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Koi Herpes Virus KHV – Carp Nephritis and Gill Necrosis Virus CNGV
Article by Ralph Knuesel, DVM, University of Stellenbosch
In the last edition of KOISA, Jim Philips gave an excellent overview on viral diseases in carp focussing especially on Koi Herpes Virus (KHV).
As many of you may be aware, the first confirmed case of KHV in South Africa has been reported from the Western Cape in December 2003. This finding changes the situation for the local Koi hobbyist and industry insofar as KHV not being an exotic disease anymore but a reality we have to deal with also in South Africa.
This article serves to provide more information on this probably most deadly and economically important disease to the Koi industry worldwide and to avoid further spread of KHV. It is important to notice that we still don’t understand everything about the virus and the disease but hopefully, research will give some answers within the next few years on how to deal more successfully with KHV.
1. Disease history
Koi Herpes Virus (KHV), also more correctly known as Carp Nephritis and Gill Necrosis Virus (CNGV)[1], was first described in Israel in 1998. It has been reported in most countries where food carp and/or Koi are bred or kept including Europe, Indonesia, Taiwan, the USA and recently, Japan.
The name KHV is probably not the best name for the virus because on one hand the disease causes more problems in the massive food carp industry worldwide than in the Koi industry and on the other hand, latest research has shown that the responsible virus might not even be a member of the family herpesviridae at all. But KHV is still the most widely used name for this serious disease and as long as the taxonomy of the virus is not clear, that name will be used.
2. Some information about viruses/KHV
Viruses are tiny, extremely specialized organisms that cannot multiply outside a host-cell. Some viruses are able to survive for days and months without host, but fortunately, KHV seems to be quite fragile outside the fish. The virus was shown to survive at least 4 h in water without fish but not more than 21 h at about 25°C. However, we don’t know the influence of water temperature and organic material, etc. on the survival time of the virus, so rather wait for several days before considering water as ‘safe’.
Because viruses measure only a few nanometers (the billionth part of a meter) in diameter they can not be seen by light microscopy but only electron microscopy which makes diagnosis of viral diseases quite difficult. Bacteria, in comparison, are about 1000 times larger than viruses and about the limit of high power light microscopy.
There is no treatment against viral diseases in fish. Therefore prevention is the only option.
3. What do we know about KHV and the disease?
Koi Herpes Virus is species specific, which means it only infects Cyprinus carpio (common or ‘food’ carp and Koi carp). Even closely related species such as goldfish, cannot act as carriers of the virus.
KHV is typically spread by introduction of infected fish, but possibly also by contaminated nets and other pond equipment, water and on people’s hands.
The virus is released from infected fish into the water and most likely infects healthy fish via the gills. It multiplies within the epithelial cells of the gills before spreading via the blood to the kidneys, which are the other main target organs, and later also to the skin and internal organs.
Incubation period (time between first contact with the virus and outbreak of disease) is between 7 – 14 days, depending mainly on water temperature.
KHV is known to be active (“virulent”) in the temperature range between 18 to about 26°C. This temperature window is crucial in the understanding and management of the disease. Below 18°C the virus is presumed to be dormant (i.e. does not multiply within the fish) and infected fish will show no signs of disease, while above ca. 28°C the virus is heat-sensitive and is thought to be destroyed. Infected fish may thus carry the virus through the cool winter temperatures, only to break with the disease in spring once the water temperature reaches 18°C or more constantly for several days.
It has been suggested that a stress trigger (poor water quality, low oxygen levels, handling, treatment, etc.) is needed to activate the disease outbreak, but in some cases it has been found that even fish kept in pristine conditions readily succumbed to the disease.
Typical mortality rates are 70 – 100% (small fish and very large fish seem to be more susceptible than medium size fish) which means the disease has the potential to literally wipe out whole ponds or dams within a few days to weeks.
4. Triad of disease
It is extremely important to realise that the health status of a fish is never static but a delicate, dynamic equilibrium influenced by various factors. The so-called “Triad of disease” model classifies these factors into three categories: fish (“host”), pathogen and environment. With regard to KHV, the host is carp/Koi; the pathogen KHV and important environmental factors include water temperature, dissolved oxygen and fish density. When factors regarding any of these circles of the triad are out of balance, the circles move closer together in the middle. This middle region represents the potential for disease outbreaks.
Following conditions must be fulfilled for a successful infection and subsequent disease outbreak in a healthy Koi by KHV:
Ø Environmental conditions favourable for the virus (e. g. water temperature between 18 and 26°C)
Ø Enough virus particles (“infectious dose”) present in the water to establish the infection
Ø The Koi must be susceptible to KHV (no “natural immunity”)
Ø A trigger (stress, chemical treatment, etc.) will accelerate a disease outbreak
5. Typical clinical signs of a KHV outbreak include:
Ø Gill necrosis: gill tissue breaks down with pale – brownish patches
Ø Increased secretion of sticky mucus (whitish or pale patches) on the gills and the skin leading to exhaustion of the mucus cells and finally patches of dry skin (“sandpaper”)
Ø Mass-mortalities of 80 – 100% within a few days to weeks
Ø Fatigue, weakness and loss of appetite
Ø Gasping, increased gill movements
Ø Superficial skin haemorrhages, ulcerations and body sores
Ø High susceptibility to bacterial and/or parasitic infections
Ø Sunken eyes (advanced or chronic stage of disease)
Please note that not all of the symptoms are usually seen at the same stage. Individual fish may show a slightly different pattern of symptoms. KHV sometimes even causes mortalities without obvious clinical symptoms.
Most of the symptoms listed above are non-specific for KHV and can indicate bacterial and/or parasitic infections and/or poor water conditions, without KHV even being present. It is important not to jump to conclusions and make hasty decisions until a correct diagnosis is made.
6. What happens if a Koi is exposed to KHV?
Theoretically, there are 4 possibilities:
a) Not infected (natural immunity, not enough virus particles to infect the fish, sub-optimal water temperature for the virus, etc.); fish remains “naïve”
b) Infected and dies
c) Infected, survives but becomes asymptomatic carrier (?)*
d) Infected, survives and eliminates the virus (?)*
* Fish that do survive a KHV outbreak (up to 20% of infected fish) become immune to the disease, but it is not known yet how long this immunity protects these fish from re-infection nor whether they are subsequently asymptomatic carriers of the virus or not. Extensive testing still needs to be done in this field and in the interim, all survivors should be treated with caution and kept separate from other fish.
Several centres are reportedly working on a vaccine against KHV and this remains a hope for the future control of the disease.
7. Testing/diagnosis of KHV
The only reliable diagnostic test for the virus is the PCR (Polymerase Chain Reaction) technique. This molecular method is based on the detection and amplification of a defined, unique part of the viral genome. The specificity of PCR is therefore 100% (a positive result is 100% correct positive, if all necessary controls were included). However, due to the hidden nature of the virus in healthy carrier fish, the sensitivity of the currently used PCR is not yet close to 100% (a negative result is not 100% correct: it is possible to get false negative results).
Therefore, to have a more reliable result, at least three fish need to be tested. Pieces from the gills of up to three fish from the same pool/batch can be pooled into one sample, stored in 75% Ethanol and shipped to a specialised laboratory for analysis. The PCR for KHV is presently not available in South Africa and samples are currently sent to Germany (cost per sample including transport ca. 60 Euro).
Another diagnostic method is histopathology: Very thin sections of formalin-fixated gills and kidneys are prepared and stained at a specialised laboratory. These sections are then investigated by light microscopy for signs of presence of virus (“inclusion bodies”, see picture 5). Unfortunately, this method has disadvantages:
Ø Inclusion bodies are not always present in KHV-infected fish
Ø Inclusion bodies are not specific[2] for KHV
Ø Only a well trained pathologist is capable to identify inclusion bodies
8. Superheating
There is a ‘superheating’ treatment, which has been employed extensively in the USA that obviously cures affected fish of the symptoms of KHV and saves valuable fish lives. However, there is no guarantee at this stage that post-superheated fish are not still virus carriers. Basically fish are heated and kept at over 30°C for at least 4 – 7 days.
Important:
Ø Prior to the superheating, the fish should be screened for external parasites (skin and gill smears) and appropriate treatment should be considered since most opportunistic parasites/bacteria thrive at high temperatures.
Ø Aeration is crucial since at higher temperatures the Koi’s metabolism needs more oxygen but the water carries less dissolved oxygen.
9. Prevention: How to protect your Koi from KHV
Buy fish only from reputable dealers, breeders and hobbyists who understand KHV, quarantine their fish properly and have the interests of the Koi industry and hobby at heart.
Quarantine any new Koi yourself regardless of their origin !
Quarantine is the single best method of preventing infectious disease - including KHV - from entering your Koi pond.
Set up a completely separate facility with own water supply, nets, bowls, cleaning gear, etc.
1. Routine quarantine procedure:
Ø If the fish are very weak from the transport, you may add salt (5 kg per 1000 l) for a few days
Ø Add one or two fish from your pond (the resident fish can also infect the newcomers; in that case you have to treat your fish/pond before you add the new fish)
Ø Quarantine for a minimum of 3 weeks
Ø Temperature doesn’t have to be maintained at a specific level
Ø Check routinely for external parasites
Ø Keep records
2. Cohabitation[3]/Quarantine for KHV:
Ø Add at least 3 cheap fish (carp/Koi) from a known KHV unexposed source / from your pond, which can be tested for KHV in case of a disease outbreak
Ø Important: Maintain water temperature at 23°C (+/- 1°C)
Ø Don’t add salt or anything that relieves stress. Remember: you may even have to stress the fish to trigger an outbreak of KHV in carrier fish (e.g. keep the oxygen supply rather low)!
Ø Check routinely for external parasites
Ø Keep records
At first sight, it may doesn’t make sense to stress fish in quarantine. But remember: Healthy fish can cope with slightly sub-optimal conditions but carriers will break out with the disease and we want to find out if there are carriers among the newcomers.
!!But don’t kill your fish in quarantine by playing too rough!!
If all fish are still healthy after 3 weeks, you can be fairly confident that your new fish are not infected with KHV.
This method is not fail-proof, but so far, there is no laboratory method of detecting carrier fish.
10.What is the situation regarding the local outbreak?
In December 2003 there have been the first confirmed outbreaks of KHV in South Africa at two places in the Western Cape, which had been in contact through aquacultural activities.
These sources have acted responsibly by sending appropriate samples for analysis to a specialized laboratory overseas and in conjunction with veterinary experts have taken appropriate measures to deal with the disease and control it’s spread.
Already two meetings have been held involving representatives of the local breeders, dealers, SAKKS members and veterinary experts to discuss the implications of the KHV outbreak, measures to contain it and how to protect the Koi industry and hobby in the future.
However, at the beginning of January 2004, a few more mass-mortalities with typical clinical symptoms for KHV were reported. These mass-mortalities are unrelated to the two original outbreaks and include at least one from KwaZulu/Natal. Samples for KHV have been taken and sent overseas for analysis but so far we don’t have results yet.
11.What to do if you suspect you have KHV in your pond
If you are experiencing sudden mass mortalities in your Koi collection, ask yourself these questions:
Ø Are all my water parameters OK? (Ammonia, Nitrite, Nitrate, dissolved oxygen, pH, alkalinity - check each one!)
Ø Have I ruled out any possibility of toxins (pond construction, insecticide washed into the pond by rain, etc.) running into my pond?
Ø Have I added any new fish without proper quarantine within the last few weeks or possibly during last winter (water temperature then below 18°C, has now recently reached 18°C)?
Ø Have I taken a skin scraping of several fish and confirmed microscopically that there are only normal levels of parasites present?
Ø Was the water temperature of the pond within the range of 18 - 26°C?
If you have answered ‘yes’ to all these questions then you may have KHV in your pond. In this case or if you would like to have your fish tested for KHV, contact: Mike Harvey.
[Mike Harvey is the Chairman of the KwaZulu Natal Chapter of the SAKKS. In countries other than South Africa, contact your local Koi Health Officer.
12.Acknowledgements
Centre for Fish and Wildlife Health, University of Bern, Switzerland, for general assistance and picture 4
Centre for International Agriculture (ZIL), Switzerland, for funding the research project about fish diseases in South Africa
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