Studies On Etiology And Histopathology Of Spotted Knifejaw(Oplegnathus Punctatus) Iridovirus Disease

In August 2017,an outbreak of unknown etiology occurred in the spotted knifejaw?Oplegnathus punctatus?cultured in cages offshore of a marine fish farm in Shandong province.The disease investigation showed that this disease occurs in summer?from August to October?with water temperature of 26-28°C.The infected fish were mainly juveniles at the age of 4 months.The total length of the diseased fish was generally16.3±1.6 cm and the body weight was generally 156.9±37.0 g.The disease has a short course and a high mortality rate.The cumulative mortality of juvenile fish reached 90-95%within two weeks after the onset of the disease,resulting in heavy economic losses.Three-year-old adult fish cultured in recirculating aquaculture system in the same farm also suffered from the same disease,but the cumulative mortality was lower than that of juvenile fish,reaching 10-40%.Clinic examination showed that there was no obvious damage to the appearance of diseased spotted knifejaw,but it had poor vitality and shortness of breath.Necropsy findings included swollen and brittle spleen,erosion of kidney,hemorrhagic spots with liver.No parasites were detected and no pathogenic bacteria were isolated from the liver,spleen and kidney in the diseased fish.Histopathological sections showed that there were a large number of enlarged cells about 20?m in diameter in the spleen and kidney tissues of diseased fish.In addition,a large number of hexagonal virus particles with a diameter of about 145 nm were observed in the electron microscope sections of the spleen and the kidney,and the morphology was similar to that of the reported iridovirus.Spleen homogenate of naturally infected fish was filtered.Healthy spotted knifejaw was infected with the homogenate by intraperitoneal injection at 26°C.After 10 days,the infected fish died and the cumulative mortality rate reached 95%within 14 days.Artificially infected fish showed the appearance symptoms similar to those of naturally infected fish,and a large number of enlarged cells and similar virus particles could be observed in spleen and kidney tissue sections.Severe infection of iridovirus was detected in the spleen and kidney tissues of naturally and artificially infected fish using specific PCR primers.The major capsid protein?MCP?gene with 1362 bp in length of the iridovirus was cloned and sequenced,and the phylogenetic tree of the family Iridoviridae was constructed.The results showed that the virus belonged to the red sea bream iridovirus?RSIV?of genus Megalocytivirus in the family Iridoviridae.Based on the results of epidemiological investigation,clinical symptoms observation,etiological detection,histopathological observation,artificial infection experiment,molecular biology diagnosis and viral phylogenetic analysis,it is confirmed that the disease causing the mass death of spotted knifejaw was iridovirus disease,and the pathogen was red sea bream iridovirus.In order to understand the pathological characteristics of iridovirus disease in the spotted knifejaw,histopathological and ultrapathological observations were carried out in the diseased fish.The results showed that the most prominent histopathological feature of diseased fish was the presence of a large number of enlarged cells with basophilic,homogeneous cytoplasm and diameter of about 15-20?m in many organs.On ultrathin sections,a large number of hexagonal virus particles were observed in the enlarged cells of diseased fish,and some virus particles were free between tissues and cells due to the breakup of enlarged cells.The virus inclusion body occupies most of the space of the enlarged cells,and the organelles and nucleus of the host cells are squeezed to the edge of the enlarged cells.Only some mitochondria were found at the edge of the enlarged cells,and other organelles were rare.This indicates that the enlarged cells have been specialized as"virus factories"and their normal functions have been seriously affected.It is particularly noteworthy that the cell membrane of the enlarged cells appeared irregular invagination,forming a large number of dendritic sulcus gyrus structures with several microns in length,and the whole cell presents a"wrinkled"shape.It has also been observed that many enlarged cells were adhered by a single layer of lymphatic endothelial cells.Compared with naturally infected fish,the tissues and cytopathic lesions of artificially infected spotted knifejaw are more serious.The above pathological studies provide an important reference for the diagnosis and pathogenesis of RSIV in spotted knifejaw.In this study,the SYBR green real-time PCR detection method for RSIV in spotted knifejaw was established and applied.Firstly,the MCP gene of RSIV in spotted knifejaw was cloned and the standard plasmid was prepared.Then,the specific primers MCP-152F and MCP-152R were used to establish the standard curve for quantitative detection.The detection sensitivity of the method was about 10² virus copies,and the melting curve of the target amplification product showed a single peak at 84°C.The specificity of the method was strong.Using the method established above,the load of iridovirus in various organs?tissue?of natural and artificially infected fish was determined,and it was found that the viral load of artificially infected fish was about10 times that of natural diseased fish.This result is consistent with the observation of histopathology.Through artificial infection experiments,this study determined the median lethal dose(LD₅₀)of the virus and explored the relationship between water temperature and virulence of the virus.When the water temperature was 28°C,the LD₅₀0 of RSIV in spotted knifejaw was calculated to be about 3×10⁶ copies based on the cumulative mortality of the experimental fish on the 14th day after infection.In addition,the study found that the higher the water temperature,the earlier the death time of the infected fish,and the higher the cumulative mortality rate.When the water temperature is higher than 25°C,the infected fish all died within 14 days.When the water temperature is22°C,the cumulative mortality rate is 40%.When the water temperature is 19°C,the infected fish does not die.Using the SYBR green real-time PCR method established above,it was found that RSIV load in dead fish was high,with an average of 1.8×10⁸copies/?L,while the viral load of the survival fish was 4.0×10⁴ copies/?L?22°C group?and 6.3×10⁶ copies/?L?19°C group?.This study shows that controlling the water temperature can effectively inhibit the outbreak of RSIV disease in spotted knifejaw.The results of this study have a good guiding effect on the prevention and control of RSIV in spotted knifejaw.