Submitting samples for Japanese encephalitis testing

Where to submit samples

Submit samples from clinically consistent cases directly to the Biosecurity Sciences Laboratory (BSL), using the specimen advice sheet and the specimen advice sheet — Japanese encephalitis supplement.

Laboratory testing of pigs and horses with signs consistent with clinical Japanese encephalitis (JE), where appropriate samples have been submitted, will be fully subsidised by the Queensland Government.

Samples required

Complete and suitable samples support a thorough diagnostic work-up and improves the likelihood of reliable and meaningful results.

Post-mortem

Post-mortem specimens should be collected from animals with neurological signs killed in the acute stage of the disease or from recently dead animals.

1. Pigs less than 6 months or aborted foetuses:
   • fresh specimens (collected into separate, clearly labelled containers)
     • cerebrospinal fluid (CSF)
     • brain (aseptically removed if possible)*
     • spinal cord*
     • tonsil*
     • spleen*
     • liver*
     • kidney*
     • lung*
     • heart*
     • placenta (from aborted foetuses)*
     • abdominal and/or thoracic fluid.
   • formalin-fixed specimens (*as listed above) in 1 container per animal necropsied
   • whole fresh aborted or stillborn foetuses are also suitable for submission where samples can't be collected on the farm.
2. Horses with neurological signs:
   • appropriate specimens for Hendra virus exclusion. Hendra virus should be excluded before progressing to invasive sampling or post mortem
   • CSF (in sterile container)
   • fresh and formalin-fixed tissues (including brain and spinal cord).

Ante-mortem

• From animals in the acute stage of disease, collect whole blood (EDTA) and serum samples for virus detection and serology. For serum, collect at least 7–10 mL of blood from animals in the acute phase and convalescent stage of disease. Collect paired serum samples 2–4 weeks apart.
• CSF should be collected from animals presenting with neurological signs. Hendra virus should be excluded first for horses.

Ideally, separate serum from clot before shipment and submit both.

Fresh semen from boars with sperm abnormalities may be considered as an additional sample for virus detection in pigs.

Transport of samples

• Chill blood samples and unpreserved tissue samples at 4°C. Note: Do not freeze samples
• Send samples in esky with frozen gel packs
• Formalin fixed tissue can be sent at room temperature
• If mummified, stillborn or abnormal piglets are submitted they should be double bagged using strong plastic to avoid leakage of sample and sent chilled (on ice or with ample frozen gel packs)
• Ensure the necessary paperwork is submitted in hardcopy alongside the specimens (place in separate zip-lock bag).

Contact BSL if you have any queries about sample submission.

Laboratory testing

A diagnosis of JEV infection can be made through these methods.

1. Isolation and identification of JEV.
2. Detection of JEV by nucleic acid testing.
3. Immunohistochemical detection of JEV antigen in association with appropriate histopathological lesions.
4. Seroconversion by testing paired serum samples or a significant increase in antibody level (a fourfold or greater rise in titre) to JEV in a virus neutralisation test.
5. Detection of elevated levels of JEV–specific antibody (IgM or IgG) in cerebrospinal fluid.
6. Detection of elevated levels of JEV–specific antibody (IgM) in serum.

There is a high degree of serological cross-reactivity between flaviviruses, care must be taken in interpreting results in areas where related flaviviruses co-circulate, which is the case in Queensland.

Of the antibody tests available, the plaque-reduction neutralisation test is the most specific and can be used to resolve cross-reactions.

Molecular tests (reverse transcriptase-PCR) are available, however have low sensitivity as the acute viraemic phase is short (up to 3 days in horses) and may precede clinical signs so diagnosis of JEV infections is often reliant on detection of antibodies.

When fresh samples are available, molecular methods can be performed on a range of samples, including infected tissues and CSF.

Infection with JEV can also be detected in fixed tissues using immunohistochemistry diagnosis.