WAVLD Symposium Handbook_V4.indd - csiro

World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
EQUINE INFLUENZA IN AUSTRALIA – A HIGH THROUGHPUT LABORATORY RESPONSE:
ACHIEVEMENTS AND CHALLENGES.
PD Kirkland* 1 ,
1 Virology Laboratory, Elizabeth Macarthur Agricultural Institute, NSW DPI, Menangle, NSW Australia
On 24 August 2007, the Chief Veterinary Officer of New South Wales was advised of clinical signs that were
consistent with influenza in horses in a large equestrian centre. Over the next 2 days, a multi-focal outbreak
was identified on properties scattered over a 700 km range extending from the Sydney region through the
Central Coast and Hunter Valley regions and north along the NSW northern tablelands and slopes into
southern Queensland. The Virology Laboratory at EMAI provided the laboratory support for NSW during this
outbreak with confirmatory testing of selected samples at the Australian Animal Health Laboratory (AAHL) at
Geelong, Victoria.
Nasal swabs collected into viral transport medium and clotted blood were submitted to the laboratory for the
confirmation of influenza virus infection in horses with suggestive clinical signs or from those that had had
possible contact with clinical cases. Samples were also collected from horses during surveillance in buffer
zones at the time of vaccination and for movement testing once the initial standstill/movement restrictions
were eased. Influenza A group reactive assays previously developed at AAHL were used for both the
detection of viral RNA and antibodies to the virus. The viral transport medium was tested in an Influenza A
group reactive real time reverse transcriptase PCR (qPCR) assay to detect a sequence of the matrix gene
while serum samples were tested in an Influenza A group reactive blocking ELISA (bELISA) and in a
haemagglutination inhibition (HI) assay to detect antibodies specific to the H3 subtype.
The qPCR assay had been established as a broadly reactive test for influenza viruses in birds but had not
been applied to testing of equine samples. Similarly, the bELISA had not been validated for testing of horse
sera. Nevertheless, these assays were successfully applied to testing of equine samples with parallel testing
taking place to support validation of the assays. The existing high throughput PCR capability in the EMAI
Virology Laboratory was further refined during this rapidly evolving outbreak in response to variable but
increasing workloads driven by a rapidly changing field situation. The test procedures were streamlined to
minimise repetitive tasks and maximise reproducibility from day to day. The system being employed is based
on a 96 well plate format utilising magnetic bead based total nucleic acid extraction chemistry (MagMAx
Viral, Ambion) in conjunction with a magnetic particle processing system (Kingfisher 96, Thermo). The qPCR
chemistry consisted of a complete master-mix to which the primers and probe had been previously been
added. The only component added at the time of set-up of the assay was the enzyme mix and the sample
RNA. Assays were run on either an ABI 7500 Fast or an ABI 7900HT Fast thermocycler, both run in standard
mode. This combination of RNA extraction and qPCR equipment allowed a throughput of more than 1,000
samples per day and a “turn-around” time of about 3 hours for a batch of samples. Urgent samples could be
completed in about 2 hours. Over the first 2 months of the outbreak the assay system has proven to have
extremely high sensitivity and specificity, combined with ‘same day’ completion of testing.
While sample test procedures were streamlined and a large number of samples could be tested with a
relatively small number of staff, numerous challenges arose in other areas. These rarely involved complex
technical issues and usually were associated with the timely delivery and receipt of specimens. Due to the
diverse nature of the horse industry, there were many small properties and hence individual laboratory
accessions, resulting in an extremely heavy workload and delays in specimen receival and cataloguing.
From time to time, delays in specimen delivery also caused frustration. Similarly, sub-optimally collected or
packaged specimens had a major impact on throughput, sometimes doubling the number of specimens to be
handled and resulting in significant additional processing in the laboratory. Availability of consumables for
specimen collection (eg swabs and sample vials) often caused problems and the high throughput placed the
manufacturers of PCR reagents under pressure on occasions.
While the need for a sustained output placed demands on staff, additional pressure arose when investigating
sites with a high public profile (eg thoroughbred racing stables) due to the intense media interest. At times
there was public monitoring of progress with specimen receival and anticipated times for release of results.
At the scientific level, the application of new assays also raised other questions. In particular, the application
of qPCR and the inevitable detection of residual virus and RNA at times longer than previously described
generated concern when a property was due for release from quarantine. Finally, the detection of infection in
dogs, questions about the role that birds might play and the potential for human infections all added extra
dimensions to providing laboratory support for this outbreak. And, of course, the test needs for the influenza
outbreak have been fully met on a daily basis in a virology laboratory that routinely has a high workload
which has been maintained throughout this outbreak
Mon 12 November
13th International World Association of Veterinary
Laboratory Diagnosticians Syposium
11 - 14 November 2007
Tuesday 13 November