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 KHV From the SAKKS Health Guide

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Chris Neaves

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PostSubject: KHV From the SAKKS Health Guide   KHV From the SAKKS Health Guide Icon_minitimeMon Oct 26, 2009 8:31 pm


Sounds like a very interesting seminar in January 2010 - I will definitely be attending.

In the mean time here is the section on KHV that I am writing for the new SAKKS Health Guide. SAKKS issues the Health Manual / Health Guide free of charge to members.

This is a cut and paste so I see the pictures did not come out and some of the text may be a little jumbled as it is associated with pictures.

It is not finished - however this may answer some of your questions and stimulate other questions for the seminar.

A virus (from the Latin virus meaning toxin or poison) is an ultramicroscopic (many times smaller than bacteria - they are about 1/100th the size of bacteria) infectious agent that can reproduce only inside a living host cell.

Viral populations do not grow through cell division because they are acellular. They use the machinery and metabolism of a host cell in the body to produce multiple copies of themselves and they assemble in the cell.
Viruses are found in almost every ecosystem on earth.
Viruses are the most feared type of disease as they are mostly untreatable.
Very few vaccines are available for viral diseases of fish. For many koi and carp farms the only remedy for some viral infections is complete slaughter of stock and disinfection of the ponds
Viruses are not considered living organisms.

One of the most important viruses to affect the koi industry is the Koi Herpes Virus – or KHV,
Other viral diseases commonly found on koi include Carp Pox and Lymphocystis.
Viral diseases are incurable. You must rely on the fishes immune system to overcome these problems.

There are many things we don't know about this virus that has become such a deadly problem throughout the koi world.
The more we can learn about this disease, the more likely we are to be able to defeat or at least control it.
I am deeply in indebted to -
• Spike Cover founder and past Director of the AKCA Project KHV, Mission Viejo, CA.
• As well as Duncan Griffiths (author of the excellent book on koi diseases xyz)
• Prof. Jim Phillips
• Mike Harvey -
for making the information freely available to me for inclusion in the SAKKS Health Guide. Many of the following facts are verbatim quotes.

Archived histological material taken from U.K disease outbreak in 1996 was later tested and KHV DNA was shown to be present in these samples (Way et al., 2004).

First found in Israel in May 1998 (Hedrick, et al., 1999).

First found in the U.S. in fish from the East Coast in August, 1998 (Hedrick et al., 2000).
Virus first isolated and partially characterized in 2000 (Hedrick et al., 2000).

In South Africa the exact date for the first case of KHV is not known. However, in late 1998 and early 1999 there was an outbreak of what we now suspect was KHV. At the time some dealers / breeders ascribed this disease outbreak to Bacterial Gill Disease (Flavo-bacterium).

The outbreak of the disease was so serious that SAKKS cancelled ALL shows in 1999.

At that time there was no laboratory in South Africa that could do PCR testing and so any testing had to be done in Germany. SAKKS was aware that some breeders and dealers sent samples for testing in Germany in the early 2000's however none of these breeders/ dealers ever disclosed any results or reported any confirmed KHV outbreaks.

Between 1999 and 2003 there were regular reports of dealers, breeders and hobbyist suffering catastrophic mortalities and by this time everyone was aware of KHV around the world. There was a strong suspicion during these years that it was KHV that was causing these massive mortalities but no one would admit to it.

In 2003 a Koi Farm suffered massive losses and Dr. Ralph Kneusel diagnosed KHV, which was confirmed in PCR tests conducted in Germany.

As a further precaution in 2004 the SAKKS decided to cancel ALL shows in South Africa due to the threat posed by KHV.

First PCR tests developed in 2002, Gilad and Gray produced different primers (Gilad et al., 2002 and Gray et al., 2002)

KHV has been found in most countries in the world where koi and/or carp are commonly raised or kept – (Pokorova, et al., 2005)

It is not found in Australia.

Is a herpes-type virus the genome of which is a double stranded DNA molecule of approximately 295 kilo base pairs (Haenen et al., 2006 – Aoki presentation). KHV is relative large as compared to other known mammal and bird herpesviruses (125 to 245 kbp), however, CyHV-1 (carp pox) has been estimated to also be about 295 kbp. KHV is proposed as a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae. (Waltzek et al., 2005)
Others resist the classification as a herpesvirus and prefer the designation CNGV. (Dishon, et al., 2005)
Is highly contagious and can produce high (80% to nearly 100%) mortality rates in diseased populations of koi and common carp (Dishon et al., 2005).
Appears to cause disease and mortalities only in common carp and koi but the virus can also infect goldfish and crucian carp (Haenen et al., 2006 –Bergmann & Tinman presentations).
Infections are transmitted via virus in the water, in fecal material, in sediments and from fish to fish (Dishon et al., 2005; Hartman, et al. 2004).
The route of infection into the fish is likely thru the gills (Dishon et al. 2005) and the gut (Haenen et al., 2006 – Bergmann presentation).
Propagates mainly in intestine and kidney of infected fish (Dishon et al., 2005).
Deaths can start within 1 to 2 days following the onset of clinical signs (Hartman et al., 2004).
Infected fish usually die within 6 to 24 days (post infection) at permissive temperatures (Dishon et al., 2005)
Permissive temperatures are variously reported as 17 - 26°C; 18 - 27°C; 18 - 25°C; 22 - 26° C and 18 - 28°C (Haenen et al., 2004; Hartman et al., 2004; Ronen, et al., 2003; Perelberg et al., 2003 & Haimi, 2003 [plus several others], respectively)
Temperature ranges for optimum virus growth in cell cultures tend to be 2 - 3°C wider than those in fish (Gilad et al., 2003).
Can produce latency and/or a persistent low-level infection such that survivors can later become infectious (St-Hilaire et al., 2005).
Can survive off the fish for weeks, probably in sediments and/or filters (Haenen et al., 2006 –Bergmann presentations).
Survives in water for 4 hours but less than 18 hours (Perelberg et al., 2003)
Clinical signs seem to be arrested at above 30°C and fish raised to this temperature can often survive the disease (Ronen et al., 2003).
Clinical signs seem to be arrested at below 13°C (Hedrick et al., 2005)
Does not appear to cause disease at or below 13°C (55° F) – (Gilad et al., 2003). However, researchers in Europe have shown the disease may be adapting to lower temperatures.
Antibodies have been found in survivors up to a year post infection (Haenen et al., 2006 –Dixon presentation).
Virus found up to 7 months post infection in the base of the gills, the kidney, the spleen and the leukocytes (Haenen et al., 2006 – Bergmann presentation).
The entire viral genome has been sequenced (Haenen et al., 2006 – Aoki presentation).
There are several different strains (mutations) of the virus (Haenen et al., 2006 – Ito presentation).
There is no concern with infections to humans with KHV. (Hartman et al., 2004; Southard et al., 2006) .
Sven Bergmann in Germany, has found the viral DNA in blood (white cells) for up to a year post infection.
Some Japanese investigators have found KHV in river water several months after the apparent cessation of the active disease in carp in that river.
Researchers in Weymouth (England) found that non-cleaned filters from systems that contained fish with active disease could infect naive fish for up to two weeks after all disease fish were removed from the system.
Hobbyists in Atlanta Georgia (USA) had a KHV outbreak in the summer (late June-early July), heat-treated (30°C) the diseased fish, many survived. However, naive fish placed in the pond broke with KHV the next spring.
Sophie St-Hilaire et al have authored two papers in which she and the others showed that survivors can re-break with the disease.
St-Hilaire et al and Adkison et al and others have found that some survivors develop antibodies to KHV and that (ELISA) tests for the presence of these antibodies are possible (both Weymouth and U.C. Davis offer such tests).
Bran Ritchie of Univ. or Georgia (USA) developed a serology test for inferring the presence of KHV antibodies - the test shows if dilute serum from suspect fish will inhibit the growth of KHV in a culture of Koi/carp cells.
Both the (ELISA) antibody tests and the serology tests have been used to detect survivors of past KHV infections with the inference that fish that test positive are very likely carriers of the disease.

If and where the KHV virus exists for periods more than 1 year inside the fish when it’s not causing disease.
If and where the KHV virus exists for long periods when in the environment (outside the fish).
At the moment there is no testing that can determine if a specific fish carries the (internal) potential to become diseased or infectious (even if it “passes” quarantine), i.e. a test to determine latency.
If there is a safe and effective way to rid latent or persistently infected fish of the virus or the potential to become infective.
If there is a vaccine possible that will confer long term protection against KHV.
An effective field test for KHV.
An effective field test for KHV antibodies.
If there is vertical transmission.
We don't know for sure where it hides when it's not causing active disease. We don't know what triggers it's re-emergence to "awake" and cause disease.
All this provides clues but we still don't know if the disease forms a true latency, i.e., inserts its DNA into a cell and waits for a later opportunity to replicate and re-emerge as a complete viral particle and cause trouble, and/or forms a persistent low-level infection (the entire viral particle replicating very slowly) that goes unnoticed for a time then resurfaces as full blown disease.
Several years ago the Israelis "made" what they called "naturally immune fish" (or NIFs), by intentionally exposing Koi to KHV then heating them to 30°C for a time. It is not known if this has produced carriers.

Not all diseased fish exhibit all symptoms.
Pale and necrotic gills (gill rot) is the most common symptom – see Figure 1

Hyperplastic gills observed with or w/o bacterial infection (gill rot).
Lethargy and “hanging” in the water toward the end-stage of the disease.
Sunken eyes.
Erratic swimming.
A notch in the “nose” of the fish.

Heavy mucus at the start of disease.
Lack of mucus later as disease progresses – skin feels like sandpaper.
Patches of discolored skin.

At the moment the easiest way to test for KHV here in South Africa is to take a swab from the gills and send the sample to the lab in Durban that does all the testing for KHV.
KHV typically found in the kidneys first and longest over the course of the active disease so this is a good tissue to test on dead koi.
Gills, spleen, fins and gut are also good tissue to test if the disease is suspected.
Tissue samples (e.g., gill, kidney, etc.) may be placed in rubbing alcohol (70% or 90%) for later PCR testing – (Goodwin, 2005- pers. comm.)
Droppings of infected fish have been shown to contain KHV that can be detected by PCR testing (Dishon, et al., 2005).
Use a swab on the gills for live koi.

As can be seen from the facts in this KHV section the virus can be latent in koi, it can be brought into a collection on or in a carrier koi, it can be found in un-cleaned filters after KHV koi have been removed from the system.
A koi in your collection from the previous season may have a latent KHV infection. Something may trigger the disease and you are now confronted with KHV outbreak.
This latency makes it virtually impossible to accurately identify the source. A koi keepers natural instinct is to identify the last fish brought into the system as the source but the facts are that it is virtually impossible to identify the true source of a KHV outbreak.
A lot is been done around the world to try to combat this viral disease.
Buying from reputable sources that adequately quarantine all fish arriving at their facilities.
Add koi from your collection (presumably KHV naïve) to the quarantine group is probably helpful as naïve fish are more likely so show disease symptoms (Haenen et al., 2004)
Observe fish in quarantine system noting symptoms and behaviors, particularly those typical of KHV.
Test for KHV.
Use separate equipment to house and handle quarantined fish.
Thoroughly disinfect equipment if it is used between systems and/or between separate lots of fish. Disinfecting, or otherwise verify the safety (non-infectivity), of everything that comes in contact with water of existing pond/system or new fish. Household bleach is a good disinfectant.
Supporting efforts to educate the koi community and to find new ways to control and ultimately eradicate this disease.

If KHV is confirmed, depopulation (killing all the infected and exposed fish) should be considered as carriers may exist in any survivors.

There are currently 6 groups reported to be working on vaccines for KHV. Immunizing “agents” are reported to include killed-virus, attenuated-live virus and DNA. Delivery techniques include oral, immersion and injectable. The groups include:
● Yoshimura & Miyazaki, Mie University, Japan,
● Aoki, Tokyo University, Japan,
● Perelberg & Kotler, Hebrew University-Hadassah Medical School, Israel,
● Shivappa & Levine, North Carolina State University, CVM, USA,
● Ritchie, University of Georgia, USA, and
● Novartis, Canada
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PostSubject: Re: KHV From the SAKKS Health Guide   KHV From the SAKKS Health Guide Icon_minitimeMon Oct 26, 2009 8:56 pm

Hi Chris

Thank you for your input. I will make sure that I will send your post to him and mybe he could help you refine your information.

I look forward to see you again at the seminar.


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