Investigation on the diagnostic sensitivity of molecular tools used for detection of koi herpesvirus
Introduction
Koi herpesvirus (KHV), technically “Cyprinid herpesvirus 3 (CyHV-3)”, is a notifiable, lethal, highly contagious pathogen, causing KHV disease (KHVD) with mortality rates between 50 and 100% in koi and common carp (Cyprinus carpio) populations world-wide (Hedrick et al., 2000, Perelberg et al., 2003, Sano et al., 2004, Sunarto et al., 2005, Hedrick et al., 2006, Waltzek et al., 2005, Hutoran et al., 2005, Bergmann et al., 2006).
KHVD outbreaks were first observed in Germany in 1997 (Bretzinger et al., 1999). The agent was originally isolated in the USA and in Israel (Hedrick et al., 2000, Ronen et al., 2003). KHV has spread world-wide by infected but healthy looking carp or koi (Haenen et al., 2004). Such latent or persistent infections are characteristic of this herpesviral infection. During this phase of infection, only very weak virus loads are found in infected animals (Gilad et al., 2004, Goodwin et al., 2006). While the method of choice for diagnostics is PCR, the sensitivity of the diagnostic assay with regard to latent or persistent infections is largely unknown since it has never been investigated under standardized reaction conditions.
In this study quantitative investigations were carried out to explain false-negative results obtained from latently/persistently infected fish, using a combination of KHV fragment bearing plasmids as a “gold standard” and total DNA purifications obtained from KHV-I infected CCB cells. Selected PCRs were then retested with field samples for confirmation of the results.
Section snippets
Controls and plasmids
“Gold standard” quantitation of KHV genome equivalents in diagnostic materials and positive external controls, used a plasmid preparation (vector pGEM®-T Easy: Promega, Mannheim, Germany) with a 484 bp KHV fragment from open reading frames (ORFs) 89–90, accession no. AF411803 (Gilad et al., 2002) with known plasmid concentrations and fragment copy numbers (Table 1). Additionally, an internal control system (IC2) according to Hoffmann et al. (2006) was included, using duplex real-time PCR
Results
The gold standard showed an association between plasmid concentration and resulting ct value. With the known relation between ct values and copy number from the plasmid, the ct values obtained with the different dilutions from the DNA derived from infected CBB cells could be assigned to genomic KHV equivalents. Real-time PCR with undiluted material resulted in ct values of 12–14, corresponding to about 109 genomic KHV equivalents while with DNA at a dilution of 10−7 (10 fg) ct values of 38–40
Discussion
KHVD has recently become one of the most important threats to aquacultured carp and koi. A worldwide increase of KHVD is noticed in both aquacultured carp and koi. While the identification of KHV in acute outbreaks is not a problem with published PCRs and other PCRs used additionally in different laboratories, the diagnostic sensitivity of these tests with regard to the detection of latently/persistently infected carriers has not yet been compared. In the past, negative PCR results have been
Conclusion
Recently, EU legislation and legislation in some member states such as Germany has focused on combating KHVD. For this purpose the most sensitive diagnostic tests must be used to identify latently or perhaps persistently KHV infected carriers with a virus load between 5 and 10 KHV copies. According to this study these are the real-time PCR (Gilad et al., 2004), the nested PCR (Bergmann et al., 2006) and the newly established sn PCR.
Acknowledgements
We thank Irina Werner for her excellent technical assistance, Dr. Keith Way (CEFAS, UK), Roy Palmer (EFB, Ireland) and Annette Beidler (FLI, Germany) for their critical review of this article as well as Dr. Runge (LAVES, Lower Saxony) for testing of the koi samples in his laboratory by real-time PCR.
References (26)
- et al.
Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR
Journal of Virological Methods
(2009) - et al.
A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses
Journal of Virological Methods
(2006) - et al.
Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio
Vaccine
(2003) - et al.
Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide
Journal of Virology
(2007) - et al.
Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis
BMC Microbiology
(2005) - et al.
First detection, confirmation and isolation of koi herpesvirus (KHV) in cultured common carp (Cyprinus carpio L.) in Poland
Bulletin of European Association of Fish Pathologists
(2006) - et al.
Similarities and heterogenicity of koi herpes virus (KHV) genome detected in ornamental fish without clinical signs
Aquaculture
(2007) - Bergmann, S.M., Sadowski, J., Kiełpiński, M., Bartłomiejczyk, M., Fichtner, D., Riebe, R., Lenk, M., Kempter, J....
- et al.
Detection of koi herpes virus (KHV) genome in apparently healthy fish
Bulletin of European Association of Fish Pathologists
(2009) - et al.
Mass mortalities in koi, Cyprinus carpio, associated with gill and skin disease
Bulletin of European Association of Fish Pathologists
(1999)