Identification and Characterization of Megalocytivirus Type 3 Infection with Low Mortality in Starry Flounder,Platichthys stellatus,in Korea

MVSF‐12 belonging to megalocytivirus type 3 was isolated from cultured starry flounder; Platichthys stellatus, at the moribund or subclinical stage with low mortalities in Korea. Of 20 apparently healthy fish in the farms, 17 were also confirmed in nested polymerase chain reaction to be infected by megalocytivirus. When starry flounder; rock bream, Oplegnathus fasciatus; and olive flounder, Paralichthys olivaceus, were artificially infected by MVSF‐12 or iridovirus sachun‐1 (IVS‐1, megalocytivirus type 1), starry flounder and olive flounder showed no mortality until Day 24, without any clinical signs including enlarged spleen, while rock bream showed 100% mortality by IVS‐1 infection within 11 d but no mortality by MVSF‐12. Although it was not pathogenic, MVSF‐12 in infected fish at Day 24 was viable when successfully cultured in vitro using primary rock bream embryo cells and produced a cytopathic effect (CPE) with the viral copy numbers between 1.76 × 10⁷ and 5.23 × 10⁷/mL of culture supernatant. In conclusion, this study demonstrates the low pathogenicity of MVSF‐12 and low susceptibility of starry flounder and olive flounder to both MVSF‐12 and IVS‐1. Indeed, MVSF‐12 at the subclinical stage could be replicated with CPE in vitro, indicating a possibility to induce pathogenic effects and mortality under adverse environment or physiologic conditions.     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.     

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL‐8 and COX‐2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL‐1β, IL‐8 and COX‐2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)‐induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation‐related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.     

Low pathogenicity of flounder iridovirus (FLIV) and the absence of cross‐protection between FLIV and rock bream iridovirus

The genus Megalocytivirus is known to infect a wide range of cultured marine fish. In this study, we examined the pathogenicity of FLIV (Megalocytivirus from olive flounder, genotype III) and RBIV (Megalocytivirus from rock bream, genotype I) to their homologous and heterologous host species. Olive flounder (7.5 ± 1.3 cm) injected with FLIV [major capsid protein (MCP) gene copies, 6.8 × 10³–6.5 × 10⁶/fish] at 24 °C did not die until 90 days post‐infection (dpi). The average virus replication in the spleen peaked (1.27 × 10⁶/fish) at 20 dpi. Rock bream (6.5 ± 1.5 cm) injected with FLIV (8.8 × 10⁵ and 6.5 × 10⁶/fish of MCP copies) showed no mortality until 50 dpi. The rock bream that survived after FLIV infection were rechallenged with RBIV at 50 dpi had 100% mortality, showing that there is no cross‐protection between FLIV and RBIV. Temperature shifting (26 °C and 20 °C at 12 h intervals) did not cause FLIV‐specific mortality into olive flounder, but higher virus copies were observed in the fish exposed to higher stocking density. This study demonstrates that FLIV and RBIV have different antigenic and pathogenic characteristics and that FLIV has low pathogenicity to olive flounder.     

Effects of water temperature on mortality in Megalocytivirus‐infected rock bream Oplegnathus fasciatus (Temminck et Schlegel) and development of protective immunity

Rock bream iridovirus (RBIV) causes huge losses, especially in rock bream Oplegnathus fasciatus. Rock bream injected with RBIV and held at 29, 26, 23 or 20 °C had 100% mortality. Conversely, all infected fish held at 17 °C survived even after the temperature was progressively increased to 26 °C at 100 dpi. Rock bream exposed to virus and held for 2, 4 and 7 days at 23/26 °C before the temperature was reduced to 17 °C had mortality rates of 26.6/73.2%, 66.6/100% and 93.4/100%, respectively, through 100 dpi. When surviving fish had the water temperature increased from 17 to 26 °C at 100 dpi, they did not exhibit signs of disease and had low virus copy numbers (below 10³). To investigate the development of a protective immune, rock bream were infected with RBIV and held at 23 °C before shifting the water temperature to 17 °C at 4 dpi. All injected fish survived until 120 dpi. While 100% of the previously unexposed fish died, 80.2% of the previously infected fish survived. When the survivors were rechallenged again at 160 dpi, no further mortality occurred. The high survival rate of fish following rechallenge with RBIV indicates that protective immunity was established in the surviving rock bream.     

A natural infection by the red sea bream iridovirus‐type Megalocytivirus in the golden mandarin fish Siniperca scherzeri

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)‐subgroup I. Additionally, this train clustered with the ehime‐1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV‐type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 10² to 7.95 × 10⁶ copies/mg of tissue individually, suggesting that the infected fish were in the disease‐transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV‐type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.     

Codon‐optimized expression of fish iridovirus capsid protein in yeast and its application as an oral vaccine candidate

Fish iridovirus causes systemic disease with high morbidity and mortality in various species of wild and farm‐raised fish, resulting in severe economic losses. Recently, frequent outbreaks of iridovirus infection have occurred among cultured fish in many Asian countries, emphasizing the need for a protective vaccine programme or the development of a suitable therapy. In this study, we expressed a recombinant major capsid protein (rMCP) of rock bream iridovirus (RBIV) from yeast using codon optimization. The rMCP in yeast was added to feed in an attempt to induce intestinal mucosal immunity for protection against and/or to reduce the severity of fish iridovirus infection. We found that fish immunized orally with rMCP underwent a successful induction of antibodies (P < 0.05) and were protected (P = 0.0001) against viral challenge. Based upon these results, oral administration of immunogenic protein as an antigen can be considered a useful method for implementation of vaccine programmes against iridovirus as well as other marine viral diseases.     

Non‐invasive fast real‐time PCR assay for detection of the enteric parasite Enteromyxum scophthalmi in cultured turbot (Scophthalmus maximus L.)

Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real‐time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non‐invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.     

Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus)

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South‐East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV‐infected fish sharing a common water source.     

Disease resistance and immune‐relevant gene expression in golden mandarin fish,Siniperca scherzeri Steindachner,infected with infectious spleen and kidney necrosis virus‐like agent

Infectious spleen and kidney necrosis virus (ISKNV), family Iridoviridae, genus Megalocytivirus, may cause high mortality rates such as those seen in mandarin fish, Siniperca chuatsi. ISKNV has attracted much attention due to the possible environmental threat and economic losses it poses on both cultured and wild populations. We have investigated the pathogenicity of ISKNV‐like agent Megalocytivirus, isolated from infected pearl gourami, in golden mandarin fish, Siniperca scherzeri – a member of the Percichthyidae family – and in another Percichthyidae species, S. chuatsi. Fish were challenged with four different doses of ISKNV‐like agent Megalocytivirus (1, 10, 100 or 1000 μg per fish) over a 30‐day period, and cumulative fish mortalities were calculated for each group. No significant mortality was observed for fish challenged with the lowest dose (1 μg per fish) relative to a control group. However, all other challenged groups showed 100% mortality over a 30‐day period in proportion to the challenge dose. Quantitative real‐time PCR was performed to measure mRNA expression levels for six immune‐related genes in golden mandarin fish following ISKNV‐like agent challenge. mRNA expression levels for IRF1, Mx, viperin and interleukin 8 significantly increased, while mRNA levels for IRF2 and IRF7 remained constant or declined during the challenge period.     

Immunohistochemical characterization of polyclonal antibodies against Enteromyxum leei and Enteromyxum scophthalmi (Myxozoa: Myxosporea), intestinal parasites of fish

The enteric myxozoan parasites Enteromyxum leei (Diamant, Lom et Dyková) and Enteromyxum scophthalmi Palenzuela, Redondo et Álvarez‐Pellitero are responsible for high weight loss in infected fish, which leads to subchronic disease and low mortality rates in gilthead sea bream (GSB), Sparus aurata L., and to high mortality rates in turbot, Psetta maxima (L.). The detection of initial parasite stages in histological sections is particularly difficult, but can be simplified by means of specific antibodies. Rabbit polyclonal antibodies (pAbs) were raised against E. scophthalmi and E. leei, and direct enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry were used to characterize their sensitivity and specificity. Both pAbs were adsorbed (apAb) with non‐infected intestines to avoid non‐specific labelling of fish tissues and to improve their specificity. The highest titre obtained in ELISA was 1: 32 000 for apAb‐Eleei and 1:16 000 for apAb‐Escoph. Working dilutions in immunohistochemistry were 1:1000 for apAb‐Eleei and 1:8000 for apAb‐Escoph. Both apAbs labelled proliferative and sporogonic stages with high specificity. apAb‐Escoph was very specific, whereas apAb‐Eleei cross‐reacted with Sphaerospora dicentrarchi Sitjà‐Bobadilla et Álvarez‐Pellitero and Sphaerospora testicularis Sitjà‐Bobadilla et Álvarez‐Pellitero, suggesting the presence of shared antigens. These pAbs stand as new tools for antigenic characterization and the diagnosis of both Enteromyxum species.     

Expression and serological application of a capsid protein of an iridovirus isolated from rock bream, Oplegnathus fasciatus (Temminck & Schlegel)

Iridoviruses infect a wide variety of wild and cultured fish. Those iridoviruses belonging to the genus Ranavirus, in the Iridoviridae family, cause systemic disease in infected animals with a high morbidity and mortality. This paper reports the cloning, sequencing, and expression of the rock bream iridovirus (RBIV) major capsid protein (MCP) in an Escherichia coli expression system for subsequent immunological studies. The completeness of the expressed protein was confirmed by peptide mass fingerprinting (PMF) analysis using MALDI-TOF MS. The recombinant MCP (rMCP)-specific mouse polyclonal antibody reacted with the viral 52 kDa protein, indicating that this rMCP induces an immunological response. Fish antibodies induced against iridovirus infection were also detected using ELISA when rMCP was used as an antigen. As a result, it was found that many cultured rock bream (92.5%) were naturally infected with iridovirus and that the rMCP might be useful for serological tests.     

Susceptibility of isogeneic ginbuna Carassius auratus langsdorfii Temminck et Schlegel to cyprinid herpesvirus‐2 (CyHV‐2) as a model species

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus‐2 (CyHV‐2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV‐2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV‐2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non‐permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV‐2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV‐2 infection and immunity.     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.     

Vaccination strategies with oral booster for surubim hybrid (Pseudoplatystoma corruscans × P. reticulatum) against haemorrhagic septicaemia

This study evaluated the efficiency of differently prepared vaccines against Aeromonas hydrophila in the hybrid surubim (Pseudoplatystoma corruscans × P. reticulatum). Survival and haemato‐immunological parameters were compared between the treatments: non‐vaccinated fish (C); bacterin‐vaccinated fish (B); bacterin plus oral booster vaccinated fish (B+O); bacterin and toxoid‐vaccinated fish (B+T) and bacterin, toxoid and oral booster‐vaccinated fish (B+T+O). Fourteen‐days vaccinated fish from B+O and B+T+O were fed with an oral booster for 4 days. After 1 week, the fish were intraperitoneally challenged with 2 × 10⁸ CFU mL^?1 of A. hydrophila. Fish from the treatment B+T+O showed the lowest cumulative mortality (11.36%) 96 h after challenge, compared with other treatments (22.72–44.04%), and a relative survival of 74%. Serum immunoglobulin in B+T+O fish was higher than in other treatments. All vaccinated fish showed an increased agglutination titre when compared with non‐vaccinated fish, both before and after challenge. Fish fed with oral booster showed an increase in phagocytic percentage before and after challenge. It can be inferred that the oral booster vaccination was efficient in reducing mortality in hybrid surubim by enhancing the response against haemorrhagic septicaemia due to A. hydrophila infection.     

Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia,Oreochromis niloticus (L.)

Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non‐A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport‐induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 10⁶ CFU fish^?1) and A. veronii (8.9 × 10⁶ CFU fish^?1) killed 100% of experimental fish within 24 h, while a 10‐fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100‐fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.     

Effect of dietary lipid oxidation with vitamin C and E supplementation on fillet quality of red sea bream,Pagrus major (Temminck & Schlegel) during storage

An experiment was conducted to determine the effects of different levels of dietary vitamin C (VC) and E (VE) supplementation on fillet quality of red sea bream fed oxidized fish oil (OFO). Fish with an average body weight of 205.0 g were fed four test diets for 9 weeks. Control diet contained fresh fish oil (FFO) with 100 mg kg^?1 of VE and 500 mg kg^?1 of VC (FFO100E/500C). The other three diets contained OFO with varying levels of VE (mg kg^?1) and VC (mg kg^?1) (OFO100E/500C, OFO200E/500C and OFO200E/1000C). After feeding trial, two fillets from each fish by hand filleting were stored in a refrigerator at 4°C for 96 h during analyses. Results showed that fish fed OFO increased fillet thiobarbituric acid reactive substances (TBARS) and K‐value, and decreased fillet VC and VE concentrations during storage time. Supplementation of VC did not have any detectable effect on fillet quality. Increasing dietary VE supplementation increased fillet VE concentrations, reduced fillet TBARS and K‐value values of red sea bream. Therefore, we suggest that dietary supplementation of 200 mg kg^?1 of vitamin E could improve fillet oxidative stability of red sea bream fed OFO.     

Occurrence of trypanosomiasis in net‐cage cultured groupers (Cromileptes altivelis and Epinephelus fuscoguttatus) in Nanshan port of Sanya,Hainan province,China

In the present study, reported was an unidentified trypanosome that caused massive mortalities of cultured humpback grouper, Cromileptes altivelis (Valenciennes, 1828) and brown‐marbled grouper, Epinephelus fuscoguttatus (Forsskål, 1775) in the net‐cages in Sanya, China, and some general pathological features of E. fuscoguttatus after experimental infection of the trypanosome. Before dying in the net cages of diseased fish, listlessness and anorexia were the observable symptoms. The blood of the diseased fish was light‐coloured in comparison with that of healthy one. E. fuscoguttatus experimentally infected with this trypanosome are with different degrees of engorgement in gill lamella, liver, spleen and kidney. Tissue lesion and necrosis were observed in liver and spleen of diseased fish.