Isolation and identification of carp interstitial nephritis and gill necrosis virus (CNGV) in fingerling common carp (Cyprinus carpio L.)

Abstract

High mortality in traditional carp farms and warm water cage farms was observed in Poland. Mortality rates were consistently above 50-80% in ponds. A preliminary study suggested that carp nephritis and gill necrosis virus/koi herpesvirus (CNGV/KHV) is the pathogen inducing this high mortality. The diagnosis of CNGV infection in common carp fingerlings based on viral isolation and current PCR assays has limited sensitivity. The aim of the current study was to develop more sensitive methods for the isolation and identification of CNGV in common carp, Cyprinus carpio L., fingerlings by cloning the virus gene encoding thymidine kinase (TK) and then using it for highly sensitive PCR diagnosis. Common carp fingerlings weighing 60 g that had survived mass mortality at a temperature of 22°C were obtained from two carp farms. The gills and kidneys from 20 diseased carp from each farm were used for virus isolation and identification. Koi fin cell (KFC) cultures were used for virus cultivation and propagation. DNA extraction was performed using thermal lysis. The supernatant containing the DNA was used in PCR assays. The virus isolated and identified from diseased carp fingerlings was used to experimentally challenge healthy carp fingerlings weighing 20 g by injection or bathing. The results of this study indicated that the TK-based PCR assay is simple to perform and is sensitive in detecting CNGV infection. The cytopathic effect (CPE) in KFC from 5 to 7 days after infection was observed. Cells became enlarged and developed abundant endoplasmic vacuoles. The results of the challenge test indicated that the isolated virus induced the clinical symptoms of CNGV infection. The mortality percentage was higher in the group of fish infected by injection (93%) in comparison to that infected by bathing (72%). Until the conclusion of the experiment (day 30), mortality was not observed in the control groups. The results of this study verified that CNGV is highly pathogenic for carp fingerlings and causes high mortality.

CORRESPONDING AUTHOR:

Prof. dr hab. Andrzej K. Siwicki, Instytut Rybactwa Śródlądowego, Zakład Patologii i Immunologii Ryb, Żabieniec, ul. Główna 58, 05-500 Piaseczno; Tel./Fax: +48 22 7562490; e-mail: aksiw@infish.com.pl