Hendra virus sampling, submission and testing

Take strict precautions for yourself, and anyone assisting you, when collecting samples for Hendra virus (HeV) testing. Only take samples if the risk of exposure to yourself and others can be adequately managed.

Samples may need to be taken from live and dead horses, so a veterinary assessment of the associated risks is required. Dead horses can be sampled adequately for HeV testing without conducting a complete necropsy, which is a very high risk activity - particularly on recently deceased animals.

It's the horse owner's responsibility to pay for transporting samples to Biosecurity Queensland's Biosecurity Sciences laboratory (BSL). Biosecurity Queensland will meet all laboratory testing costs to test samples for HeV in diagnostic cases (Note: charges apply for HeV health testing).

Private veterinary practitioners should make the final decision about whether to collect samples and submit them for laboratory testing and, where required, get advice from Biosecurity Queensland.

Taking samples for diagnosis

Preferred samples for diagnosis (from most preferred to least preferred) of suspect cases are:

• EDTA blood: ensure tubes are filled to reduce the risk of anticoagulant concentration interfering with test results.
• swabs: nasal, oral, rectal and vaginal. Note the following
  • Nasal swabs may show infection earlier than blood or other clinical samples and together with EDTA blood is the minimum sample requirements for exclusion in diagnostic cases.
  • Swabs should be transported in virus transport medium (VTM). A small amount of saline (1–2ml) can be used to prevent swabs from drying out if VTM is not available.
  • A urine-soaked swab taken from the ground immediately after urination can also be helpful.

If whole blood in EDTA is not available, polymerase chain reaction (PCR) testing may be conducted on the blood clot.

Dead horses can be adequately sampled for HeV exclusion without conducting a complete necropsy. We recommend the following samples

• swabs (as above)
• blood clot obtained by cutting down onto the jugular vein
• submandibular lymph node (chilled).

Submitting a combination of EDTA blood, nasal, oral and rectal swabs should be sufficient for detecting HeV infection in a very high proportion of HeV-infected horses.

BSL doesn't undertake routine biochemistry/haematology testing. It's best to collect duplicate blood samples that you can keep safely and securely at your veterinary clinic for in-house or private laboratory testing, once HeV is excluded.

How to submit samples

HeV testing is conducted at the Biosecurity Sciences Laboratory (BSL) in Coopers Plains. Samples for HeV exclusion testing should be submitted directly to BSL as this will achieve the shortest turnaround times.

Specimen advice sheet

A fully completed specimen advice sheet (SAS) must accompany all submissions. If you want to receive diagnostic testing for HeV at no charge, you must include a full clinical history and vaccination status of the animal. Ensure that the SAS is packed on the outside of the samples and that it clearly indicates a request for HeV exclusion.

Cut-off and turn-around times

Typically, 1 round of HeV PCR laboratory testing is run each weekday at BSL, starting at 2pm. In most circumstances, samples received at BSL before this time will achieve a result on the same day. Samples received after this time will be tested the next business day.

Phone ahead to let BSL know if you intend to submit samples so arrangements can be made to facilitate testing, and so you can be contacted if the samples are not received by the cut-off time. It is essential that you provide a direct after-hours contact number on the SAS if you want to be notified of results by phone on the same day, as the results of testing are typically available around 5pm.

Packaging and transporting HeV exclusion samples

When submitting HeV exclusion samples:

• There are packaging requirements that must be met to transport biological samples by road and rail (surface) or air. The contents should have no chance of escaping from the package under normal conditions of transport.
• Everyone responsible for packaging and transporting biological samples must be appropriately trained and their competency assessed for the task that they are to perform. Training from an accredited provider is required for the packaging of some categories of samples.
• Some courier companies will provide a complete service (i.e. they will come to a veterinary practice, pack and dispatch the samples correctly). Veterinary practices should establish an account with their chosen courier service so that samples for HeV testing can be dispatched for testing without delay.
• For transport of all pathology samples and associated material by air or surface transport methods, the contents must be triple packaged in a primary receptacle, secondary packaging and outer packaging.

Contact BSL if you have any queries on sample submission.

Urgency of HeV testing for horses

Urgent testing for HeV diagnosis/exclusion may be conducted under certain circumstances.

If urgent testing is required during business hours, veterinarians should contact the Biosecurity Queensland duty pathologist at BSL to discuss the situation.

After hours, contact the Emergency Animal Disease Hotline on 1800 675 888 and discuss the situation with the Biosecurity Queensland veterinary officer.

The decision to conduct urgent HeV testing is made following discussion between the submitter and relevant Biosecurity Queensland staff. This may include the Biosecurity Queensland veterinary officer and/or the on-duty pathologist.

Requests for urgent testing will be assessed on a case-by-case basis. Reasons that may necessitate urgent testing for HeV include:

• significant human exposure to body fluids of highly suspect HeV infected horse(s)
• exposure of large numbers of animals to body fluids of highly suspect HeV infected horse(s).

BSL specimen receipt is open for sample submission Monday to Friday only (8am–5pm). Don't send samples to be delivered to BSL outside these times without first consulting with the on-duty pathologist. Horse owners are responsible for all costs associated with weekend delivery of samples for urgent HeV testing (Note: courier charges for time-definite deliveries can be significant).

HeV tests

Polymerase chain reaction (PCR) test

This is the primary test conducted at BSL for exclusion of HeV infection, particularly in acutely sick animals. PCR tests can be conducted on blood, swabs and tissue samples. They detect the direct presence of genetic material of Hendra virus in a sample and can detect live or dead viruses but cannot differentiate between them.

A positive result indicates the presence of viral genome in the sample, but does not confirm that the virus is viable and infectious. When accompanied by relevant clinical signs, a positive PCR test result is interpreted to mean an animal has an existing HeV infection.

A negative PCR test result needs to be interpreted in relation to the health of the animal and the broader epidemiological context.

Enzyme linked immunosorbent assay (ELISA) test

This is not routinely used at BSL to exclude HeV infection in acutely sick horses, particularly if they have a history of vaccination. ELISA tests are conducted on serum samples to detect the presence of antibodies and are regarded as a screening test.

A negative ELISA result is a reliable indicator that a horse has not been previously exposed to HeV.

Being a screening test, non-negative ELISA test results, including nonspecific reactor results, require confirmatory testing by virus neutralisation testing (VNT).

A positive result could be due to past infections or vaccination with the production of neutralising anti-G antibodies. Differentiating these requires differentiating infected from vaccinated animals (DIVA) testing.

Virus neutralisation test (VNT)

VNTs detect the presence of HeV antibody in a blood sample and are used to confirm non-negative results from ELISA screening tests.

A positive VNT indicates the animal is seropositive to HeV either from infection or vaccination.

The antibody response to infection or vaccination takes time to develop and this should be considered when interpreting negative results. VNT testing can only be conducted at the Australian Animal Health Laboratory (AAHL) in Geelong.

Differentiating infected from vaccinated animals (DIVA) testing

Development and assessment of DIVA tests is an area of active investigation.

Interpreting a panel of tests that detect the G glycoprotein or other HeV proteins will help us to differentiate between:

• serologically positive animals due to vaccination
• serologically positive animals due to infection.

DIVA testing can only be completed at AAHL in Geelong.

Also consider...

• Read general information about Hendra virus.