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:

1. Serum for serology (for serum, collect at least 7–10 mL of blood from animals in the acute phase and again in the convalescent stage of the disease). Collect paired serum samples (2–4 weeks apart).
2. Whole blood (EDTA).
3. Cerebrospinal fluid (CSF) and/or tissue samples
   Note: Cerebrospinal fluid (CSF) and/or tissue samples should only be taken from horses where Hendra virus has been excluded as a differential diagnosis. Place into a sterile container.

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 virus isolation and molecular diagnostic tests. Send samples with dry ice if the journey is expected to take several days.

Sample submission

AgriBio (the Victorian state laboratory) will perform testing or 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, molecular detection and/or virus isolation.

Notes on testing

1. Serological studies may aid in the diagnosis of JE but will not allow confirmation of a case without isolation or detection of the causal virus or viral antigen. Pertinent serology results include seroconversion between paired serum samples (≥ 4-fold rise in antibody titre to JEV compared to related circulating flaviviruses). Collect paired serum samples 2 – 4 weeks apart.
2. 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.
3. Of the antibody tests available, the plaque-reduction neutralisation test is the most specific and can be used to resolve cross-reactions.
4. Molecular tests (reverse transcriptase-PCR) are available, however have low sensitivity as the acute viraemic phase is short (up to three days in horses) and may precede clinical signs.(Next part of sentence removed)
   • 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 immediately 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

Investigation and testing of suitable cases is covered by the Victorian Significant Disease Investigation (SDI) Program.

Following reporting, Agriculture Victoria field staff will provide private veterinarians with direction and assistance to manage JE virus investigations, including eligibility and information requirements for SDI funding.