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KHV - The complete story
By Joop van Tol, Koi Health Officer and board member of the Nishikigoi Vereniging Nederland (Dutch Nishikigoi Association).
No part of this document may be reproduced, transmitted or otherwise disclosed in any form or by any means for any purpose except as expressly authorised in writing by the author.
One of the most current and frightening Koi diseases is caused by a virus, called ‘Koi Herpes Virus’. Unfortunately there are a lot of mumbo jumbo stories going around about this illness, which is why Nishikigoi Vereniging Nederland (The Dutch Nishikigoi Association) wants to shed some light on the matter. For this purpose, the NVN has acquired information from various well-established research institutions and has held interviews with biologists and virologists.
During a visit to Israel, we spoke to Mordi Haimi (Master of Science), a biologist working for Mag Noy, who is responsible for the health of cultivated Koi. In Jerusalem we also met up with Professor Kotler who is one of the world’s most prominent researchers in the area of HIV and also has a special interest in KHV. Also I had intensive mail contact with Dr. Arik Diamont, Head of the Department of Pathobiology of The National Center for Mariculture (NCM) in Israel, who provided me the unique photographs of the actual KHV virus.
Furthermore we visited the Central Institute for Animal Disease Control, The CIDC (Centraal Instituut voor Dierziekte Controle) in Lelystad (Holland), where we spoke to Dr. ir. O.L.M. Haenen (senior researcher in fish and shellfish diseases) and Dr. ir. M.Y. Engelsma.
On top of all this, I went through enormous stacks of research reports and publications in order to write this article on the subject at hand to be as clear and accurate as possible.
In this article the following matters will be portrayed in an as plain as possible fashion; the current status on KHV research, the reasonability of our fear for it, the means to control or the necessity to learn to live with the virus.
I’d like to emphasize that the information provided by this article is an interpretation of what we understood from the conversations we held. After all, virology is a complex matter. Nothing is black or white. Therefore no rights can be derived from this article.
What is a virus?
First of all, let’s explain what a virus is so that it becomes easier to understand what we’re dealing with. A virus is a sub-microscopic parasitic particle that infects cells in biological organisms. Typically viruses carry a small amount of genetic material either in the form of DNA (DNA virus) or RNA (RNA virus), surrounded by some form of protective coat consisting of proteins, lipids, glycoproteins or a combination. A virus is not a single-celled organism and therefore cannot be compared to bacteria for example. To give you an idea of the difference in size, a virus is about a hundred times smaller than a bacteria; a similar difference as that between a human being and a skyscraper. Viruses depend on host cells in order to multiply and such a host can be found in any living organism such as mankind, animals and even bacteria. A virus penetrates a cell wall in order to multiply within the cell and it is this process that can severely damage or even destroy the host cell. Viruses are often limited to a particular species; HIV, the virus leading to AIDS, for example cannot be carried on from human beings to fish. Nevertheless there are viruses that can successfully and dangerously make the jump from one species to another. A recent example of such a virus is the bird flu variant that now threatens human beings.
There are principally no cures for viruses!
Destroying a virus without compromising the health of a host cell, as you can no doubt imagine, is a very complicated matter. Amongst virologists there are those who have set their hopes on the developments in nano-technology, but for now the fulfillment of these hopes is more science fiction than reality. Till now the only remedy for a virus lies in the natural defense mechanism of the host itself. This defense mechanism becomes effective the moment anything ‘alien’ to the body contacts it, whether it is a dangerous bacteria, a toxic substance or a virus. The unspecific defense mechanism is the first to act, followed only after a while by the specific defense mechanism that generates the antibodies to eliminate the virus. These antibodies fit themselves around the virus like a puzzle in order to disable the virus from doings its dirty ‘job’. A problem arises when the virus replicates itself faster than the defense mechanism can produce antibodies; the host will suffer illness or even death. Vaccination is based on this very process of the defense mechanism and is in fact no more than an extra helping hand in bringing the arms race in the host’s favour. (Vaccination with attenuated virus creates a moderate disease without killing the host. In this case it helps the immune system of the fish to “Learn and study the virus”. Vaccination (like an anti-flu injection) is the process of administering a live, albeit weakened or inactive variant of an ill making virus into an organism so that it starts producing the necessary antibodies to fight the virus in question without the risk of the organism becoming seriously ill. If then the real (ill making) virus gets in contact with the organism, the defense mechanism of the immune system immediately mobilizes these stored antibodies for defending against the virus. Furthermore, the organism can generate extra antibodies more quickly because memory cells have already obtained the right knowledge on which antibodies to produce. The balance of the ‘battle’ is now shifted into the organisms favour and the flu (for example) gets no chance to develop. In other words, the organism is now resistant (or has developed immunity) against the virus. Developing a vaccine is by no means as easy as it may seem; the difficulty lies in weakening or deactivating the virus - so that the host won’t become too ill - without changing the virus) too much. If the altered virus differs too much from the original version, then the produced antibodies won’t be of much help against the natural virus in its original form. Resistance for that matter is NOT hereditary! The offspring of immune organisms are always naïve and they need to be vaccinated as well in order to obtain resistance. Naïve means that the organism is no carrier of the particular virus. It is however possible to obtain resistant populations through ongoing cultivation of organisms that are naturally resistant.
DNA viruses are less quick to mutate as they will attempt to undo the damage done to their own genetic code (Repair Mechanism), while RNA viruses on the other hand are able to mutate very fast, which possibly causes the latter to develop new characteristics. A typical example of an RNA virus is the flu; new varieties emerge constantly causing the necessity for the flu shot to be adapted every year to the flu variety at hand.
What do we know about KHV?
KHV is a DNA virus and therefore not too much variable. Viruses are divided into several categories and KHV is part of the Herpes related viruses, as can be clearly seen in the name. Professor Kotler, a prominent and in HIV specialized Israeli virologist, has spread his interest towards KHV. Even though KHV has a lot of similarities to other Herpes viruses, it is rather different. It is also very interesting from a scientific point of view as, according to Professor Kotler, it doesn’t really fit into any of the existing categories. Other prominent scientists from Europe, the U.S.A. and Japan however don’t share his opinion and consider KHV to be in fact a Herpes virus. In addressing KHV, Professor Kotler prefers to mention CNGV (Carp Nephritis and Gill Necrosis), a term referring to tissue decay in the gills and to an inflammation of the kidneys. The tissue decay in the gills is a typical exterior sign of the disease. Professor Kotler’s interest in the matter could be of good fortune to Israeli carp farmers and ultimately the consumer, but more about that will be mentioned later. Of course, the name of the virus is of minor importance. Much more important is that we learn as much as possible about the behavior of the virus and controlling it. For the sake of convenience and popular use of the term, I will use the name KHV for the rest of this article.
Like most viruses KHV is limited to a specific species; as far as we know, only Cyprinus carpio (carp) can fall victim to this virus.
KHV is a highly contagious virus that can easily spread from one carp to another; adding water from a contaminated pond to a non contaminated pond will do the trick. The virus can multiply at a temperature between over 18 and below 28 degrees Celsius. Within that range it is possible that the virus will cause an outbreak. The ideal temperature for the virus to flourish, and thus the highest risk of KHV contamination, is between 21 and 24 degrees Celsius. It is possible that Koi appearing to be in good health are carriers of the virus without showing any of the symptoms related to the illness, because the water temperature is not in the optimum range. In all cases, stress is to be considered one of the factors of acceleration of the process of becoming ill. Stress in fact severely reduces the activity of the defense mechanism (immunosupression) so that fewer antibodies will be produced. Just like with HIV, it is presupposed that KHV mainly targets the immune system.
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On day 0, 4, 9 and 18 after infection, tissue is taken from fish (from the liver, the kidneys and the gills) to contaminate other fish. The graph (source: Ayana-Benet-Perlberg) shows that the virus is longer present in the kidneys than it is in the gills or liver. Only on day nine after infection, the chance of finding the virus in the gills is greater. During the test the water temperature was kept at 24 degrees Celsius. Large concentrations of the virus are especially found in the kidneys and the gills of contaminated carps. The kidney is of vital importance to the defense mechanism. The liver and the digestive canal often provide popular habitats for the virus.
Possible symptoms of illness:
The most typical symptoms of KHV are the damaged and slime covered gills of the carp. Sunken eyes or herpes like rings on the body (like herpes zoster with humans) can also often be detected. Other symptoms are; excess of skin slime, loss of the slime coat, skin damage, loss of appetite, uncoordinated swimming and gasping for air. When the host is already weakened by the illness, secondary infections can also occur by bacteria and ectoparasite (mainly Costia). Even the entire Koi population could possibly be wiped out quickly. It is remarkable however that Koi regularly die of KHV without any of the above mentioned symptoms being detected. The process of contamination, the appearance of symptoms and the event of death can all happen in an incredibly short amount of time.
How can one diagnose KHV?
KHV can be diagnosed by means of a Polymerase Chain Reaction (PCR) in a specialized laboratory. A PCR (a technique for amplifying DNA, making it easier to isolate, clone and sequence) is based on detecting a unique piece of the viruses’ DNA sequence that differs from the Koi’s DNA, or DNA of other organisms or other viruses. To apply this technique, the CIDC in Lelystad (Holland) uses parts of the supposedly infected Koi’s kidneys or gills. Downsides to this method are that a carp needs to be sacrificed and that the diagnosis will take some time (about 1.5 to 2 days). And time is precious. For a PCR to yield a positive result, it is essential to examine tissue containing enough parts of the virus. So even though a Koi is infected, it is still possible that a ‘false negative’ result can be obtained when using tissue of a Koi that survived the virus, or tissue of a Koi that has only recently been contaminated, or tissue containing too few of the virus. Real Time PCR (RTPCR) and Nested PCR are two more accurate methods. In using both these techniques it is possible to detect even smaller amounts of the virus. It tells not only if the virus is present, but also how much of it is present. Nevertheless, a negative result from these methods is also no absolute guarantee that the carp or the population it is from is KHV free. In fact, this is the case with all viruses (including HIV). When the virus is dormant (like in winter when the temperature is not within the 18-28 degrees Celsius range) it is very unlikely that the RTPCR method will find the virus. To quote Mordi: “People send fish to labs, but have no idea what is needed to get a reliable result.” The opposite is also possible; a test can result in a ‘false positive’ just as well. It all depends on the primer, the piece of DNA sequence that is used in comparison with the sample that is to be examined. DNA sequence of KHV resembles that of carp pox (another herpes virus) or could be mistaken for other viruses or ‘disorders’. If the primer is of lesser quality, it is thus possible that another virus can be mistaken for KHV. Besides the PCR method(s), one could use a specially designed test kit called ELISA which enables diagnosis on the spot with the use of Koi droppings. Later in this article there will be more information on ELISA.
What we don’t know about KHV:
No one knows how long the virus can survive without a host, but it is thought to be a rather long period of time. How it spreads other than via carp to carp or via water still remains a mystery.
Spontaneous outbreaks have occurred in other fish from isolated ponds. If you stand next to a pond for long time you will be amazed by the amount of “visitors” to that pond. It is possible the virus could be spread by frogs, birds or insects migrating from one pond to another. It is thought to be possible for a virus to survive in a pond for quite a while without the presence of carps, maybe in the organic material in the ponds, in the mud, in the algae, in plankton, or in bacteria.
People think that the virus can survive rather longer (perhaps for several months) inside the droppings of carps, though no one knows how long exactly. In the progress of research in Israel, scientists found out that there is a great amount of virus in the droppings of the carp. They manage to infect other carps with seven days old droppings. This was a breakthrough in understanding part of the biology of the virus about how it might be able to survive in between seasons. It gives more pieces to the great puzzle, which might help to stop the virus by means of disinfecting mud ponds. For example, tests prove that raising the level of pH in the water to 11 for 2 hours, kills the virus. Maybe drying out the ponds during winter time could help to eradicate the virus.
The question whether a KHV survivor is still a carrier remains. A carrier is a fish that is infected with the virus but does not necessarily show any symptoms. After a certain amount of time it is often impossible to detect the virus in a Koi that survived, but this does not necessarily mean that the carp is no longer carrier of KHV. There could be too few parts of the virus left to detect with the (RT) PCR method. In fact, the ultimate question should not be if a survivor remains carrier, but is a survivor capable of infecting other carps! According to Mordi, even a positive PCR test does not mean that the fish is automatically a threat to other fish. It might be possible that remaining fragments of KHV DNA could result in a positive PCR, but that it is not capable of infecting other fish anymore. The real test is to grow these parts on a cell culture and try to infect naïve carp with it. Professor Ron Hedrick from Davis California is the only scientist who tried to do this and was not able to grow them on a cell culture. This means that the positive PCR was from the fragments of DNA, not from the actual virus. Many people (who are not into PCR) are only interested in “Positive” or “Negative” but it is beyond that. The beautiful thing in RT-PCR is that it tells you how many parts of the virus DNA you have, not only if the result is positive or negative. In the next stage of check, it could also show from what amount of DNA it is infectious. But this kind of research is very expensive.
Why isn’t KHV in Europe mentioned on the notification requirement list of illnesses?
Unlike swine fever or bird flu, KHV causes no immediate threat to public health and therefore Koi dealers and keepers have no notification requirement for it (yet). In the European Union and the OIE (Office International des Epizooties, http://www.oie.int) the Koi Herpes Virus is a ‘candidate’ for notification requirement. However, if KHV will be listed, it won’t necessarily mean that an outbreak in the Netherlands will be announced to the world, since the entire country is already considered to be a contaminated area; thus an outbreak automatically requires neither an isolation of the contamination nor pre-emptive killing when the area from which the virus can spread, is already too large. An outbreak of the ‘bird flu’ on the other hand will demand that anything necessary will be done to prevent it from spreading.
What about the rules in other countries outside of Europe?
In Israel, KHV is on the notifiable disease list since 1999. This is of benefit for the carp breeders, since the government pays most part of the PCR tests. In Japan, the regulation is very strict. KHV and SVC are on the notifiable disease list. The provinces in Japan however, have a different approach in dealing with the situation. In Niigata for example there is a huge research center (Niigata Prefecture Inland Water Fisheries Experiment Station) for fish diseases and KHV in particular. This governmental center controls the entire Koi breeding in Niigata. One of their main tasks is to prevent diseases from spreading and to gain as much knowledge as possible. Testing on KHV and SVC is obligatory. In the research center marked carps are bred in a sterile environment so it is a 100% certainty that they do not carry any virus. These carps are mixed with the breeders’ carp. The breeders have to raise the temperature for three weeks to 20 – 25 degrees Celsius. The breeders are randomly inspected to check if they follow the strict rules. After three weeks the marked carps are tested for KHV/SVC. Only after this the breeder can get an export clearance to European and Asian Countries. This export clearance is NOT necessary for the United States! Last year 537 breeders were under control, none of them had a case of KHV. This kind of handling of course only makes sense for the consumers when dealers do not mix these fish with other dubious stock.
What to do when KHV seems to be present:
If you think you have KHV in your pond, do not panic! Just immediately increase the temperature of your water to 30-32 degrees Celsius, even if it requires a temperature increase of more than 10 degrees Celsius within a single day. If you do not have enough heating capacity for your entire pond, then you should put your Koi in a quarantine or treatment tank so that the smaller amount of water can be heated up sufficiently. A good heating system is all you need to save your valuable fish. Make sure there is enough air circulation (this is vital) because warm water contains less dissolved oxygen than cold water. Also strictly guard the quality of the water and regularly refresh the water, especially in smaller habitats. Sacrifice one carp (if no other means of diagnosis are available) to be examined in a lab, in order to be sure that all the right measures were taken. Maintain a temperature of 30-32 degrees Celsius for the duration of three weeks and then let it slowly go down to 23. Observe your Koi very thoroughly and if again symptoms of KHV arise, repeat the entire process. If the lab confirms KHV contamination, do not sell or give away any of the surviving Koi. You could otherwise in fact risk contaminating other ponds which causes a kind of sorrow you do not wish upon another. To be on the safe side, Mordi’s advice is to heat up the pond a second time. The first “Heating Cycle” is to save the fish, then they are in the status of “Survivors”, the second “Heating Cycle” is to make them “Resistant” and safer to add new fish to the pond.
How to minimize the risk of a KHV outbreak in your pond:
Before you add new fish to your pond, it is wise to observe them elsewhere for several weeks at a temperature most suitable for the KHV. Unfortunately fish are often imported in Autumn; a time when the temperature in Koi dealers’ ponds is still below 20 degrees Celsius, which is too low to detect symptoms of a possible contamination. Then in Spring, around April/May Koi keepers will collect their new Koi from the dealers so that a possible outbreak won’t be detected any earlier than June/July when the temperature is a steady 20 degrees plus. It would be ideal if dealers quarantine new Koi for several weeks at a temperature of 22 to 24 degrees as it would decrease the risk of buying infected Koi. Some dealers even send one or more Koi to the CIDC for examination, a good thing in itself, however it offers no 100% guarantee of a virus free batch. It takes 2% of a population (with a maximum of 150 Koi) to be examined, to obtain a 95% certainty of a virus free batch. As you can imagine, this is too much for most dealers. A reasonable level of security can also be obtained in cooperation with the Koi breeder who needs to have its fish examined regularly and furthermore needs to apply the rules of integral chain liability. These rules dictate good hygiene of the breeders own business, as well as that of its suppliers. Furthermore fish batches should not be mixed with batches from any supplier outside of the chain.
However, the best method by far is maintaining newly acquired Koi in quarantine for a certain amount of time. This is not only a smart thing to do by preventing your main pond from infection with KHV, but is also very wise for all sorts of other contagious diseases. The following schedule should be adhered to:
- Fill your quarantine tank with water from the pond and hook up a quality bio filter.
- Put several Koi of lesser quality in the tank to start up the filter and keep the biomass alive.
- Regularly check the water values and refresh it often with water from a tap or from the pond. If you use water from the pond, please note that you should not reverse this process; do not use water from the tank to put back into your pond. Even the smallest amount of water from the quarantine tank should by no means be allowed to mix with the water from the pond. Therefore you should use
separate nets and other equipment for the tank and the pond.
- Make sure that the temperature of the quarantine tank is about the same as that of the dealer before you collect your new fish.
- Place your new Koi in the tank and gradually increase the temperature up to 23 degrees Celsius (with a maximum daily increase of a few degrees). A heating device in your quarantine tank is a must nowadays!
- Maintain a temperature of 22 to 24 degrees for 3 weeks and keep a close eye on the fish.
- If either the ‘old’ or the ‘new’ fish start showing symptoms of KHV, you need to contact a lab immediately and send one or two Koi to be examined. In Holland this would cost you about a hundred Euro for up to two fish, which is likely only a fraction of the total value of your Koi population.
- If the result is positive, there is KHV in your quarantine tank, but fortunately not necessarily in the pond. The safest option in this case would be to get rid of the Koi in the tank and disinfect the tank thoroughly before using it again.
The Great Outbreak
In discussing the origin of KHV, people often point the finger at Israel because that is where in 1998 ‘the great outbreak’ took place. Nevertheless, in 1990, already the illness struck in the US and in 1996 the first known outbreak in England took place. The first known case in the Netherlands was in 1998, but the virus is possibly a lot older than all this. It is likely that keeping a lot of fish together in small spaces (commercial cultivation) and the export of live fish (show fish farming) caused the illness to become so widely spread. The origin of the illness is perhaps not so important, but the fact that it is there is what counts. The problem in Israel was of gigantic proportions; isolation of various batches of fish wasn’t an option because the industry is huge and the Israeli farmers are located very close to each other. Many animals are moving in between ponds, so it was impossible to close areas for quarantine. Israel is on the way of “bird migration” routes. Twice a year, millions of birds pass through Israel on their way to Africa and back. The breeding ponds are popular forage places. Also nobody could assure the farmers that if they will disinfect the ponds it will not come back after a year or two.
The different lines of approach
There are two different ways to approach the problem: you can try to prevent the virus from spreading or you can accept the problem and learn to live with it. Examples of trying to prevent it from spreading are: Quarantine measures, disinfecting cultivation ponds, sales tanks and nets, strictly separating fish batches and limiting export. Any of these measures, of course, are wise to take even if merely to prevent other diseases from spreading. The other way of dealing with KHV is the way the largest Israeli Koi exporter (eight million fish a year), Mag Noy, does. Mag Noy and professor Kotler are convinced that there is another way to deal with the problem. Destroying all the carps and Koi in Israel will be a drastic interference in nature and is un-ecological. As mentioned before, to disinfect mud ponds, this means killing every creature that comes in contact with ponds and is impossible to do. Secondly they decided to focus on the fish. Meaning creating fish that will survive if it “Meets” the virus. Another point is “Human Nature”, if the virus is already well spread in Europe, there is a lot of regulations and measurements needed to impose it. As we already know, not all people are following the instructions, and how to control the “New Europe” with all the open borders? They would not be surprised if a long and hot summer would cause enormous outbreaks in Western Europe. According to professor Kotler it is even naïve to assume that we could eradicate the virus simply by measures of isolation and disinfection. Look at the flu, hepatitis or AIDS; research budgets for these illnesses are much larger and yet they are still there. Viruses that target animals, like bird flu or swine fever emerge every now and then again as well, despite of all measurements.
The old approach of Israel (Mag Noy):
After the great outbreak of 1998, that caused the production to crash entirely because of the massive death rates, and the after effects in 1999, Mag Noy tried desperately to find a solution for the problem at hand. With the help of Professor Kotler, they realized quickly that it was a virus that caused entire Koi populations to perish. Research then showed that the virus became inactive at water temperatures of above 28 degrees Celsius. ‘Overheating’ became the key factor in the salvation of the production. At a water temperature of between 18 and 28 degrees the virus seemed to multiply much too rapidly for the defense mechanism to be able to keep up. Above 28 degrees the multiplication of the virus is slowed down enough for the defense mechanism to keep up. This is why, for quite some time, the following method was used to make the fish resistant:
- Around April, cultivated fish of a minimum of ten grams (three months old) would be exposed to Koi suffering from KHV. The Koi are thus not injected with the virus (like many people like to believe). Then Koi were randomly picked for a PCR test to see if the virus had nested in the population they were trying to get immune.
- After this the temperature would be increased to above 30 degrees for the duration of several weeks. Loss of fish in this stage could be as much as 40 to 50%! Losses are mainly caused because the fish are very small. The bigger the fish, the better it goes through over heating.
- Surviving fish would then be placed into cultivation ponds from May till September to be exposed again to the virus (where it naturally derived from). The temperatures in these ponds are in the danger zone of 18-28 degrees Celsius for a large part of the growing season.
- In September and October the fish would be harvested and exposed again to several fish suffering from KHV.
If, after being exposed for the third time, even one fish died of KHV, the entire batch was rejected and all of the above measurements were repeated. This ‘old method’ required contaminated Koi to be kept ‘in stock’, so that new batches could be exposed. Storing Koi that are KHV infected is done with a carrousel: a round vat divided into several non water proof compartments. One compartment is filled with KHV infected Koi. The next compartment is filled with naïve Koi. After five days, (when the virus is most contagious, see diagram) naïve Koi will be placed into the next compartment so that the fish in the first compartment can be used to contaminate another group of fish. This compartment is filled with naïve Koi and so on. Throughout this process dead fish are removed daily. The ‘carrousel of death’ is a rather bizarre but effective way to have contaminated Koi at one’s disposal non stop.
It may be clear that they paid a very high price for this ‘overheating’ method; a loss of fish up to 50% was a very costly business. In the mean time, Mordi had established a good connection with Professor Kotler who quickly started to develop a vaccine.
The vaccine:
To our enormous surprise there appeared to be an effective KHV vaccine for quite some time! The vaccine was created by cloning the virus from one culture medium of living carp cells to another. Some of the clones were slightly weaker than the original and so these weaker versions were used to clone with further. On top of this, the clones were exposed to UV light to partially damage the DNA. Eventually this process delivered a much weaker version of the virus that yet still possessed the essential characteristics of the original virus. P36 (it took 36 generations of clones to create a satisfactory vaccine) was the result. Please note:
- The risk that P36 mutates back into it’s original dangerous form is very small and close to Zero.
- P36 is hardly dangerous for carps.
Why don’t we have it then?
Unfortunately such matters are rather complicated; P36 is a living virus (vaccine) and the export of such a thing is subject to strict regulation. So far no one has managed to create a working vaccine with an inactive (dead) virus. Someone is likely to someday succeed in doing so, but it will take more time and research. Besides, facilities have to be created to produce such a vaccine on a large scale. Another practical problem lies in PCR tests; all vaccinated Koi carry a ‘close relative’ to the original aggressive virus and therefore the test results will be positive. The most important issue however will be that of liability; how could one even prove that the vaccine itself would not be responsible for a KHV outbreak in a pond where not all Koi are treated with the vaccine? For the issues with PCR and liability a solution has been found in the shape of a ‘marker’. More about that will follow later on in this article.
The new (current) approach of Israel (Mag Noy):
Elaborate testing revealed the ideal amount of vaccine needed to vaccinate a Koi with P36. For two hours fish will be put into water containing P36. Again, the Koi themselves are thus not injected with the vaccine. These Koi must be above a minimum of ten grams, which is the equivalent of an age of about three months. The reason for this age minimum is that younger fish have hardly developed an immune system yet and for some strange reason also are not (yet) susceptible to the virus. The Koi then will be kept at 21 degrees Celsius for the duration of 21 days. ‘Over heating’ is no longer a method used in Israel! After this the same procedures as in the ‘old method’ will be followed. The fish are put into cultivation ponds where they will be exposed to the natural (aggressive) virus for the first time. Some of their cells have already been occupied by the P36 virus and the number of antibodies in these Koi is already high. The fish are thus well ‘equipped’ to deal with the hostile intruders and have become resistant to the virus. After having spent their summer in these cultivation ponds, every year, a sample of 100 kilogram of fish from each pond is tested by exposing them to the wild type virus, to check if the group is resistant. According to the standards: zero mortality is required, otherwise the whole pond is going through the vaccination process again. This third exposure becomes increasingly important because the virus slowly perishes in the cultivation ponds, which is a logical consequence of the ponds being only populated with fish that are vaccinated with P36, leaving little or no room for the original virus. The loss of fish in this method is up to 15%, when using of course only three months old fish. When larger fish are vaccinated with P36 (this time by means of injection) no Koi are lost.
Difficulties in vaccination with P36:
Problem 1: How long do vaccinated Koi stay resistant?
We visited Israel eleven months after the first fish were vaccinated with P36. Since then the fish have been regularly exposed to KHV and up till now they have proven to be resistant. How long this resistance will last, is yet unknown. It is possible that once vaccinated a Koi will remain resistant for life (as is the case with a smallpox vaccination for children). If the resistance turns out not to last a life time, a repeat of the vaccination, a ‘booster’, is needed.
Problem 2: Is it possible that a vaccinated carp can contaminate a non vaccinated carp?
Mag Noy and Professor Kotler are firmly convinced it is not! The fish no longer secrete the virus. In the USA and in Europe however, they aren’t too sure of this. To take away any doubts, vaccinated populations of fish are sent to laboratories outside of Israel to test the fish. At the moment the German Veterinarian Services is checking the vaccine. Up to two years ago Mag Noy tested resistant fish for this important question. They co-habitat them with naïve fish, they stress them with naïve fish and even immuno-suppressed them (shutting down the immune system with cortizol), with no mortality at all!
The question whether vaccinated carp can contaminate non vaccinated carp is of course more pressing when using the old method of vaccination because the aggressive form of the virus is used to create resistant Koi. With the current method it could even be desired for vaccinated fish to be contagious. However cruel this may sound, it is not; since P36 is hardly dangerous, it would by spreading make other fish resistant against the aggressive form of the virus without any fish getting ill. But as said above, according to Kotler and Mag Noy it is impossible that vaccinated Koi are contagious, also not with P36!
Problem 3: You can easily say it isn’t possible, but can you prove it?
One cannot really blame people for questioning Mag Noy in its conviction that vaccinated Koi are not contagious, as Mag Noy thrives on export of Koi. Despite of all their testing, it was not enough and it left many people with question marks. If an outbreak of KHV occurs in any pond occupied with vaccinated fish and non-vaccinated fish, the finger will be easily pointed at Israel. It’s their resistant fish that survive the outbreak! To make sure that P36 cannot be falsely blamed, and because some countries demand that a vaccine is traceable, Professor Kotler created a ‘marker’. A marker is a unique piece of DNA sequence inside the genetic code of P36 that differs from the original virus. A PCR test with it will be able to distinguish P36 from the original virus. By coincidence the announcement that Professor Kotler and his crew succeeded in creating a marker was made to Mordi during our visit to Professor Kotler. It was very exciting to have witnessed the announcement of such a breakthrough! By using a PCR test on KHV infected Koi, it can now be proved that it is the original virus and not P36 has caused the illness. Mag Noy is so keen on the success of vaccination that they are willing to go as far as guaranteeing that vaccinated fish are not contagious. Of course this can not be proven until P36 has passed all tests without fail. Mag Noy voluntarily offers batches of, P36 vaccinated, Koi to various research laboratories (in the USA, Germany and Netherlands CIDC) to do whatever test these labs wish to do. The CIDC in Lelystad however has to decline this offer because it is a non commercial institution. Being non commercial would make them perhaps more objective, but it ‘disables’ them from testing a vaccine created by a commercial enterprise.
Problem 4: I am convinced, so can I get the vaccine?
Unfortunately, it is not that easy, which in another way is just as well. There are very strict regulations regarding the import of vaccines. After all, we are dealing with a living virus that needs to be tested thoroughly if we want to prevent any possible catastrophe from happening. No country would take such a thing in unless it is absolutely clear that it is safe. In Indonesia and Poland however, the extremities of the problems (especially with consumer carps) are of such an extent, that they had to call upon the Israeli vaccine for help (they conducted their entire test on the vaccine very fast as an urgent National need procedure) .When approved, a full scale production still needs to be started. All the testing and all the paperwork will take up around two years. Koi keepers in Western European countries will most likely have to wait untill 2008 before they can get their hands on the vaccine. Furthermore, at this moment the vaccine has to be maintained at a temperature of minus 70 degrees Celsius, which makes transport and distribution of it costly and complicated. And then, of course, there is the matter of the ‘booster’. If necessary, after how long will it be necessary to repeat the vaccination?
Problem 5: What about that “booster”, my fridge only goes to minus 20 degrees Celsius.
At the moment there is a research going on in trying to create a “Dry Booster” (Powder). If the research succeeds, then it is another step forward. The meaning of this is that it can be kept in a normal fridge (4° Celsius) in the hobbyist hands. And if needed, once a year or so, to collect all your fish into a small vat, spray the vaccine into the water for an hour or so, and put them back to the pond. In this case it will be as if you are vaccinating all your stock.
Question 6: What would such a vaccination cost?
The US and Japan are also separately trying to create a vaccine. Israel made several attempts to cooperate with Japan, so far without success. Finding a vaccine and testing it, as you can no doubt imagine, is a very costly matter and therefore it is desirable to work together. On the other hand, if P36 turns out to be the solution, then Israel would apply for a patent and if no other countries by then have an alternative to offer, the vaccine could become ‘big business’. Mag Noy expects the vaccinating a Koi would increase its production price by 25% and because Israel produces mostly ‘cheaper’ Koi, the price increase would not be too high. The price increase for expensive Koi would be relatively less.
ELISA
As could be read throughout this article, the most used method in detecting KHV is the PCR test. There are however some disadvantages to it:
- It takes valuable time to get the test result.
- At least one fish from each batch or pond has to be sacrificed for it’s tissue.
- Only a specialized lab can perform a PCR test which is why time is lost in transporting the fish.
- PCR is relatively expensive.
For these reasons the ELISA Test kit was developed. It had to comply with the following demands:
- User friendly.
- Reliable.
- Quick.
- No need to kill or damage any fish.
- Cheap.
Various laboratories now use this ELISA kit that isn’t based on finding a unique piece of the viruses’ DNA, but on detecting a specific protein of the virus. The test kit requires some of the carp’s droppings and provides an answer within several hours. Before, blood samples were used, but then it became apparent that the droppings contained a higher concentration of the protein. A positive result can only be obtained if the virus is present in large quantities (ideally after four to eleven days after contamination) and if indeed there is an outbreak. If a Koi is carrier of the disease, it can not be detected with ELISA. According to Mag Noy, the test result obtained with ELISA is just as reliable as that of a PCR (if done in the period of illness).
A substantial advantage of the ELISA test kit is that it is easy to transport. I expect that it won’t be long until Koi doctors bring the kit along with the rest of their standard equipment when visiting various Koi keepers’ ponds. I also think that less death cases supposedly caused by KHV, but not confirmed by a lab, will be reported. You can often read on forums that there are many deaths caused by KHV while in reality only one or a few fish died. It is, of course, always wise to have a PCR test to confirm a positive result.
Mag Noy today and tomorrow:
The Koi production at Mag Noy is currently almost back at the level it was before the great outbreak. Mag Noy is the name of the umbrella organisation of five Koi producers and on the night after the successful development of the marker was announced, they all decided together to vaccinate all Koi with P36 from that moment on. Another ‘scoop’ for us during our stay! Mag Noy aims to have only Koi that are vaccinated with P36 by the 1st of January 2007. All Koi currently exported are vaccinated either with P36 (86%-89%) or by the ‘overheating’ method (11%-14%). Mag Noy claims that all fish are safe. If you study their export numbers, you can conclude that the rumours and bad reputation are in contrast with the increasing demand of Mag Noy fish. Why buy fish that are causing problems? To support these arguments even more, Mordi tells us that even vaccinated carps are being exported to England (a country known for very strict regulations) for sport fishing. CEFAS (Centre for Environment, Fisheries & Aquaculture Science) and the British Environmental Agency has done research on the vaccine and granted permission for import of vaccinated carps. That these carps are placed in England’s open waters, I must say, generates great confidence.
The high costs of P36 vaccination might cause Mag Noy to alter their working method; production might decrease in quantity, but increase in quality. It has become profitable to start Culling (selecting) earlier, since it requires less vaccine.
Carrier or not, is that the question?
The most urgent question remains whether a Koi that survived a KHV outbreak is not whether it remains a carrier, but if it is a potential threat to other carps. Survivors are now mostly subjected to pre-emptive killing, which causes both great emotional and material damage. Perhaps such pre-emptive killing isn’t necessary, but it remains nevertheless the only safe way to prevent major contamination and spreading. It is odd though, that we entirely ‘clean out’ our ponds, while, till recently, Mag Noy contaminated Koi with the aggressive virus on purpose to export them after that to other countries. When I asked clarification on this matter, Mordi replied in the following manner: For us the most important thing is to have safe fish, as we know that at the end many people mix fish in their private ponds. As for that we are doing our best to insure that. We work under very controlled and calibrated conditions when applying the ‘overheating’ method. Fish are exposed three times to the virus. The meaning of this is that the final “product” is “Resistant fish” . We can then be 100% sure that the fish are no longer ill when we sell them. Furthermore we are convinced that these carps are no longer contagious to other fish. When a non planned outbreak occurs in any private pond, it is “an un-calibrated event” meaning that you do not know what is the status of those fish which survive the outbreak. The final “product” here is no more than “Survivor fish”. One can’t be sure if the virus is eradicated or perhaps just dormant (because multiplication of the virus might be slowed down due to a temperature drop). According to Mordi, fish in such ponds are of utmost danger to non contaminated Koi and for that they must be destroyed.
In conclusion:
Clearly KHV is a highly contagious and lethal disease. It is however also obvious that there is hope. Unlike HIV, a virus I often use in comparison, there is a vaccine for KHV. Could Israel become the proverbial Promised Land after all? Cooperation between Koi producers and importing countries is of utmost importance. CIDC in Lelystad has excellent relations with the rest of Europe, the US, Israel and Japan and also with the NVN (Nishikigoi Vereniging Nederland, The Dutch Nishikigoi Association). This of course offers great potential. We will definitely maintain our pleasant connections and keep you posted on any developments. It is important that people will be more open about the subject and that any outbreak will be reported. It is therefore that I highly appreciate the openness shown by Mag Noy (Israel), but also by Momotaro (Japan) and Koikas Bemmel (The Netherlands). These are three fine examples of businesses that have been confronted with KHV and have dealt with it in all openness. Especially the openness of Mordi Haimi has positively surprised us. To be honest, I did not expect them to be so extremely forthcoming, but in the three days we spent with them, Mag Noy has clearly shown that they choose the “open visor” strategy. In our eyes, that is a very brave decision. If you would ask me if it is sensible to buy an Israeli Koi, I would, without hesitation, say that after all export fish are vaccinated with P36, the answer is “yes”. Of course the choice and responsibility is yours!
Joop "GinRin" van Tol
Health Officer
Nishikigoi Vereniging Nederland
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