Persistence of viral RNA in fish infected with VHSV-IVb at 15°C and then moved to warmer temperatures after the onset of disease.

Smallmouth bass, Micropterus dolomieu Lacepède, bluegill, Lepomis macrochirus Rafinesque (coppernose strain), koi carp, Cyprinus carpio L., and channel catfish Ictalurus punctatus (Rafinesque), were infected by intraperitoneal injection with viral haemorrhagic septicaemia virus genotype IVb (VHSV-IVb) at 15 °C. When clinical signs of disease developed, one-third of the fish was moved to 20°C and one-third to 25°C. Mortality in challenged fish at all three temperatures ranged from 25 to 45% in smallmouth bass and from 70 to 90% in bluegill. No koi carp or channel catfish died during the study. Viral copy numbers detected by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in fish dying at 20 and 25°C decreased over time. In survivors of the challenge, viral copy numbers were higher in the more susceptible species (smallmouth bass and bluegill) than in the more VHSV-IVb disease-resistant species (koi carp and channel catfish). In fish surviving 28days post-infection, prevalence of infection was 66-100% depending on species and temperature, and VHSV-IVb was detected at 10(3) -10(5) copies µg(-1) host RNA. Our results show that qrt-RTPCR is a useful tool to investigate fish kills even 28days after temperatures are elevated above those known to be permissive for VHSV replication.

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms.

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus, caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10(7) copies microg(-1) DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10(6)-10(8) copies of CyHV-2 microg(-1) DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22-10 degrees C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (+/-SE) of 7.3 +/- 11 to 394 +/- 55 microg(-1) DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.

Detection of the herpesviral hematopoietic necrosis disease agent (Cyprinid herpesvirus 2) in moribund and healthy goldfish: validation of a quantitative PCR diagnostic method.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen of goldfish Carassius auratus auratus L. that causes herpesviral hematopoietic necrosis (HVHN) disease. The disease is associated with necrosis of hematopoietic tissues and anemia with high mortality. We have developed a real time 5'-nuclease PCR method (Taqman) that quantitatively detects CyHV-2 with a linear response over 8 logs of target concentration. The coefficient of variability on replicate samples tested on different days was 13% and the calculated sensitivity approached 1 target molecule per reaction. The assay does not cross-react with other similar fish herpesviruses, including CyHV-1 (carp pox) and CyHV-3 (koi herpesvirus), but reliably detects known CyHV-2 positive fish. The assay detects CyHV-2 not just in clinical cases of HVHN but also in apparently healthy 1 yr old goldfish fingerlings and even in 3 to 5 yr old broodfish.