Abstract

Until an effective koi herpesvirus (KHV) vaccine is developed and approved for use by the USDA, diagnostic testing in conjunction with strict quarantine procedures will remain our only methods of reducing the spread of KHV. To assist with these testing efforts, the Emerging Diseases Research Group at the University of Georgia College of Veterinary Medicine developed and validated PCR-based^a and in situ hybridization^b assays to detect target samples of viral DNA in the tissues of koi. Using stock reagents provided by Dr. Ron Hedrick, we have developed and validated an assay to detect neutralizing antibodies in the serum of suspected survivors of KHV infection.

When choosing a diagnostic assay, it is best for the clinician to determine specifically the question for which an answer is desired and then use the test that best answers that question. For example, if one wishes to determine if koi has been infected with the currently identified strain of KHV in its immunologically detectable past, one would choose a serologic based assay. If one wishes to determine if a koi with clinical signs suggestive of KHV may be shedding the virus, then one would choose a PCR-based assay. If one wishes to determine with KHV may have contributed to the death of a koi, then would choose in situ hybridization as a follow-up test for tissues in which suggestive lesions have been described.

Antibody Tests

The detection of organism-specific antibodies requires that the host survive an infection, the immune response of the host produces a detectable quantity of antibodies and that the antibody titer remains high for a sufficient period of time for detection. As a herpesvirus, it has been assumed that KHV causes a latent infection with periodic shedding of virus in response to triggers that have yet to be identified. If latency does in fact occur, a positive antibody test would indicate that the host has responded to an infection in its immunologically detectable past and should be consider a risk for shedding of virus in the future. Demonstrating a four-fold or greater increase in antibody titer in at least two serum samples collected two to four weeks apart would be necessary to confirm an active infection. Because antibody levels may decline over time (yet to be defined for a sufficient number of KHV survivors) to undetectable concentrations and some hosts mount a poor immune response, a negative antibody test does not mean a host is not latently infected. One should also consider that most serologic assays do not distinguish between natural infection and vaccination, and the widespread use of modified live vaccines overseas could complicate the interpretation of serologic testing during a quarantine program.

Nucleic Acid Amplification and Detection (PCR and DNA Probes)

Organism-specific DNA probes can be used to detect a target strand of nucleic acid that is extracted from a sample and attached to a membrane or it can be used to detect organism-specific nucleic acid in a section of formalin-fixed tissue that has been processed for histologic evaluation. The process of using nucleic acid probes to detect organism-specific nucleic acid sequences in tissues is called in situ hybridization. In situ hybridization using organism-specific nucleic acid probes is particularly valuable in diagnosing an infection when an organism is present in small numbers or produces lesions that microscopically resemble those induced by other viruses.

Use of DNA probes to detect the presence of an organism's nucleic acid in infected tissues, where high numbers of the organism are usually present, is fairly simple. In contrast, detection of an organism in excretions or secretions where numbers of the organism may be limited requires additional processing. To increase the likelihood of finding an organism in a diagnostic specimen (improved sensitivity), a sample to be tested is often subjected to a group of reactions that will amplify the number of DNA molecules in the sample that originated from the target organism. Increasing the number of target molecules in a sample is the purpose of the polymerase chain reaction (PCR) step of many nucleic acid detection tests. Theoretically, amplification procedures can use one copy of a nucleic acid sequence from an organism to produce 1,000,000 replicates of the same sequence.

The primary advantage of PCR is its extreme sensitivity in detecting the presence of small quantities of target nucleic acid. The major disadvantage of PCR is its extreme sensitivity in detecting the presence of small quantities of contaminating or irrelevant target nucleic acid, which may or may not have originated from an organism within a host and may or may not have been recovered from a viable organism. When interpreted correctly, PCR technology provides indispensable information. When interpreted incorrectly, PCR technology can be extremely detrimental.

Any sample collected for PCR should be treated at least as carefully as a sample collected for culture. It is of interest that clinicians frequently discuss the recovery of certain bacteria from the surface of fish as contaminants, but it is rare to hear clinicians discuss the detection of a target segment of nucleic acid in similarly collected samples as contamination, even though PCR is magnitudes more sensitive and thus more prone to contamination error than culture. For this reason, one should consider that that any positive PCR-based test from the surface of a koi is a representative of the animal's immediate environment and does not necessarily represent the detection of nucleic acid from an ongoing infection. Likewise, PCR-based assays detect only a target segment of an organism's nucleic acid; they do not routinely differentiate between nucleic acid that originated from a viable or non-viable organism.

Diagnostic Tests Mentioned in the Text

 Virus neutralization and PCR-based assays--Infectious Diseases Laboratory, University of Georgia, College of Veterinary Medicine, Athens, GA 30602. 706-542-8092.

 In situ hybridization--In situ Hybridization Laboratory, University of Georgia, College of Veterinary Medicine, Athens, GA 30602. kpennick@vet.uga.edu