CSIRO livestock researchers explore potential of new technologies

Two animal-virus hunters at CSIRO are exploring the potential of advanced genetic and proteomics technologies to make more accurate and rapid diagnoses of viral diseases and other microbial infections of livestock.

Protein microarrays

For more than two decades, ELISA (enzyme-linked immunosorbent assay) technology has been the gold standard for rapid and accurate disease diagnosis of livestock diseases.

But ELISAs are a do-little technology; a single ELISA test can do no more than confirm or disprove that an animal has a particular virus disease. If none of the usual suspects tests positive differential diagnosis can only be achieved via a series of one-shot ELISA tests -- a time-consuming and expensive process that ultimately may prove inconclusive.

Dr Sandra McKean, from CSIRO's Australian Animal Health Laboratory in Geelong, last week received a 2003 Science and Innovation Award for Young People in Agriculture Fisheries and Forestry from Agriculture Minister Warren Truss.

McKean will use her award, which involves a research grant of up to $8000, to determine whether protein microarray technology can provide faster, more rapid diagnoses than ELISAs.

She plans to use protein microarrays -- microscope slides spotted with tens to hundreds of proteins purified from infectious agents. The location and identify of each antigenic protein is known, and recorded in a database.

A sick animal that has contracted an infection from a particular virus, bacterium or parasite, makes antibodies to the infectious agent; the protein microarray selects any matching antibodies from the animal's serum.

The protein-antibody complex is then labelled with a secondary antibody, linked to fluorescent marker dye, and the microarray is placed in an automatic scanner, which records the location of any fluorescent spots and compares them to patterns stored in a diagnostic database to make a diagnosis.

McKean plans to use the limited number of diagnostic antigens already available, existing ELISA tests as a starting-point, to determine whether the technique performs better than one-shot ELISAs.

In practice, serum from sick animals would be collected in the field and sent to a central laboratory with an automated scanner - McKean says that protein microarrays could potentially scan simultaneously for hundreds, even thousands of different antigens.

PCR meets flow cytometry

Real-time PCR (polymerase chain reaction) revolutionised viral diagnostics, by providing virologists with the ability to identify a particular virus, with exquisite precision, in just four hours.

But like ELISA antibody-based diagnostic tests, real-time PCR tests for only one virus at a time. CSIRO researcher Dr Tim Bowden, of the Australian Animal Health Laboratory in Geelong, is testing a new system that can test simultaneously for multiple virus targets in a single reaction by combining PCR with the familiar technology of flow cytometry.

Bowden was one of two young researchers to receive a 2003 Science and Innovation Award for Young People in Agriculture Fisheries and Forestry from Agriculture Minister Warren Truss in Canberra last week.

Bowden says the technology, developed by the Luminex Corporation, employs tiny polystyrene beads, each labelled with one of 10 different concentrations of 10 different fluorescent dyes. With 100 permutations of colour and intensity, researchers can simultaneously test for up to 100 different viruses, and discriminate between multiple strains of the same virus.

The polystyrene beads fish for a specific virus with a unique 'hook' -- a short oligonucleotide complementary to some unique sequence in each target virus.

The beads are passed through a flow cytometer, like cells. Any bead that has found a match will fluoresce in a particular colour, and at a certain intensity, when it is hit with a laser beam, and the detection is recorded and identified in a database.

Bowden says the technology is very flexible -- differential diagnosis is performed simply by mixing beads for each of the candidate viruses in a suspension. "It's a fairly compact machine and it can test a range of samples- blood, serum tissues -- anything from which you can amplify viral genes."

Bowden plans to test the new technology on differential diagnosis of a group of rare, potentially deadly viruses, the Henipaviruses, which include Hendra virus, which caused the death of Brisbane racehorse trainer Vic Rail, and the devastating Nipah virus, which devastated piggeries and killed more than 200 people in Malaysia in the late 1990s.