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.     

Meiotic gynogenesis with heterologous sperm in the mandarin fish Siniperca chuatsi and evidence for female homogamety

The mandarin fish Siniperca chuatsi is a historically important aquaculture species in China and exhibits sexually dimorphic growth. However, sex determination of this fish remains unclear so far. In this study, we induced meiotic gynogenesis in S. chuatsi using irradiated heterologous sperm from spotted mandarin fish (Siniperca scherzeri) to uncover its mechanism of sex determination. Up to 7.52% diploid progeny were obtained among three gynogenetic families in this study. Molecular analysis of female and male donors and sampled young gynogens by seven microsatellite loci further confirmed no genetic contributions from the ‘father’ S. scherzeri. After 8 months of culture, external morphology of adult fish showed that all gynogens were cloned from their mothers. Gonads of the gynogenetic progeny were examined by histological observations and the sexing results showed that they were almost 100% females, strongly supporting an assumption of female homogamety in mandarin fish. By this study, we obtained pure lines of S. chuatsi and elucidated its genetic mechanism of sex determination, providing a basis for possible sex control breeding in this species.     

Effects of live and artificial feeds on the growth,digestion, immunity and intestinal microflora of mandarin fish hybrid (Siniperca chuatsi ♀ × Siniperca scherzeri ♂)

How to acclimate mandarin fish to eat artificial feeds has been always a challenge for researchers. The mandarin fish after hybridization (Siniperca chuatsi ♀ × Siniperca scherzeri ♂) could be fed artificial feeds which solved the problem to some extent. However, the growth performance, digestibility, immunity and intestinal microflora of mandarin fish hybrid fed artificial feeds need further study. One hundred and twenty fish of similar size (average weight, 19.5 ± 0.9 g) were randomly stocked into six 250‐L aquariums and separately fed live baits (the control) or artificial feeds in triplicate for 70 days. The weight gain and special growth rate of fish fed artificial feeds were significantly lower than those of fish fed live baits (P < 0.05). The protease activities of stomach, liver and intestine in fish fed live baits were all significantly higher than those in fish fed artificial feeds. The activities of catalase and lysozyme, the content of glutathione in serum of fish fed live baits were all significantly higher than those in fish fed artificial feeds. However, the content of malondialdehyde in liver of fish fed artificial feeds was significantly higher than that in fish fed live baits. The dominant bacteria in both groups were Proteobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria, Verrucomicrobia. However, live baits greatly affected the amount of beneficial and harmful bacteria of intestine in mandarin fish hybrid and broke the balance of intestinal flora.     

Pre‐challenge and post‐challenge haemato‐immunological changes in Oreochromis niloticus (Linnaeus, 1758) fed argan oil against Lactococcus garvieae

The present study investigated the effects of argan oil, obtained from Argania spinosa, on pre‐ and post‐challenge immuno‐haematological and biochemical responses of Nile tilapia, Oreochromis niloticus. For this purpose, the fish were fed diets containing 0, 0.5%, 1% or 2% argan oil for 45 days. Following 45 days of feeding, fish were challenged with Lactococcus garvieae and mortality was recorded for 15 days. During the pre‐challenge period, significantly higher respiratory burst activity, total white blood cell (WBC), serum lysozyme activity and myeloperoxidase activity were determined in the argan oil‐fed groups. The serum glucose and cholesterol levels decreased whilst total protein and albumin did not change in the groups fed with argan oil‐supplemented diets. After challenge with Lactococcus garvieae, the percentage survival (%) was found to be the highest in the 1% and 2% argan oil‐supplemented feeding groups. Also, there was a significant increase in weight gain, specific growth rate and feed conversion ratio in those fish fed argan oil. The results of this study indicated that after the supplementation of fish diets with argan oil, especially at 1% and 2% concentrations, the immunological, haematological and biochemical values remained similar in both the pre‐ and post‐challenge periods and the immune response against L. garvieae in Nile tilapia was modulated.     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

Virulence assay of rhizoid and non‐rhizoid morphotypes of Flavobacterium columnare in red tilapia,Oreochromis sp., fry

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris‐like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non‐rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non‐rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post‐challenge as determined by whole fish bacterial loads. At 24 and 48 h post‐challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non‐rhizoid morphotype. The results suggested that an inability of the non‐rhizoid morphotype to persist in tilapia fry may explain lack of virulence.     

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.     

Nucleotide promotes feed intake and protein utilization via regulating the gene expression of feeding and nitrogen metabolism in juvenile Chinese perch (Siniperca chuatsi)

Nucleotide (NT) could enhance growth, feeding and immunity in higher vertebrate and fish. Chinese perch (Siniperca chuatsi) refuse artificial diet in natural water, whereas the NT might promote its feeding of domestication diet. The present study was designed to investigate the effect of dietary NT on growth, feed intake, feed utilization, body composition, serum biochemistry and nitrogen metabolism in juvenile Siniperca chuatsi. 0, 0.5, 1.0, 1.5, 2.0 and 4.0 g NT kg^?1 diet were supplemented in each diet, respectively. Triplicate groups of Chinese perch (36.21 ± 0.98 g) were fed twice a day to apparent satiation for 8 weeks. The results showed that fish fed with 1.5 g NT kg^?1 diet had the highest weight gain (WG), specific growth rate, feed efficiency, feed intake, protein efficiency ratio and protein retention efficiency. The crude protein content in whole body was highest in fish fed the diet containing 1.5 g NT kg^?1 diet. Dietary NT supplementation significantly increased the neuropeptide Y gene expression and significantly decreased the agouti‐related protein and pro‐opiomelanocortin gene expressions in brain. NT supplementation decreased urea nitrogen content, aspartate aminotransferase (AST) and alanine aminotransferase activities in serum. The hepatic AST activity was increased first and then decreased, and the highest activity was observed in fish fed with 1 g NT kg^?1 diet. Inversely, the activity of glutamate dehydrogenase (GDH) in liver and adenosine 5′‐monophosphate deaminase in muscle decreased first and then increased, and the lowest activity was observed in fish fed with 1.5 g NT kg^?1 diet. Similarly, the gene expression levels of GDH and arginase in liver were lowest in fish fed with 1.5 g NT kg^?1 diet. Based on the broken‐line regression analysis of WG in the present experimental condition, 1.44 g NT kg^?1 diet was the optimum supplementation level in juvenile Siniperca chuatsi.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

传染性脾肾坏死病毒_ConA和L_省略_对体外鳜的头肾淋巴细胞转化的影响

采用MTT颜色反应法研究传染性脾肾坏死病毒（ISKNV）、伴刀豆球蛋白A（concanavallin A,ConA)和脂多糖(lipopolysaccharide,LPS）在离体状态下对健康和ISKNV感染后存活30d或60d的鳜的头肾淋巴细胞转化的影响。结果表明，ConA和LPS能促进健康鳜头肾淋巴细胞的转化，而对感染后存活30d或60d的鳜头肾淋巴细胞没有作用；纯化的ISKNV对健康鳜头肾淋巴细胞没有促进淋巴细胞转化的作用，但对ConA的作用有抑制，对感染后存活的30d或60d的鳜头肾淋巴细胞，纯化的ISKNV有一定的促进转化的作用，感染存活的鳜头肾中有对ISKNV的免疫记忆性T细胞。另外，用建立的ISKNV PCR检测方法对健康和ISKNV感染后存活的鲜组织进行了检测，结果显示，健康鳜为阴性，而ISKNV感染后存活的鱼，头肾、后肾、脾脏和部分鱼的心脏为阳性。ISKNV在鳜体内形成潜伏感染。     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Ammonia nitrogen excretion in Mandarin Fish (Siniperca chuatsi) and Grass Carp (Ctenopharyngodon idellus) fed practical diets: the effects of water temperature

To understand the actual production of fish culture about the utilization of dietary protein and excreta impact on the environment between mandarin fish (Siniperca chuatsi) and grass carp (Ctenopharyngodon idellus), the study to investigate the effect of temperatures (19 ± 0.5°C, 24 ± 0.5°C and 29 ± 0.5°C) on ammonia‐N excretion in mandarin fish and grass carp under fed and fasted states was conducted. These two species were fed a practical diet containing 325.2 g kg^?1 crude protein at 3% body weight per day. The ammonia‐N excretion rate was significantly increased when temperature increased from 19 to 29°C, and a linear relationship between ammonia‐N excretion rate and temperature. The maximum ammonia‐N excretion levels of mandarin fish and grass carp were observed at 4–8 h and 2–4 h after feeding, respectively, and the minimum values for both species were observed at 24 h after feeding. Under the feeding condition, mandarin fish had a lower ammonia‐N excretion level compared to grass carp at 24°C and 29°C. The average amount of ammonia‐N excreted by mandarin fish at 24 h is significantly higher than grass carp under fasting conditions, except 19 ± 0.5°C. These results indicated that mandarin fish might make better use of protein at higher temperature than grass carp when fed practical diets in commercial production. These results of this study suggested that mandarin fish had a lower ammonia‐N excretion level compared with grass carp, making a less contribution to environmental loading in an intensive fish culture.     

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron‐like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1‐TRBIV‐MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT‐PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT‐PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post‐vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon‐induced antiviral molecules were observed in the vaccinated fish 60 h post‐vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.     

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.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

鳜IRAK4基因的克隆、组织表达及病毒感染后表达分析

为了研究鳜IRAK4生物学特性及其在抗病毒免疫应答中的作用,根据鳜转录组数据中筛选出的IRAK4 unigene序列设计引物,利用SMART-RACE技术克隆得到CDS全长为1389 bp的c DNA(命名为Sc IRAK),编码462个氨基酸,含有1个N端死亡结构域和1个保守的中央蛋白激酶结构域。采用荧光定量RT-PCR方法分析了Sc IRAK4在健康鳜各组织中的表达差异及病毒感染后在脾脏中的表达变化,结果显示,健康鳜中Sc IRAK4在肝脏中表达量最大,与其他组织差异显著,而在血液、脑和胃中表达量最低;传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)感染鳜后Sc IRAK4的表达量呈现下调趋势,24 h脾脏中的表达量达到最低,为对照组的45%;而鳜弹状病毒(siniperca chuatsi rhabdovirus,SCRV)感染鳜后Sc IRAK4的表达量呈现上调趋势,12 h脾脏中Sc IRAK4的表达量达到最高,为对照组的8.17倍,表明Sc IRAK4在抗ISKNV和SCRV的免疫应答中可能发挥不同的作用。本研究为进一步揭示Sc IRAK4的抗病毒免疫反应机制提供了依据。     

Symbiotic supplementation on the hemato‐immunological parameters and survival of the hybrid surubim after challenge with Aeromonas hydrophila

This study evaluated the effects of dietary supplementation with two symbiotics in the hybrid surubim (Pseudoplatystoma corruscans × P. reticulatum) prior to and after challenge with Aeromonas hydrophila. The fish were divided into unsupplemented fish, supplemented fish with Weissella cibaria and inulin, and supplemented fish with Lactobacillus plantarum and inulin. Twenty days after being fed symbiotics, the fish were sampled for immunological, haematological, and microbiological analysis pre‐challenge. After this period, they were challenged with Aeromonas hydrophila, and after 96 h the fish were sampled for blood collection and survival. Fish fed W. cibaria and inulin from the pre‐challenge showed the highest values of lysozyme activity. Fish fed L. plantarum and inulin presented a higher number of thrombocytes and granular leukocyte PAS⁺ than the other fish. After challenge, fish fed both symbiotics presented an increased red blood cells number. Fish fed L. plantarum and inulin showed an increased total leucocyte count (WBC), neutrophils, and monocytes number as well as total immunoglobulin. The W. cibaria‐ and inulin‐supplemented fish showed improved haematological and immunological parameters and reduced cumulative mortality after the challenge.