse’s digestive systems but many of the larval stages of worms invade the tissues of the horse and some types of worm actually live just under the skin.

To help protect against worms the immune system has to use one of its most powerful weapons, known as the “mast cell”. These mast cells coat themselves in IgE antibodies and lie in wait just under the skin or in the lining of the intestine. When the IgE binds its target, the mast cell releases a cocktail of chemicals that cause a severe inflammatory reaction and attract other immune cells which can injure or kill the parasite.

Unfortunately mast cells, like other weapons of mass destruction, can cause a lot of collateral damage. Allergies occur when the immune system makes a mistake and mounts an anti parasite response to the wrong thing. For example, in people this could be a food like peanuts, or in hay fever it is often pollen, and in some cases people even develop an allergy to horse hair. But in horses themselves the commonest allergy is to midge saliva.

When we looked for IgE antibodies that bound to midge saliva glands we only found them in the serum taken from horses with sweet itch, confirming that this disease is an allergic response to midge bites.

What is it in midge saliva that the horse’s immune system reacts to?

The next task in our research, which was funded by The Horse Trust, was to identify all the different proteins in midge saliva. One way of doing this is to isolate the relevant genes that contain the instructions for making the proteins in midge saliva. In animals, every cell contains DNA that carries the code for making an entire animal, but only those genes that are needed by a particular cell are switched on.

From midge saliva glands, we isolated the switched-on genes which code for the saliva proteins and put them into a special type of bacteria in the laboratory. When grown overnight on a dish of agar gel each individual bacteria forms a colony that will contain only one extra gene from midge saliva.

We can then pick each colony of bacteria, isolate the midge gene and read its coded message. By reading lots of coded messages we can work out which ones are most common in midge saliva and are most likely to be the genes of the proteins that cause sweet itch.

The second approach is to look at the proteins themselves. Using a combination of methods, proteins can be separated in a polyacrilamide gel according to their size and acidity. The individual proteins form spots and each spot is then punched out of the gel and digested into fragments. The sizes of the fragments are then measured by a mass spectrometer. To identify the proteins a computer program is used that compares the pattern of the fragment sizes with those that we would expect to find based on the genetic codes. After putting all this information together we can work out what the commonest proteins in midge saliva are.

We can also investigate which of the spots bind IgE antibodies in the serum of allergic horses. All horses with sweet itch have IgE antibodies that bind midge saliva proteins but individual horses will recognise a different pattern of spots.

Once the genes or the relevant proteins have been isolated we can put them into cultures of insect cells which will make the protein in an identical way to a midge saliva gland. Each culture can be over a litre in size and makes only one midge protein so we can produce a pure protein equivalent to the contents of several million midges’ saliva glands.

What are we planning to do with all that protein from midge saliva?

We can in theory use it to re-programme the immune system of an allergic horse to act like that of healthy horses. First we need to better understand why the immune systems of only some horses react with an allergic response. Although Icelandic horses do not get sweet itch in Iceland where there are no midges, when brought to mainland Europe, more tan one in four Icelandic horses may eventually develop the condition.

So are Icelandic horses as a breed genetically more likely to get sweet itch? Scientists and vets from Iceland and European countries have looked at this in detail and the answer is no. Although there are genes in some horses that make them more likely to get sweet itch, these are not more common in Icelandic horses compared to other breeds, and Icelandic horses born on mainland Europe do not get sweet itch any more often than other horse breeds.

Other studies on the development of the foal’s immune system have shown that they do not make IgE antibodies until they are about six months old. We think that when foals are exposed to midges before this age their immune system usually becomes programmed not to make IgE antibodies to midge bites. But Icelandic horses first exposed to midge bites as adults are very susceptible to developing sweet itch because as foals their immune system was not programmed to ignore midge bites.

A case of mistaken identity

Remember how the B-cell attaches a different handle to its antibodies – how does it know what kind of handle it needs to use? Well, the B-cell is told what kind of handle to use by chemical messages sent by another group of immune cells called T-cells. So how do the T-cells know what chemical messages to send?

When the proteins in midge saliva are separated in a polyacrilamide gel according to their size and acidity, the individual proteins form spots like those seen in the top picture. Proteins are made up of long chains of amino acids, like a string of beads. To identify the proteins each spot is punched out of the gel and the amino acid chain is cut into pieces. The size of the bits is always the same and this can be measured by a mass spectrometer. The result is called a peptide mass fingerprint which uniquely identifies each protein. The bottom picture shows midge saliva proteins that have been separated in a gel then stained with IgE antibody from a horse with sweet itch. In this process the darker grey smudges show where IgE has bound to some of the midge proteins, indicating that they are the ones to which this horse is allergic.
When the proteins in midge saliva are separated in a polyacrilamide gel according to their size and acidity, the individual proteins form spots like those seen in the top picture. Proteins are made up of long chains of amino acids, like a string of beads. To identify the proteins each spot is punched out of the gel and the amino acid chain is cut into pieces. The size of the bits is always the same and this can be measured by a mass spectrometer. The result is called a peptide mass fingerprint which uniquely identifies each protein.
The bottom picture shows midge saliva proteins that have been separated in a gel then stained with IgE antibody from a horse with sweet itch. In this process the darker grey smudges show where IgE has bound to some of the midge proteins, indicating that they are the ones to which this horse is allergic.

The T-cells are told what to do by yet another type of cell called a dendritic cell which detects foreign substances and can tell if they are from bacteria, from viruses or are of parasite origin. This seems to be the root of the problem – the dendritic cells recognise bacteria because they are made of different materials, and when cells are infected by viruses they send out distress calls that alert the dendritic cells to the virus’s presence. But what about parasites?

A migrating parasite has to break down the tissue. To do this it secretes enzymes similar to those the midge uses to break down the skin, and like midges, the migrating parasites also release factors that can interfere with blood clotting. The horse’s immune system “detects” these effects and interprets them as an invading parasite when in fact it is only a midge bite. So it looks like sweet itch is a case of mistaken identity leading to the wrong messages being sent down the chain of command to the B-cell which responds by making IgE antibodies which trigger an allergic reaction.

Scientists at the Veterinary School in Berne analysed the chemical signals made by the immune system of horses with sweet itch, and showed a clear difference in the messages made by the T-cells from healthy horses responding to midge saliva and the T-cells from horses with sweet itch.

Can we re-program the immune system of horses with sweet itch to be like that of healthy horses?

The term immunotherapy is used to describe treatments that can be used to re-program the immune system of people (or animals) with allergies; usually this involves repeatedly exposing the immune system to small amounts of the allergen. Originally this was done by daily injections but it is unlikely that this method would be suitable for use in horses. There are several newer ways that re-programming could be attempted but it will take some time to carefully work out how this can be safely done.

For example, would mixing the midge proteins with some parts of bacteria fool the immune system’s chain of command into giving orders that divert the B-cells from making IgE? Or could feeding midge protein convince the immune systems that this is really a harmless food? Should all foals be inoculated with midge proteins at an early age?

We don’t yet know. It has taken almost 10 years to get this far but we are making progress. In research, there are always new surprises that await, but one thing is certain in science – it always take longer that you think. Yet if everyone works together, one day we will indeed be able to “do something about sweet itch” and develop an effective cure.

Doug Wilson
Doug Wilson

This article was first published on Horsetalk.co.nz in July, 2010.

Dr Doug Wilson (Doug.Wilson@bristol.ac.uk) is a Lecturer in Virology at the University of Bristol School of Veterinary Sciences. His main area of research, which has been funded by a research grant from The Horse Trust, is the immunology of horses with a special interest in the immunopathology of Insect Bite Hypersensitivity (Sweet Itch).

The project, which finished last year, succeeded in isolating the various proteins in midge saliva that could cause sweet itch. “I would like to thank The Horse Trust for funding my recent research into sweet itch. Their funding enabled me to carry out vital research to better understand the causes of sweet itch and has moved us one step closer to finding a cure for this unpleasant condition,” Dr Wilson said.

The Horse Trust

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