How can KHV be diagnosed?

In the same way as the H1N1 flu (also called swine flu) shares many similar symptoms with other flu caused by viruses, all the symptoms mentioned above are too ambiguous for KHV diagnosis. Through hard work by many researchers, several diagnosis methods have been developed since the first scientific report of the KHV. The following chart is adapted mostly from Adams and Thompson to show the advantages and disadvantages of current diagnostic methods for detecting KHV.

Chart 1: KHV diagnosises



Histopathology is a very important tool to understand a new disease and recognize its causative agent. When the KHV disease emerged in late 1990’s, researchers also applied electron microscopy to study the morphology of KHV and temptatively categorized the virus in the family Herpesviridae. However, these two methods, Histopathology and microscopic examination, require sacrificing fish and skilled personnel. Hobbyists who have diseased koi fish would rather wait and let their beloved pet die naturally than kill the fish to be examined. Even if they are willing to sacrifice some fish for the examination, the rest of the fish may die during the time of sample shipping and laboratory investigation. Being highly contagious and with a short incubation time, KHV definitely needs to be detected with a fast, accurate, and reliable approach.

Current diagnostic methods for KHV include enzyme-linked immunosorbent assay (ELISA), loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) which all do not involve sacrificing fish. Viral DNA can be extracted from mucus or droppings. Each method will be explained as follows:

A. ELISA, developed in Israel, is based on specific antibody to KHV. It is a simple and rapid diagnosis well suited for large-scale screening. However, positive ELISA cannot distinguish between ongoing and past KHV infection.

Figure 6: Illustration of ELISA detection

Note: The figure is modified from the journal written byRose C. Gergerich & Valerian V. Dolja to suit the topic of KHV diagnosis (Source: www.apsnet.org/education/IntroPlantPath/PathogenGroups/plantviruses)

In addition, a rapid commercial immuno-diagnosis kit for KHV is available and requires no specialized equipment. From sampling to results interpretation only takes about 5 minutes. Yet the sensitivity of this kind of assay is not acceptable as it cannot detect low levels of antibodies or antigen.

B. KHV PCR assay, first developed in 2002, seems to be the most efficient method for virus detection. The PCR method can detect viruses from samples of fish’s mucus, droppings, and fresh and frozen fish tissues. Viral DNA extracted from fish can be amplified in a PCR machine. The amplified DNA fragments are non-infectious and can be easily detected by gel electrophoresis. At the end, the results are compared with positive and negative controls. The whole procedure usually takes about 4 to 6 hours.

Figure 7: The procedures for PCR detection


A real-time TaqMan PCR assay was developed to not only diagnose KHV, but also quantify the amount of KHV. This more sophisticated method has proven sensitivity and reproducibility in laboratories throughout the world, not to mention it can reduce the risk of cross-contamination and eliminate the need of running gel electrophoresis.

The main drawback of PCR is the cost for the facilities, including a laboratory divided into three rooms in accordance with PCR procedures, along with devices and machines. In addition, trained professional staff to operate the PCR test is also a critical element for reliable test results.

C. LAMP, developed in 2000 in Japan, is a novel method based on the principle of auto cycling strand displacement DNA synthesis. Unlike the PCR test which needs an expensive delicate machine, LAMP requires only a water bath or a hot block to produce a very large amount of the DNA. Another advantage of this method is its rapid reaction which only takes 40 to 60 minutes. Positive test result can be recognized by bare eye, although it is easier to interpret the result with UV light after adding a fluorescent dye to the solution. For the sake of its convenience, accuracy, specificity, relatively low cost, and simplicity, LAMP has been widely adopted for molecular diagnosis of many pathogens.

Although the methods mentioned above have been proven to be effective and some commercial kits are available, most can only be operated in laboratories with trained personnel. As a result, koi farmers and hobbyists often still rely on observation of the fish’s behavior and symptoms.



Bibliography

Adams A., and Thompson K.D. (2008), Recent applications of biotechnology to novel diagnostics for aquatic animals. Revue scientifique et technique, vol.27, no. 1, 197-209

Dishon Arnon, Davidovich Maya, Ilouze Maya, and Kotler Moshe (2007), Persistence of cyprinid herpesvirus 3 in infected cultured carp cells. Journal of Virology, vol.81, no. 9, 4828-4836

Gilad O., Yun S., Zagmutt-Vergara F.J., Leutenegger C.M., Bercovier H., Hedrick R.P. (2004), Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis Aquat Org, vol. 60(3), 179–187

Gray W.L., Mullis L., LaPatra S.E., Groff J.M., and Goodwin A. (2002), Detection of koi herpesvirus DNA in tissues of infected fish. Journal of Fish Diseases, vol. 25, 171-178

Ilouze M., Dishon A., Kahan T., and Kotler M. (2006), Cyprinid herpes virus-3 (CyHV-3) bears genes of genetically distant large DNA viruses. FEBS Letters, vol. 580, 4473-4478

Kalupahana A.W., and De Silva D. P. N.(2009), Application of polymerase chain reaction (PCR) technique to detect koi herpes virus (KHV) infection in carps. Proceedings of the Peradeniya University Research Session, vol. 14, 68-72

Mackay I., Arden K., and Nitsche A. (2002), Survey and summary Real-time PCR in virology. Nucleic Acids Research, vol. 30, no. 6, 1292-1305

Notomi T., Okayama H., Mashubuchi H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., and Hase T. (2000), Loop-mediated isothermal amplification of DNA, Nucleic Acids Reasearch, vol. 28(12), E63

Pokorova D., Vesely T., Piackova V., Reschova S., and Hulova J. (2005), Current knowledge on koi herpesvirus (KHV): a review. Veterinary medicine-Czech, vol. 50(4), 139-147

Soliman, H., Midtlyng, P.J., El-Matbouli, M. (2009), Sensitive and rapid detection of infectious pancreatic necrosis virus by reverse transcription loop mediated isothermal amplification. J. Virol. Methods, vol. 158, 77-83.

What are the signs and symptoms of KHV?

Diseased fish become lazy and disoriented, and remain near the water surface prior to death. Among many symptoms, gill necrosis is the most common clinic sign could be noted as early as 2 days after infection and becomes more evident after 6 days and beyond. Other clinical symptoms include sunken eyes, pale patches on skin and gill (See figure 3 and 4). Mucus secretions increase at the early stages, but decrease as the disease progresses. However, all these signs are not specific to the KHV disease. Furthermore, diseased fish are vulnerable to secondary infections caused by parasites and bacteria. These often worsen the fish’s sickness and alter the symptoms, leading to a misdiagnosis.

Figure 3: Patches on the skin


Figure 4: Patches on the skin


(You can see picturs of gill necrosis and sunken eyes in http://koiclubsandiego.org/library/khv/)

Internal lesions and damages can be found in many organs such as the gills, kidneys, spleen, liver and the gastrointestinal system. The earliest and most prominent changes usually concentrate in gills and kidneys though. Gilad et al. claimed that viral DNA can be detected in various tissues as early as 1 day post virus exposure with real-time TagMan PCR assay.



Bibliography

Gilad O., Yun S., Zagmutt-Vergara F.J., Leutenegger C.M., Bercovier H., Hedrick R.P. (2004), Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis Aquat Org, vol. 60(3), 179–187

Gray W.L., Mullis L., LaPatra S.E., Groff J.M., and Goodwin A. (2002), Detection of koi herpesvirus DNA in tissues of infected fish. Journal of Fish Diseases, vol. 25, 171-178

Hartman K.H., Yanong R.P.E., Pouder D.B., Petty B.D. Francis-Floyd R., and Riggs A.C. (2004). Koi herpesvirus (KHV) disease, Fact sheet VM-149, Extension service, Institute of Food and Agricultural Sciences, University of Florida 2004

Hedrick R. P., Marty G. D., Nordhausen R.W., Kebus M. J., Bercovier H., Eldar A. (2000), An herpesvirus associated with mass mortality of juvenile and adult koi Cyprinus carpio. Fish, Health Newsletter, Fish Health Section, American Fisheries Society, vol.12 (1), 44-57

Ilouze M., Dishon A., and Kotler M. (2006), Characterization of a novel virus causing a lethal disease in carp and koi. Microbiology and Molecular Biology Review, vol.70(1), 147-156

Pokorova D., Vesely T., Piackova V., Reschova S., and Hulova J. (2005), Current knowledge on koi herpesvirus (KHV): a review. Veterinary medicine-Czech, vol. 50(4), 139-147

Why are some infected fish not diseased?

As long as KHV enters the fish’s habitat, there is a good chance for the fish to get infected. However, a small proportion of the infected fish doesn’t fall ill and show symptoms. There are two likely explanations. First, the infected fish may have a good immune system against the virus and stay healthy. However, they can only survive from low-level infection at the permissive temperature. Second, the environment is not suitable for KHV propagation. The most critical condition for KHV breeding is water temperature. As there is slight difference among various reports, the permissive temperature for KHV is from 18°C to 28°C. This is probably the reason why most of the KHV outbreaks have happened in spring and autumn. Out of the permissive temperature, then the virus is suggested to stay dormant (latent) in its host cell for a period of time. Such a period of time could be at least 200 days at 12°C. Dishonet el al. also suggest that koi herpesviurs could persist in fish body, based on his finding that KHV-infected cells became “healthy” after the temperature was elevated to 30°C, but low levels of virus infection was shown to be maintained this way for 30 days. Notably, virus infection was reactivated after the temperature was switched to permissive temperature.



Bibliography

Dishon Arnon, Davidovich Maya, Ilouze Maya, and Kotler Moshe (2007), Persistence of cyprinid herpesvirus 3 in infected cultured carp cells. Journal of Virology, vol.81, no. 9, 4828-4836

Gilad O., Yun S., Zagmutt-Vergara F.J., Leutenegger C.M., Bercovier H., Hedrick R.P. (2004), Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis Aquat Org, vol. 60(3), 179–187

Gray W.L., Mullis L., LaPatra S.E., Groff J.M., and Goodwin A. (2002), Detection of koi herpesvirus DNA in tissues of infected fish. Journal of Fish Diseases, vol. 25, 171-178

Haenen O.L.M., Way K., Bergmann S.M., Ariel E. (2004), The emergence of koi herpesvirus and its significance to European aquaculture. Bull Eur Assoc Fish Patho, vol. 24, 293–307

Hartman K.H., Yanong R.P.E., Pouder D.B., Petty B.D. Francis-Floyd R., and Riggs A.C. (2004). Koi herpesvirus (KHV) disease, Fact sheet VM-149, Extension service, Institute of Food and Agricultural Sciences, University of Florida 2004

Ilouze M., Dishon A., and Kotler M. (2006), Characterization of a novel virus causing a lethal disease in carp and koi. Microbiology and Molecular Biology Review, vol.70(1), 147-156

Perelberg A., Ilouze M., Kotler M., and Steinitz M. 2008), Antibody response and resistance of Cyprinus carpio immunized with cyprinid herpes virus3 (CyHV-3). Vaccine, vol 26, 3750-3756

Pokorova D., Vesely T., Piackova V., Reschova S., and Hulova J. (2005), Current knowledge on koi herpesvirus (KHV): a review. Veterinary medicine-Czech, vol. 50(4), 139-147

Soliman H., and El-Matbouli M. (2009), Immunocapture and direct binding loop mediated isothermal amplification simplify molecular diagnosis of Cyprinid herpsvirus-3. Journal Virological Methods, vol. 162(1-2), 91-95

St-Hilaire S., Beevers N., Way K., Le Deuff R. M., Martin P., and Joiner C. (2005), Reactivation of koi herpesviurs infections in common carp Cyprinus carpio. Diseases of Aquatic Organisms, vol. 67, 15-23

How do fish get infected with KHV?

As you already know that the KHV disease is caused by a virus; therefore, a contact with the virus is required for infection. According to reports available, there are a variety of sources for KHV spreading.

Figure 2: The sources for KHV spreading



A. Infected fish: As long as a fish is infected, no matter if the fish is showing symptoms and if the viruses are small in number, KHV can ruthlessly spread out. KHV was presumed to invade vulnerable fish through gills, because viral DNA is firstly detected in gills in many cases. Yet, Costes et al. proclaimed that KHV is transmitted by a “skin-to-skin” mode when infected fish rubbed other individuals or objects. Uninfected fish can also get infected by pecking skin lesions off or droppings from infected fish.

B. Carriers: Researchers applied various detection methods to support that KHV has a wider range of host besides koi and common carp. Goldfish (Carrassius auratus), Crucian carp (Carassius carassius), grass carp (Ctenopharyngodon idellus), and tench (Tinca tinca), seemed to carry and spread KHV, although all appear not to be affected KHV.

C. Water and mud: KHV remains infectious in water and mud for at least 4 hours and up to three months in a water body according to the report by Minamoto et al. in 2008. Water from the Yura River which had a KHV outbreak in one section was collected for examination. High levels of KHV were still detected after 3 months.
D. Other media: Anything, such as a net or a filter, which had contact with the virus, could possibly transmit the virus.



Bibliography

Costes B., Sta;om Raj V., Michael B., Fournier G., Thirion M., Gillet L., Mast J., Lieffrig F., Bremont M., Vanderplasschen A. (2009), The major portal of entry of koi herpesvirus in Cyprinus carpio. Journal of Virology, vol. 83 no. 7, 2819-2830

El-Matbouli M., Saleh M., and Soliman H. (2007), Detection of cyprinid herpesviurs type 3 in goldfish cohabiting with CyHV-3-infected koi carp (Cyprinus carpio koi), Vet Rec vol. 78, 23-28

Hartman K.H., Yanong R. P. E., Pouder D. B., Petty B. D. Francis-Floyd R., and Riggs A. C. (2004). Koi herpesvirus (KHV) disease, Fact sheet VM-149, Extension service, Institute of Food and Agricultural Sciences, University of Florida 2004

Pokorova D., Vesely T., Piackova V., Reschova S., and Hulova J. (2005), Current knowledge on koi herpesvirus (KHV): a review. Veterinary medicine-Czech, vol. 50(4), 139-147

What is KHV?

KHV (Koi herpesvirus) is a highly contagious viral disease which was first reported in 1998, after the outbreak in Israel. However, analysis of archived materials indicated that a koi farm in the United Kingdom was infected by KHV in 1996. Since then,
KHV has been found in most countries that cultivate common carp and/or koi. The world organization for animal health (OIE) has listed KHV as a serious reportable disease since 2006.
The koi herpesvirus only attacks koi and common carp. That means the virus has no impact on other fish species, or on humans who eat the infected fish. The reason that this disease scares hobbyists and aquaculture farmers is that KHV often causes more than 80% to nearly 100% mortality in both fries and adult carp.

Some researchers recognized this unknown virus had similar shape and characters as other herpes viruses. Based on morphology and the sequential development in the host cell nucleus, the new virus was designated as koi herpesvirus. On the other hand, based on the finding of exceptional large size of dsDNA and distinct DNA sequences in KHV, it was once called as “carp interstitial nephritis and gill necrosis virus” (abbreviated as CNGV) by Ilouze et al. to reflect its clinic symptoms shown in infected fish. The dispute on designation was subsided when Waltzek et al. found the most convincing evidence to group KHV within the Herpesvirus family in 2005. KHV genome encodes several genes closely related to homology in two known cyprinid herpsviruses, namely carp pox virus (Cyprinid herpesvirus-1, CyHV-1) and hematopoietic necrosis herpesvirus (CyHV-2). Together with previous morphological and biological description, KHV officially became the third member (CyHV-3) of Cyprinid herpesviruses.

Bibliography”

Aoki T., Hirono I., Kurokawa K., Fukuda H., Nahary R., Eldar A., Davison A. J., Waltzek T. B., Bercovier H., and Hedrick R. P. (2007), Genome sequence of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide. Journal of Virology, vol. 81(10), 5258-5065

Gray W. L., Mullis L., LaPatra S. E., Groff J. M., and Goodwin A. (2002), Detection of koi herpesvirus DNA in tissues of infected fish. Journal of Fish Diseases, vol. 25, 171-178

Hartman K.H., Yanong R. P. E., Pouder D. B., Petty B. D. Francis-Floyd R., and Riggs A. C. (2004). Koi herpesvirus (KHV) disease, Fact sheet VM-149, Extension service, Institute of Food and Agricultural Sciences, University of Florida 2004

Hedrick R. P., Marty G. D., Nordhausen R.W., Kebus M. J., Bercovier H., Eldar A. (2000), An herpesvirus associated with mass mortality of juvenile and adult koi Cyprinus carpio. Fish, Health Newsletter, Fish Health Section, American Fisheries Society, vol.12 (1), 44-57

Ilouze M., Dishon A., and Kotler M. (2006), Characterization of a novel virus causing a lethal disease in carp and koi. Microbiology and Molecular Biology Review, vol.70(1), 147-156

Kalupahana A.W., and De Silva D. P. N.(2009), Application of polymerase chain reaction (PCR) technique to detect koi herpes virus (KHV) infection in carps. Proceedings of the Peradeniya University Research Session, vol. 14, 68-72

Ornamental Aquatic Trade Association (OATA). 2001. http:\\www.ornamentalfish.org

Pokorova D., Vesely T., Piackova V., Reschova S., and Hulova J. (2005), Current knowledge on koi herpesvirus (KHV): a review. Veterinary medicine-Czech, vol. 50(4), 139-147

Waltzek T. B., Kelley G. O., Stone D. M., Way K., Hanson L., Fukuda H., Hirono I., Aoki T., Davison A. J., and Hedrick R. P. (2005). Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpeviridae. Journal of General Virology, vol. 86, 1659–1667