Prevention of a Mortal Disease of Carps Induced by the Carp Interstitial Nephritis and Gill Necrosis Virus (CNGV) in Israel

Ariel Ronen, Berta Levavi-Sivan, Marina Hutoran, Yechiam Shapira,
Michael Steinitz, Ayana Perelberg, Eli Pikarsky and Moshe Kotler

Department of Pathology, The Hebrew University-Hadassah
Medical School, Jerusalem, Israel

ABSTRACT

Massive mortality of koi and common carp - Cyprinus carpio species - was observed in many farms throughout Israel, resulting in severe financial losses. This lethal disease is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. We isolated a carp nephritis and gill necrosis virus (CNGV), which is the etiology agent of this disease. The virus propagates and induces severe cytopathic effects five days post infection in fresh koi fin cell cultures (KFC). The virus harvested from KFC cultures induced the same disease with mortality of 75-95% upon inoculation of naive koi and common carp.

Electron microscopy revealed viral cores with icosahedron morphology of 100-110 nm resembling the herpes virus. Electron micrographs of purified pelleted CNGV sections, together with sensitivity to ether and Triton x 100 suggest that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA molecule of 250-300 Kbp, larger than that of known Herpesviridae members. The viral DNA seems highly divergent and bears only small fragments (16-45 bp) similar to the genomes of several DNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent as yet unclassified virus species endemic to carpinoids. 

Carps, exposed to the virus at 23⁰C for 3-5 days and then transferred to the non-permissive temperature of 30⁰C, became resistant to a challenged infection and their sera demonstrated a high level of virus specific antibodies. We have isolated attenuated non-pathogenic viruses that render virus-vaccinated carps resistant to the disease. Furthermore, vaccinated fish developed high levels of antibodies against the virus. We suggest, therefore, that this attenuated virus could be used as a live vaccine for the eradication of the mortal disease afflicting common and ornamental carp fisheries in many countries.

We examined the pathobiology of this disease in carp using immunohistochemistry. We found large amounts of the virus in the kidneys of sick fish, and lesser amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected using both immuno-flourescence and semi-quantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days post-infection, which increased in severity up to 10 days post-infection. There was severe gill disease evidenced by loss of villi with accompanying inflammation. Minimal focal inflammation was noted in livers and brains.

Two diagnostic methods for identifying the CNGV in alive fish are in use in our laboratory: Using PCR with authentic primers is applied on blood, kidney or gills DNA samples and immunological diagnostic kit. The immunological kit is design to be a simple, rapid and low cost diagnostic kit, which will be appropriate for retailers as well as hobbyists to identify the CNGV in presymptomatic fish. We believe that these means will be instrumental in preventing the distribution of sick fish worldwide.