General Guidelines for Koi Herpes Virus Testing

  • Koi Herpes virus (KHV) is extremely infectious and potentially lethal in Koi.
  • The virus can also be present in fish in a latent phase with the fish showing no symptoms of being infected. However, under certain environmental stressful conditions, such as changes in temperature or transportation, the virus can be activated resulting in a systemic infection.
  • Infected fish shed the virus into the water which in turn infects other fish that are susceptible to the virus.
  • It is possible that certain strains of KHV could be more pathogenic and infectious than others or that some fish have immunity to the virus that protects them from being infected.
  • There are many unanswered questions but the strategy at present is to control and manage the spread of all KHV in SA using a molecular testing strategy to identify KHV infected fish.
  • It is possible to detect KHV in fish that are infected with the virus using extremely sensitive molecular tests that are now available at MDS.
  • The best samples to analyze are gill clippings or a gill swab.
  • It has been shown that by raising the tank temperature to 23 C it is possible to activate the latent virus in infected fish.
  • In the warmer climates the activation temperature needs to be raised a little higher. In other words if the temperature of the pond water is at 23 C then activation might require a temperature shock of 25-27 C to activate the virus.

Koi Sample Collection Guidlines

Preparation of the fish for testing

Fish should be placed in a quarantine tank for 10-14 days before that sample is collected for testing. Obviously, if any fish start to show symptoms or die then you should take a sample immediately and send it for analysis.
Ideally the temperature of the quarantine tank water should be 23º C (or 2º –3º C greater than the temperature of the pond water if the pond water is 23º C or greater). In the warmer areas activation temp should be 25º-27º C.
At the moment the guidelines recommend that samples should be collected from 3 fish per batch.

Sample Type:

Gill Clippings
It is important that the person taking gill clippings is trained in this technique. A small section of the gills should be cut off from the anaesthetized fish and placed into the provided collection tubes. Gill clippings should be about 5mm2 in size. It is recommended that gill clippings are not preserved in Ethanol or Formalin as this can result in inhibition. Snap close the tubes and label each of the tubes. Complete the MDS fish disease request form and place the samples in the fridge.

Gill Swabs
We have shown that Gill Swabs are equally as effective as Gill Clippings to collect a sample for testing. Gill Swabs has many advantages over gill and kidney biopsies.

Transport of samples to MDS laboratory
Contact MDS to request the courier service to collect the samples for afternoon collection and overnight delivery. Swabs can also be sent by speed post or your own preferred courier service or they can be dropped off at the laboratory. Should samples not be shipped immediately then store them in the freezer.

Gill Clippings
It is preferable to send gill clippings on ice and as soon as possible.

Recommended Samples Required

For economic reasons it is suggested that 3 samples are sent per pond. However, the OIE(World Organisation for Animal Health) recommends that enough fish should be tested to cover a 95% confidence limit. The table below lists the number of samples that need to be tested to cover various confidence limits for different numbers of fish per pond. The figures are based on the assumption that 2% of the fish are infected with KHV.

Size of sample needed at prevalence of at least 2% infection.

,population,error rate,,,,,
50, 50, 35, 20, 10, 7, 5, 2
100, 75, 45, 23, 11, 9, 7, 6
250, 110, 50, 25, 10, 9, 8, 7
500, 130, 55, 26, 10, 9, 8, 7
1000, 150, 55, 27, 10, 9, 9, 8
1500, 140, 55, 27, 10, 9, 9, 8
2000, 145, 60, 27, 10, 9, 9, 8
4000, 145, 60, 27, 10, 9, 9, 8
10000, 145, 60, 27, 10, 9, 9, 8
>10000, 150, 60, 30, 10, 9, 9, 8