Pathomorphology and immunobiology of Acquired Equine Polyneuropathy (AEP); a key to the aetiology?

A disease in the peripheral nervous system of horses, Acquired Equine Polyneuropathy (AEP), has emerged in the Nordic countries since the 1990’s. The aetiology is unclear, but a toxic substance in the haylage is hypothesized to be responsible for the injuries seen at cell level in the nerves. The aim of the research project was to elucidate important steps of the pathogenesis of AEP, in order to approach the aetiology. We have clarified that AEP affects initially the Schwann cells of the nerves, with accumulation of inclusion bodies as a primary event. By that, we may establish specific cell cultures and expose them for presumed toxic substances to search for the aetiology without doing research on healthy animals. We have also successfully used a new diagnostic method, TcMS, in affected horses and we have adapted a blood test (detection of serum anti-gangliosides) for horse diagnostics. This is a cooperation between Norges miljø- og biovitenskaplige universitet (NMBU) Oslo, Norge and SVA.

Have you heard of a nerve disease in horses sometimes called “Scandinavian knuckling disease“ or "acquired equine polyneuropathy" (AEP)?

This disease causes stumbling and knuckling of the hind limbs of several horses in the same herd, with recumbency of the most severely affected horses, leading to euthanasia. No one knows the cause of AEP, but earlier research hypothesizes there might be a neurotoxic substance in certain batches of wrapped silage. In this project, we have examined the peripheral nerves during the disease course. We discovered which cells in the nerves change first, namely the Schwann cells which form the insulating myelin sheath around each nerve fibre. We were also able to show that the first alteration in the structure of these cells are the storage of so-called inclusion bodies outside the cell nucleus.

The project has focused on explaining the disease mechanisms (pathogenesis) in the nerves at the microscopic level, thereby getting closer to the root cause (aetiology) of AEP. This knowledge is important to prevent disease outbreaks. In addition, it provides insight into how sick animals could be treated.

We set up four parts of the project; to find out how the changes in the nerves correlate to the horse's movement disorder during different parts of the disease process, to clarify how the stored substance in the Schwann cells is constructed and what it consists of, to investigate the immune damage through the various stages of the disease and to determine whether diseased horses have antibodies in the blood against substances in the nerve cell membrane called gangliosides.

To accomplish this, we investigated diseased horses and blood samples from the horses, and studied nerves in detail from diseased horses that were euthanised because of AEP.

Although the clinical signs are most evident in the hind limbs, we could show that AEP is a generalized nerve disease. Several different types of nerves and from completely different body parts were attacked at the same time. Surprisingly, there was no direct correlation between either the location or the severity of the visible pathological alterations in the nerves and the degree of disability to move. The explanation may be that the actual impairment is due in part to the fact that antibodies block the electrical nerve impulses, which cannot be seen in the microscope.

The inclusion bodies in the Schwann cells proved to be protein-like in appearance. We found that the diseased nerve contained a form of aggregated protein using the luminescent conjugated oligothiophenes method, LCO. Of all about 2,000 proteins found in a normal horse nerve, there were too much of 10 specific proteins in the diseased horse's nerves, as demonstrated by mass spectrometry. These candidate proteins in the aggregate/inclusions should be investigated with other methods.

We also saw that the myelin sheath around many nerve fibres is broken down (demyelination), and there are white blood cells attached to the nerve fibres. This suggests that the signs of AEP are triggered or aggravated by inflammation and immune-mediated events in the nerves. We suggest that this is a secondary issue in the development of AEP disease.

We modified an enzyme-linked immunological assay (ELISA) to be able to analyse anti-ganglioside antibodies in horse serum. The method has been used in the diagnosis of diseases with inflammation of the nerves of humans. We found that most horses on farms with diseased animals, both with and without signs of AEP, have elevated serum levels of a specific anti-ganglioside, compared with healthy horses from other farms. The results suggest that even those horses who do not exhibit disease signs in an affected farm may have been exposed to the factor that plausibly causes nerve damage. This blood analysis can possibly be used in the diagnosis of AEP at herd level in the future.

About 1/3 of disabled horses with AEP are killed for animal welfare reasons. However, for those horses which can get up and remain standing during the disease, the prognosis is good. The nerve function is restored over time, and almost all such horses recovered from AEP. Our study shows that most surviving horses performed at the same or higher levels again after the disease, but recovery may take a long time - one can expect it to take from a few months to a year.

Although earlier research suggests that something in the wrapped forage may be toxic to the nerves, no poison has been found in the forage samples yet. An attractive way to test toxic substances without animal testing is to exploit cell cultures. Cultivated Schwann cells could be exposed to potentially toxic substances, for example, extract from hay silage bales. Thanks to our new knowledge of how the structures of these cells react, presumably to a toxic substance from the feed, we see opportunities to research further to reveal the root cause of the disease without having to attempt animal testing.