Assessment of genetic tools for detection of carp edema virus (CEV) by a laboratory comparison test in Germany

Carp edema virus (CEV) was initially detected in Japan in the 1970s as the causative agent of koi sleepy disease (KSD) in koi and common carp (Cyprinus carpio L.) populations. CEV has spread all over the world mainly by trading of asymptomatic carrier fish. In Germany KSD was detected for the first time in spring 2009 from samples collected from imported Japanese koi at water temperatures below 10 degrees C. In 2014 large outbreaks of KSD in common carp and koi became evident in Germany. Several unsuccessful investigations were carried out to isolate the agent in permanently growing fish derived cell cultures. For diagnosis, the clinical signs of KSD were difficult to differentiate from the clinical signs related with koi herpesvirus disease (KHVD), except during the advanced stage of disease, when the fish started to lie down (sleep) on the bottom of the tank. The only successful and reliable diagnostic methods were PCRs and qPCR. However, virus sequences obtained from diseased specimens demonstrated high geographical diversity. Therefore, the original primer pairs, published from Japan in the 1990s, gave negative or uncertain results in detection of CEV in samples from carp and koi in Europe, and new primers and probes had to be designed for PCR and nested PCR as well as for TaqMan and SYBR green based qPCRs. While some of them recognised CEV obtained from koi samples only, a decision was made to use only PCR and qPCR assays that detect all variants of CEV for diagnostic testing. Three molecular assays were chosen to conduct a laboratory comparison test with 10 Animal Health Laboratories all over Germany. The aim was to evaluate and standardise CEV detection with defined samples, thereby making the results directly comparable. The results show that even though all laboratories used different PCR kits and cycler systems, they were able to identify CEV in samples from koi and common carp with given primers and a probe. Only in samples diluted above 1:1000, were the results negative by PCR and qPCR in a few laboratories. Some participating laboratories also identified CEV from koi and carp samples by sequence analysis. These results confirm that accurate and reproducible CEV diagnostic results can be achieved resulting in both an improved control strategy, and the implementation of more effective biosecurity practices, for koi and carp farms. Additionally, it was shown that primers and the probe designed for PCR, nested PCR and qPCR are very sensitive and specific to CEV. The results were reproducible across multiple laboratories that utilised different manufacturers of both PCR kits and machines as well as varying assay conditions.