Investigation on the diagnostic sensitivity of molecular tools used for detection of koi herpesvirus

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Abstract

Previous and new PCRs for KHV detection were compared by estimation of their sensitivity in recognizing KHV DNA in plasmids, cell culture extracted KHV DNA and total DNA obtained from field tissue samples. A modified real-time PCR (Gilad et al., 2004), combined with an internal control system (IC2, Hoffmann et al., 2006) in a duplex assay, was used as a “gold standard”. The lowest reliably determined virus concentration between, 5 and 10 KHV DNA genomic equivalents, was found by real-time PCR (Gilad et al., 2004), nested PCR (Bergmann et al., 2006) and one-tube semi-nested PCR. All other published and unpublished PCRs, as well as the commercial Loopamp®, recognized KHV DNA at higher concentrations only. Additionally, KHV variants, newly adapted to European conditions, which could not be detected by PCR according to Bercovier et al. (2005) were found in two field samples from carp and koi from different regions of Germany. A negative influence of sample pooling was shown with field samples tested by real-time PCR.

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.

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