This excellent article by Syd Mitchell will help all Koi keepers understand what they need to know about the 2 tests used for KHV - the PCR test and the ELISA test. It is written in simple terms that are easy to understand!
Arguably, KHV represents the greatest and most insidious threat to the health of our Koi and is responsible for many complete or near complete wipe-outs in ponds. Much has been written on the subject of the disease itself and of its effects on Koi which I don’t propose to repeat here other than to make a brief recap of KHV as an introduction to the main purpose of this article which is to shed some light on two means of detection - PCR and ELISA. Read the articles on the page on this link to learn more about KHV itself.
http://www.koiorganisationinternational.org/search/node/khv
What is KHV?
KHV (Cyprinid Herpesvirus 3 or CyHV3) is a viral disease of carp (Cyprinus Carpio) and that species includes Koi. It can be carried indefinitely by a Koi that has been exposed to the virus without showing any outward signs of the disease until it becomes stressed. At that point the infected fish will begin to shed virions (infectious virus particles) in its faeces, urine, and in mucus from its gills and skin. Nearly all infected Koi will die rapidly with any survivors remaining as carriers of the disease and able to infect any other Koi that are restocked in that pond, (even if the pond and equipment has been cleaned and disinfected), or all the Koi in any other pond that an infected Koi is transferred to. This means a decision has to be made whether to keep any survivors in isolation and not restock the pond or whether to euthanase them so that the pond may be restocked.
What is the PCR test?
PCR stands for Polymerase Chain Reaction and is a technique that is used to amplify trace amounts of DNA or RNA. Every living organism including humans, animals, plants, bacteria and viruses, contains genetic material such as DNA or RNA that is unique to their species. Just as a photocopier can be used to make an unlimited number of identical copies of a single document, PCR can be used to make millions or even trillions of identical copies of DNA or RNA sequences in vitro (in a test tube or Petrie dish). This amplified sample of DNA or RNA can then be used to determine the identity of the source.
The PCR assay (simplified)
To establish whether or not the CyHV3 virus is present in a fish suspected of showing the symptoms of KHV, the first step is to take a blood sample from it.
In the PCR assay, any DNA in the blood sample is copied repeatedly, as many times as necessary, until there is a sufficiently large sample of the DNA to detect whether the CyHV3 virus is present and that the fish is testing positive for KHV. The sample is typically copied by duplicating the DNA in it between 20 and 40 times.
Duplicating DNA samples by 20 to 40 times doesn’t result in 20 to 40 copies of any DNA in it.
The first time it is duplicated there will be two identical copies of the DNA. The second duplication will double those two and result in four copies. The third duplication will leave eight copies, the fourth will result in 16 copies then 32, then 64, 128, 256 and so on until the 20th time the DNA is duplicated there will be a little over one million identical copies of any DNA in the sample. If the sample is duplicated 40 times, there will be a staggering one trillion copies.
The diagram above shows how the first stage of the duplicating process doubles any DNA strand in the sample so there will be two copies. The second stage doubles those two into four copies. Repeated duplication processes will double the number of copies each time which rapidly leads to huge numbers of copies of the DNA in the sample. This amplified sample of DNA can then be compared with DNA from known sources to determine whether it’s from a virus e.g. CyHV3 if the purpose of the PCR assay is to see if the fish is testing positive for KHV.
By binding with the antigen, the antibody “labels” it in a way that allows other parts of the immune system to recognise and neutralise it. The common feature of the various variants of the ELISA test involves taking a blood sample and adding it to a pre-prepared Petri dish.
The TK PCR assay
The TK (thymidine kinase) based PCR assay was developed specifically for the detection of KHV and is able to detect as little as 30 virions or 10 femtograms of CyHV3 DNA. This is a tiny amount; there are one quadrillion femtograms in one gram. 10 femtograms is about one trillionth the weight of a grain of rice. A TK PCR assay is the most sensitive method of diagnosing KHV.
Too good to be true?
Remembering that either the basic PCR assay or the specialised TK PCR assay can be done using a sample of blood from a Koi (or any other Carp species) and this can be taken without harming it, the PCR assay sounds perfect but are there any disadvantages? Yes, there is one big disadvantage in that both versions of the PCR assay can only confirm whether or not the fish actually has active KHV. Neither version can detect if a fish has ever been exposed to the disease or whether it’s a carrier of the virus and which may develop the active form of KHV later if it is stressed. Nor will they detect if a fish is carrying the virus asymptomatically (no outward symptoms) and will be capable of infecting other fish.
The ELISA assay
ELISA stands for Enzyme-Linked Immunosorbent Assay and, as the name suggests, the method is more complicated than the PCR test. It is further complicated by the fact that there are four main versions; Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA so this review will be confined to a basic overview of the common aspects of the test.
An antigen is any pathogen, such as bacteria and viruses, that stimulates the immune system to produce antibodies.
Antibodies are Y-shaped protein molecules that fish (and human) adaptive immune systems produce in response to pathogenic substances called antigens. Each antibody produced in response to a particular antigen has a receptor on each of its “Y” branches that will bind precisely with the part of the particular antigen that it was produced to “recognise”. A simplified diagram of how antibodies bind with antigens is shown in the diagram below.
ELISA antibody test
If a fish has been exposed to KHV, and is carrying it in its non-active or latent form, it won’t be showing any outward symptoms but may either be a carrier or it may develop active KHV later. In either case it will have CyHV3 antibodies in its blood. To test for these antibodies in the blood sample, the pre-prepared Petri dish will already contain the CyHV3 virus.
If the CyHV3 antibody is present in the blood sample it will bind to the virus in the Petri dish. A further process causes a colour change which, not only, proves that the antibody is present but in what quantity. This is similar to the way we use a reagent and a colour scale to determine the level of a particular water parameter such as ammonia in a water sample but the ELISA test does it in a more sophisticated way.
ELISA antigen test
If a fish appears to have the KHV symptoms, an antigen ELISA will indicate whether the virus is present by detecting it directly. The process is similar to the antibody test above except that the Petri dish will already contain CyHV3 antibodies and, if the virus is present it will bind to the antibodies. As above, there are further processes that cause a colour change if the virus is present. There will be little point in testing how much colour change and, therefore, how much virus is present because the presence of the virus is sufficient to confirm that the fish is testing positive for KHV.