Investigation procedures for Japanese encephalitis (JE) in horses

Sample collection

Collect initial samples from animals in the acute stage of the disease or from animals that have been dead for less than 12 hours.

For Japanese encephalitis, collect serum for serology (for serum, collect at least 7 to 10 mL of blood from animals in the acute phase and again in the convalescent stage of the disease), ideally 2 to 4 weeks, to a maximum of 6 weeks, later). Hold the initial sample and submit both samples after the convalescent sample has been collected.

Packaging of samples

Standard packing systems for dangerous goods apply (three layers between potentially infected liquids and a person).

For blood samples:

Wipe the tubes so that they are free of contamination before packing. Prevent the tubes rattling around in transit with a rubber band or polystyrene tray.

• 1st layer – Blood is contained within a sealed plastic vacutainer.
• 2nd layer – Put in a sealed plastic bag. Use a cold pack, not ice, in the esky, with absorbent material sufficient to absorb any liquids in the esky such as a disposable nappy.
• 3rd layer – sealed in an esky within a solid cardboard outer.

Accession form paperwork should be outside the sealed esky but inside the outer box, so it is the first thing seen on opening.

Transport

Chill blood samples and unpreserved tissue samples at either 4 °C, or with frozen gel packs.

Do not freeze samples unless processing and shipping is going to be delayed; freezing can reduces the sensitivity of tests. Send samples with dry ice if the journey is expected to take several days.

Sample submission

AgriBio (the Victorian state laboratory) will perform initial testing and coordinate sample packaging and consignment for delivery to CSIRO–ACDP, if needed. Veterinarians should contact their local district vet or the Emergency Animal Disease hotline on 1800 675 888 to arrange sample submission.

Diagnostic tests

Laboratory diagnosis for Japanese Encephalitis Virus (JEV) is achieved by serological tests.

Notes on testing

1. There is a high degree of serological cross-reactivity between flaviviruses, so care must be taken in interpreting results in areas where related flaviviruses co-circulate.
2. Of the antibody tests available, the plaque-reduction neutralisation test (PRNT) is the most specific and may resolve cross-reactions.
3. Molecular tests (reverse transcriptase-PCR) are available but have very low sensitivity.
   • When fresh samples are available, molecular methods can be performed on a range of samples, including infected tissues and CSF, however the need to consider Hendra virus as a differential diagnosis will often prevent collection of appropriate samples in a timely manner.
   • Infection with JEV can also be detected in fixed tissues using immunohistochemistry diagnosis.

Please consider Hendra as a differential diagnosis. For information on Hendra go to Investigation procedures for Hendra Virus webpage.

For more general resources on emergency animal diseases go to our Emergency animal diseases web page.

Disease notification

Japanese encephalitis is a notifiable disease throughout Australia. Upon suspicion of the disease in Victoria it must be reported to Agriculture Victoria through direct contact with your local District Veterinary Officer or the Emergency Animal Disease Hotline on 1800 675 888.

Significant Disease Investigation (SDI) Program

Funding support is available for investigation and testing of significant disease events under the Victorian Significant Disease Investigation (SDI) program. However, for horses, suspicion of Japanese encephalitis does not routinely meet the criteria for a significant disease investigation.

If SDI funding support is approved, PRNT testing at ACDP can occur following receipt of a complete set of samples (initial and convalescent serum samples).

The attending veterinarian should hold the initial sample, then submit both samples after the convalescent sample has been collected and with the necessary supporting disease investigation documentation.

Samples should be collected into serum separator (gel) tubes and spun after collection and refrigerated.