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.
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