Biomarker identification and development of aptamers for the diagnosis of sub-clinical facial eczema
Facial eczema is a disease caused by the ingestion of fungal spores on pasture grass, causing irreversible liver damage. The disease causes significant decreases in production (i.e. meat, milk and reproduction) for affected animals and primarily affects ruminants. As ruminants contribute enormously to New Zealand’s primary industries, facial eczema is an economically relevant disease. Anthropogenic changes in New Zealand’s climate are likely to increase the fungi’s prevalence in our pastures, driving a need for innovations in treatment and prevention of facial eczema.
A key area for improvement is the current facial eczema test, which measures the liver enzyme gamma-glutamyltransferase (GGT) to assess the degree of liver damage. This is normally employed in a small selection of the wider herd due to logistical constraints, as blood samples must be taken from each animal and returned to a veterinary laboratory. This greatly limits the number of animals in a herd that can be tested, forcing treatment to be non-specific and herd-wide. If facial eczema is detected, then herd-wide treatment measures are implemented at significant cost and with substantial side effects for the animals being treated. An alternative method could utilise aptamers in a diagnostic test that could be performed farmside at a reduced cost, and studying this possibility in greater detail formed the core of this research project.
Aptamers are single-stranded oligonucleotide sequences developed using systematic evolution of ligands via exponential enrichment (SELEX), an iterative process of selection acting upon an oligonucleotide library against a target of choice. Their properties enable them to form unique secondary structure conformations that bind the specific target molecule they are generated to. As the GGT family has four major isozymes with significant structural variation, it was necessary to develop an enzyme-linked immunosorbent assay (ELISA) using GGT isozyme-specific antibodies to identify the isozyme that is elevated during facial eczema infection.
In this study, these ELISAs were developed and used to test a wide range of serum samples collected from sheep that were known to be positive or negative for facial eczema. It was determined that GGT1 was the best candidate isozyme for a facial eczema biomarker. Due to time constraints, the target molecule used for SELEX was a combination of a crude GGT preparation, and serum that was positive for facial eczema. A counter-SELEX step using serum that was negative for facial eczema was successful in removing substantial oligonucleotide sequences that exhibited non-specific binding. The enriched oligonucleotide library was then sequenced producing 21 unique aptamer candidates.
In summary, this study identified the GGT isozyme most likely to be an accurate biomarker of facial eczema, and generated 21 potential aptamer candidates that may be used in future studies to develop a novel, cheap and easy on-farm detection test for facial eczema.