Investigation on probable viral agents associated with grass carp and silver carp mortality in Guilan province

Warm water aquaculture development along with import of eyed eggs, Broodstocks and ornamental fish also transferring of fingerlings and broodstocks from one province to another one has led to spreading of some unwanted diseases. According to the Gilan Fisheries Department report, mortality rate of silver carp(Hypophthalmichthys molitrix) in the summer of 2010 caused losing of silver carp production more than 1162 tons which was valued over than twenty-three milion rials that was increased to about 40 million rials in 1391. Also grass carp had a less mortality rate in some farms. Observed symptoms and severity of losses increased the probability of viral diseases such as spring viremia of carp, koi herpes virus and grass carp reovirus. In this research, totally 411 silver carp and grass carp with 5 to 2300 gram weight from 83 farms were sampled during 2011-2012. Clinical symptoms was different in various farms and subcutaneous hemorrhages on the flanks, red fin base or total fin and Red belly and operculum, mild exophthalmia, hemorrhaging in the abdominal cavity (yellow, transparent or red fluid) and petechial haemorrhages of skin and viscera and Ascites in abdominal cavity and some silver carps and grass carps were observed with palied liver and gill. In some cases swelling muscle was observed in Silver carp. Several fries with the severe emaciation were observed. Difference between clinical symptoms can indicate the presence of different causes of mortality in different farms. Various water colors from completely transparent to dark green, yellow or slightly brown observed in different pool, which shows the difference in overall culture management including water quality management. The minimum Water turbidity using secchi disk was 40 centimeters to one meter page in the fields. Source semi-deep wells or deep pools that were used as water source. Average water temperature, oxygen and pH in fileds 26/5-31 °C , 4/3-5/7 mg / L and 7/4-9/38. In order to virological surveys, sampling of internal organs, including kidneys, and spleen were done.and Homogeneous target tissues after spending 0/45 micron filter on EPC and BF2 cell line were inoculated to observe cell damage(CPE) in case of confirmation of antibody detection methods brilliant, RT-PCR and PCR done. In cell culture examination, a kind of cell damage (CPE-like changes) was observed after inoculating of 3 samples of Silver carp those were suspected to SVC, but in the second and third passages there were no sign of cell damage. It may be because of toxic effect of tissue filtrate on the cell monolayer. Concerning sample of Grass carp on two EPC and BF2 cells no evidence of cell damage was found. In indirect fluorescent antibody test, 72 hours after inoculation of tissues filtrates of of silver carp and grass carp samples on EPC, no positive reaction was observed. PCR and RT-PCR tests using specific primer pairs were done to test all of the silver carp and grass carp samples for SVC and KHV diseases Also for GCRV in grass carp samples. In addition, simultaneously commercial PCR kits applied for testing of SVC and KHV (IQ2000. Taiwan). The results of PCR and RT-PCR tests showed no evidence of robdovirus Carpio and grass carp reovirus RNA and no sign of koi herpesvirus DNA in tested samples. In conventional PCR and RT-PCR tests Using specific primer pairs none of positive bands related to SVCV(470 bp) and GCRV(292bp, 697bp and 320bp) were not confirmed in the tested samples. Also in electrophoresis of PCR products using IQ2000 kit revealed 471 bp and 640 bp negative bands in all samples and the positive bands were not observed. For confirmation of the results, three suspected samples were sent to Europe Union Reference Laboratory and none of mentioned diseases were confirmed. Simultaneously bacteriolocical examination applied for 26 using blood agar. In bacterial culture and related analyzes 6 isolates of Pseudomonas and 21isolates of Aeromonas hydrophila were detected from 26 farms while no virus was confirmed in the same samples. Based on the results of cell culture, PCR, RT-PCR and IFAT tests no one of SVCV, KHV and GCRV viruses were confirmed in tested samples. Therefore, the etiological reason of the mortalities in the tested farms was not viral diseases and should be prevent and control by biosecurity and health management in the farms.