The virus appears at various times during infection. The virus may be detectable in the gills or blood for only a few hours or days. In the liver and especially the kidney, infected fish will usually have detectable virus.

So, not all tissue types are “equal” diagnostically.

Best: Harvest and homogenize kidney and liver. Ship frozen or near freezing for PCR. Formalin fix for In Situ Hybridization.

Processing Tissues

Long-dead (rotting) fish will not be useful as the DNA integrity can be compromised.

You should use live fish or VERY recently dead fish for best results and use kidney, liver and gill.

PCR test looks for specific DNA markers which are usually present in dead and dying fish organs. Can be frozen.

Live virus will not be recoverable from frozen specimens.

Gill “swabs” are all but useless unless there is overt (visible) disease among fish being tested.

Tests to Run

For now, the best method to find virus is a PCR test.

PCR = Polymerase chain reaction test. It detects an exact sequence of unique nucleic acids only found in the Koi Herpes Virus.

http://www.koilab.com

In Situ Hybridization – Looking for virus in the cells. (Proving infection)

There is also an agglutination (antibody) test for fish blood which would indicate past infection, and help screen live fish.

http://www.koilab.com

In Situ Hybridization

Run on Tissues Looking for Carriers.

Detects virus DNA actually IN the tissues, which is more specific and sensitive than the PCR test and the fish don’t have to be infected or dying. But they probably have to die for sample collection.

Tissues are prepped for histopathology, are stained with a marked protein which binds specific viral KHV DNA, then the protein/DNA complex shows up blue under the microscope.

This test is available at UGA and is good for virus detecting “carriers”

http://www.koilab.com

Serological Assay

This test uses blood so the fish don’t have to die

They’re introducing a marked KHV virus particle which causes antibodies in the blood to agglutinate.

If the blood proteins agglutinate, it means the fish has antibodies, and this means the fish was, in the last 8 months, infected.

Antibody “memory” to KHV seems only to last 6-8 months.

This is a good life sparing test to tell if a fish was ever infected with KHV.

http://www.koilab.com

Do the tests “Play” together?

PCR looks for the virus LIVING in tissues. Generally this virus test cannot be relied upon to show carrier states.

In Situ Hybridization looks for virus “HIDING” in the tissues and is far more sensitive, just as specific, and good for finding carriers.

The Serology test proves a fish survived an infection. It doesn’t identify carriers. It helps when used WITH in situ hybridization –

To wit: If the serology shows ‘infection survivor’ then the in situ hybridization test becomes colossally important to HELP determine latency or carrier state.

In situ hybridization on a fish negative by serology is of far less value. Why?

Because if the fish is negative on serology it would suggest it's never been exposed or infected.....So the second test looking for virus PARTICLES is irrelevent.

http://www.koilab.com