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Emerging pathogen, carp edema virus (CEV) causes koi sleepy disease (KSD) in Koi and common carp causing severe mortalities worldwide. In the present study, a total of 150 fish species belonging to eight different families were sampled from the ornamental fish retailers and farms, located in Karnataka, India. The OIE protocol viz., level-I, II and III diagnoses confirmed the infection of CEV in 10 koi fish. Interestingly, other fish species belonging to different fish family including cyprinidae family were negative to CEV. Further, CEV infection was confirmed by sequencing (partial 4a gene); it showed the similarity with that of CEV reported from India and Germany strains with similarity of 97.4–99.94% and belonged to genogroup IIa. TEM analysis of purified CEV, in vivo cohabitation and tissue infection experiments confirmed the CEV infection. In addition, viral load was significantly higher (106−7 copies) in koi collected from Dakshina Kannada than of Bengaluru (103−4 copies). To understand the host–pathogen interaction, different organs such as gill, kidney, liver and spleen from naturally (CEV) infected koi were used to study the immune gene responses by using eight innate and one adaptive immune response. Results indicated that TNF-α, RohTNF-α, iNOS, IFN-γ and IL-10, and catalyze β-2M of MHC class I pathway genes were upregulated in koi. Higher expression of immune genes during the CEV infection may have inhibited viral replication and mount an antigenic adaptive response. Similar to other viral infections, interferon-γ play an important role during poxvirus infections. Quantification of immune genes in infected fish will provide insights into the host responses and provide valuable information to devise intervention strategies to prevent and control disease due to CEV. • Ornamental fish species were screened for CEV, only ten koi were found to be positive. • TEM analysis and in vivo experiments confirmed the CEV infection. • Viral load was high in koi from Dakshina Kannada followed by Bengaluru. • Upregulation of immune genes such as TNF-α, RohTNF-α, iNOS, IFN-γ and IL-10 in koi. [ABSTRACT FROM AUTHOR]

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. However, few cases of A. veronii infection were reported in Odontobutis potamophila , which has been becoming a promising fish species in China in recent years. In this study, the dominant bacteria were isolated from diseased O. potamophila showing signs of hemorrhage on fins, ulceration on the dorsal and abdomen. The representative isolate Stl3-1was identified as A. veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. The median lethal dosage (LD 50) of the isolate Stl3-1 for O. potamophila was determined as 4.5 × 105 CFU/mL. Histopathological analysis revealed that the isolate Stl3-1caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Detection of virulence-related genes showed that A. veronii Stl3-1 was positive for exu, ompA, lip, flaH, hlyA, aer, flgM, tapA, act, flgA, gcaT and flgN. Additionally, quantitive real-time PCR (qRT-PCR) was also undertaken to analyses the host defensive response in O. potamophila infected by A. veronii. The immune-related gene expressions in O. potamophila during experimental infection were monitored at different point of time, and the results showed that the expression levels of MHC II , Myd88 , TLR , and SOD were significantly up-regulated in liver, gill, spleen, and head kidney. The results revealed that A. veronii was a pathogen causing mass mortalities of O. potamophila and will contribute to better understanding the host defensive response against A. veronii infection. • Mass mortalities of cultured O. potamophila with characteristic symptoms of skin ulceration were reported. • Pathogenesis and histopathological analysis related to O. potamophila with Aeromonas veronii infection were studied. • A. veronii infection induced a wide defensive response of various immune related genes in O. potamophila. [ABSTRACT FROM AUTHOR]

The protective role of β-glucan (BG) on liver function, histopathology, immune and antioxidant related gene expressions in Nile tilapia exposed to subacute deltamethrin (DLM) was investigated for 30 days. Fish (28.18 ± 1.34 g) of the 1st and 2nd groups fed the control diet, while the 3rd and 4th groups fed BG at 0.5 g/kg and the 2nd and 4th groups were exposed to DLM (15 μg/L) in rearing water. DLM-treated fish displayed a considerable increase in blood biochemical parameters (creatinine, urea and bilirubin) as well as hepatic enzymes (ALP, AST and ALT) (P < 0.05). Blood total protein, globulin, albumin, WBCs, RBCs, Hb, phagocytic index, phagocytic and lysozyme activities were significantly decreased in fish subjected to DLM (P < 0.05). Fish fed BG showed significantly the lowest cortisol and glucose levels, while fish exposed to DLM without feeding BG showed the highest cortisol and glucose levels (P < 0.05) after 15 and 30 days. Additionally, DLM toxicity caused downregulation in antioxidant (CAT and GPx) and immune (IL-1β and IL-8) related gene expressions, while and IFN-γ , HSP70 and CASP3 were upregulated. The histopathological examination of Nile tilapia exposed to DLM revealed damage in gills, intestine, spleen and liver which confirmed the toxic effects. Conversely, BG presented protective effects and restored the above-mentioned parameters when fish exposed to DLM and fed BG. Thus, BG supplementation exhibited defensive effects against DLM toxicity in Nile tilapia through improving blood biochemical responses, immune, and antioxidant related gene expressions as well as histopathological effects. • Deltamethrin (DLM)-treated fish showed increased blood creatinine, urea, bilirubin, ALP, AST and ALT. • Blood immune responses were decreased in fish exposed to DLM. • DLM downregulated CAT , GPx , IL-1β , IL-8 and IFN-γ genes, while HSP70 and CASP3 were upregulated. • Nile tilapia exposed to DLM revealed abnormal histopathological alterations. • β-glucan presented protective effects and restored the aforementioned parameters. [ABSTRACT FROM AUTHOR]

The high morbidity and mortality of Macrobrachium nipponense occurred in several farms in China, with cardinal symptoms of slow swimming, loss of appetite, empty of intestine, reddening of the hepatopancreas and gills. The pathogen has been confirmed as Decapod Iridescent Virus 1 (DIV1), namely DIV1-mn, by molecular epidemiology, histopathological examination, TEM observation, challenge experiment, and viral load detection. Histopathological analysis showed severe damage in hepatopancreas and gills of diseased prawns, exhibited few eosinophilic inclusions and pyknosis, and TEM of diseased prawns revealed that icosahedral virus particles existed in hepatopancreas and gill, which confirmed the disease of the farmed prawns caused by the DIV1 infection. Besides, challenge tests showed LD 50 of DIV1 to M. nipponense was determined to be 2.14 × 104 copies/mL, and real-time PCR revealed that M. nipponense had a very high DIV1 load in the hemocytes, gills and hepatopancreas after infection. Furthermore, qRT-PCR was undertaken to investigated the expression of six immune-related genes in DIV1-infected M. nipponense after different time points, and the results revealed UCHL3 , Relish , Gly-Cru2 , CTL , MyD88 and Hemocyanin were significantly up-regulated in hemocytes, gills and hepatopancreas, which revealed various expression patterns in response to DIV1 infection. This study revealed that DIV1 infection is responsible for the mass mortality of M. nipponense , one of the important crustacean species, indicating its high susceptibility to DIV1. Moreover, this study will contribute to exploring the interaction between the host and DIV1 infection, specifically in terms of understanding how M. nipponense recognizes and eliminates the invading of DIV1. • Mass mortalities of cultured Macrobrachium nipponense caused by DIV1 was reported. • The pathogenicity of DIV1 to M. nipponense was determined by histopathological examination, LD50 and LT50. • DIV1 infection induced a wide defensive response of various immune related genes in M. nipponense. [ABSTRACT FROM AUTHOR]

Outbreaks of mass mortalities occurred in Macrobrachium rosenbergii farms in Gaoyou county, Jiangsu Province of China. The bacterial isolates from M. rosenbergii exhibited the same phenotypic traits and biochemical characteristics, and were identified as non-O1/O139 Vibrio cholerae according to biochemical characteristics and molecular identification. In challenge test, M. rosenbergii infected with non-O1/O139 V. cholerae GXFL1-4 developed similar pathological signs to the naturally diseased prawns, and LD 50 of the strain to M. rosenbergii was 4.5 × 106 CFU/mL at 96 h post-infection. Histopathological analysis revealed that hepatopancreas and intestines of diseased M. rosenbergii exhibited obvious inflammatory responses to non-O1/O139 V. cholerae infection. Detection virulence factors of the strain GXFL1-4 showed that the bacteria produced caseinase, lipase, amylase, lecithinase and hemolysin, and carried toxR , hlyA , ompW , ompU , hap , rtxA and rtxC virulence related genes, supporting the strong virulence to M. rosenbergii. Additionally, the immune related gene expression in M. rosenbergii evaluated by qRT-PCR analysis showed that HSP70 , Crustin , Lysozyme , TRL1 , ALF1 , Lectin , Peroxinectin , proPO and SOD immune related genes were significantly up-regulated at 6 and 12 h after infection with GXFL1-4. The results of our study suggested that non-O1/O139 V. cholerae was an etiological element in the mass mortalities of M. rosenbergii and this study provided preliminary insights into the diversity in the immune response of M. rosenbergii to the bacterial invasion. • Mass mortalities of cultured M. rosenbergii from several farms in Gaoyou country of China was reported. • Pathogenesis and histopathological analysis related to M. rosenbergii with non-O1/O139 V. cholerae infection were studied. • Non-O1/O139 V. cholerae infection induced a wide defensive response of various immune related genes in M. rosenbergii. [ABSTRACT FROM AUTHOR]

Abstract Outbreaks of mass mortalities among cultured Macrobrachium nipponense occurred in a commercial hatchery during the autumn of 2017 in Jiangsu province, P. R. China, and non-O1 Vibrio cholerae was isolated and identified as causal agents of M. nipponense , with a LD 50 value 4.09 × 104 CFU/mL. Detection of virulence-associated genes by PCR indicated that XL1 was positive for Mp , HlyA , RtxA , OmpU , Ace , Zot and T6SS. Furthermore, the results of extracellular enzyme analysis revealed that the strain can produce lecithinase, amylase, gelatinase and hemolysin. Histopathological analysis revealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger, and the brush border disappeared in the hepatopancreas. Quantitive real-time PCR (qRT-PCR) was undertaken to measure mRNA expression levels for thirteen immune related genes in M. nipponense after non-O1 V. cholerae infection. The transcriptional analysis of these immune related genes demonstrated that the expression levels of dorsal, relish, p38, crustin1, crustin2, crustin3, hemocyanin, i-lysozyme, anti-lipopolysaccharide factors 1, anti-lipopolysaccharide factors 2, prophenoloxidase were significantly up-regulated in hemolymph of M. nipponense post-infection. These results revealed varying expression profiles and clear transcriptional activation of these immune related genes in hemolymph, which will contribute to better understand the pathogenesis and host defensive system in non-O1 V. cholerae invasion. Highlights • Mass mortalities of cultured Macrobrachium nipponense from several farms in Yangzhou city of China is reported. • Non-O1 Vibrio cholerae XL1 carry Mp , HlyA , RtxA , OmpU , Ace , Zot and T6SS by PCR detection. • Non-O1 Vibrio cholerae triggers a wide defensive response of various immune related genes in Macrobrachium nipponense. [ABSTRACT FROM AUTHOR]

Abstract White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar) , are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii. Highlights • Hematopoietic tissue responded to viral infection by upregulating immune-related genes. • Novel immune related genes in M. rosenbergii are found during their viral response. • Cell-growth regulatory genes were highly expressed to produce more hemocytes for fighting against MrNV. [ABSTRACT FROM AUTHOR]

Abstract In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum , that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm , inos , tlr2 , tlr4 , pcna and cytokeratin. Infected gills resulted positive for igm , inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1 , il-8 , il-10 , hep , cox-2 , cla , cat, casp9 , and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures. Highlights • Gills from naturally infected ESB emphasized the presence of round/pear-shaped AO trophonts. • Immunohistochemical labelling confirmed positive for ESB igm , inos , pcna and cytokeratin but negative to tlr2 , tlr4. • Inflammation (il-8 , cc1 , cox-2) triggered novel molecules (Hep). • First demonstration of igt expression in ESB infected with AO. • Hepcidin , cc1 and igt could be potential markers for AO infection in ESB. [ABSTRACT FROM AUTHOR]

Abstract Hemorrhagic septicemia of mandarin fish (Siniperca chuatsi) was mainly caused by Aeromonas hydrophila which was an opportunistic pathogen. In recent years, the disease has caused tremendous economic loss with high morbidity and mass mortality in the mandarin fish breeding industry. Histopathological analysis and the immune related gene expression profiles of mandarin fish (S. chuatsi) infected with A. hydrophila were investigated in this study. Transmission electron microscopy (TEM) images showed that the cells of A. hydrophila densely covered with a mass of fimbriae. Histopathological analysis revealed that inflammation, vacuolization and extensive necrosis existed in the gill, liver, spleen and head kidney of the diseased fish. Quantitative real-time PCR was performed to measure mRNA expression levels for six immune related genes in mandarin fish after A. hydrophila infection. The transcriptional analysis of these immune related genes demonstrated that the expression levels of major histocompatibility complex class II (MHC II), T cell receptor α (TCRα), tumor necrosis factor α (TNFα), CC chemokine 3, interleukin 8 (IL-8) and Hepcidin were strongly up-regulated in spleen and head kidney of mandarin fish post-infection. These results will contribute to further study on the pathogenesis and host defensive system in A. hydrophila infection. Highlights • The morphological characteristic of Aeromonas hydrophila isolated from the diseased mandarin fish was observed. • Histopathological analysis related to mandarin fish with Aeromonas hydrophila infection were studied. • Aeromonas hydrophila triggers a wide defensive response of various immune related genes in mandarin fish. [ABSTRACT FROM AUTHOR]

Abstract Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes. Highlights • Sessile hemocytes of gills, heart and lymphoid organs were enriched. • Transcriptome analysis between circulating and sessile hemocytes was conducted. • Sessile hemocytes may have a different type of hemocytes from circulating hemocytes. [ABSTRACT FROM AUTHOR]

The effects of inulin and mannan oligosaccharide (MOS) at different doses (2.5, 4 and 10 mg/g) in singular or combined diet on growth rate, immune related genes expression, and resistance to white spot syndrome virus (WSSV) and Vibrio alginolyticus in Pacific white shrimp ( Litopenaeus vannamei ) were investigated. At the end of 28-day singular feeding experiment, the highest values of specific growth rate (SGR) and the expression of toll-like receptor1, 2 and 3 (TLR1, 2, 3), signal transducer and activator of transcription (STAT), crustin, anti-lipopolysaccharide factor (ALF) as well as prophenoloxidase (proPO) were observed in shrimp individually fed with 5 mg/g dietary inulin or MOS, respectively. Compared with individual treatments, diet containing combined prebiotics (5 mg/g inulin and MOS) significantly improved the expression of TLRs, STAT, proPO, crustin and ALF in L. vannamei after four-week feeding. Additionally, Pacific white shrimp fed with combined dietary prebiotics showed significantly higher expression of immune related genes and lower cumulative mortality in WSSV and Vibrio alginolyticus challenges, compared to the singular feeding groups and control. These results in the present study demonstrated that the combined supplementation of inulin (5 mg/g) and MOS (5 mg/g) remarkably enhanced innate immune response and pathogen resistance of shrimp, and should be considered as a promising immunostimulatory additive for the culture of Pacific white shrimp. [ABSTRACT FROM AUTHOR]

Red Sea bream iridovirus (RSIV) was initially isolated from marine fish, which belongs to Megalocytivirus , Iridoviridae . It can cause great economic losses in fish culture with high morbidity and mortality. In the present study, the pathogenicity and immune response associated with a RSIV genotype megalocytivirus infection were determined in mandarin fish ( Siniperca chuatsi ). Fish challenged showed typical clinical signs of iridovirus infection, including acute haemorrhages and enlarged visceral organs. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the stomach, spleen, kidney and liver of the diseased fish. Blood cells counting and phagocytic assay indicated that the numbers of the red and white blood cells in the peripheral blood of infected fish increased significantly compared to the control group and the phagocytic percentage of leukocytes peaked at day 6 post infection. Quantitive real-time PCR (qRT-PCR) was also undertaken to analyse the host defensive response in mandarin fish challenged with RSIV. The expression level of ten genes including interferon-related factors (IRFs) IRF1 and IRF7 , Mx , Viperin , JAK1 , STAT1 , TCRα , TNFα , IL-1β and IL-8 during experimental infection were monitored at different point of time in liver, spleen and head kidney. Results revealed varying expression profiles and clear transcriptional activation of these immune related genes in different tissues, which will contribute to better understand the pathogenesis and host defensive system in iridovirus invasion. [ABSTRACT FROM AUTHOR]

A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei ). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKβ and IKKε, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus ( V. parahaemolyticus ) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection. [ABSTRACT FROM AUTHOR]

The outbreak of mass mortality of M. salmoides occurred in an aquaculture farm in Jiangsu province of China, showing signs of skin ulceration and haemorrhages. The bacteria were isolated from diseased largemouth bass, and identified as Plesiomonas shigelloides based on morphological, physiological and biochemical features, as well as 16S rRNA gene sequence analysis. The pathogenicity of P. shigelloides was determined by challenge experiments, and the median lethal dosage (LD 50) of the isolate NJS1 for M. salmoides was calculated as 1.6 × 105 CFU/mL at 7 d post-infection. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the kidney, liver and gill of the diseased fish. Detection of virulence-related genes showed that P. shigelloides NJS1 was positive for astA, astB, astD, astE, actP and 6 ahpA. Additionally, the host defensive response of M. salmoides infected by P. shigelloides was analyzed by quantitive real-time PCR (qRT-PCR), and the results showed that the expression levels of Cas3 , Hep1 , HIF , IgM , IL15 and TGF were significantly up-regulated in head kidney, liver and spleen in different hours post-infection, which revealed varying expression profiles and clear transcriptional activation of immune related genes. The results suggested that P. shigelloides was an etiological element in the mass mortalities of M. salmoides and this study provided deeper insights for the pathogenesis and host defensive system in P. shigelloides invasion. • Mass mortalities of cultured Micropterus salmoides with symptoms of skin ulceration and hemorrhage were reported. • Pathogenesis and histopathological analysis related to M. salmoides with Plesiomonas shigelloides infection were studied. • P. shigelloides infection induced a wide defensive response of various immune related genes in M. salmoides. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of various levels of multi-strain probiotic on the immune related gene expression, digestive enzyme activity, growth performance, body chemical composition and survival of Litopenaeus vannamei post-larvae. After transferring post-larvae of L . vannamei to indoor conditions and subsequent acclimation to laboratory condition for 14 days, the shrimp were fed multi-strain probiotic at four different doses of 0, 0.25, 0.5 and 1.0 g kg −1 for eight weeks. Shrimp fed 0.5 and 1.0 g kg −1 probiotic PrimaLac ® exhibited significantly (p < 0.05) higher weight gain, specific growth rate, body crude protein as well as lower FCR compared to other groups. Feeding on 0.5 and 1 g kg −1 dietary multi-strain probiotic significantly (p < 0.05) increased the level of body crude protein. Oral administration of 0.5 and 1.0 g kg −1 multi-strain probiotic significantly (p < 0.05) decreased body crude lipid and body moisture respectively. 30 days after feeding, protease, amylase and lipase activity increased in groups fed 0.5 and 1.0 g kg −1 probiotic PrimaLac ® . However, on the 60th day, specific protease and amylase activity in all treatment groups were significantly higher than control group (p < 0.05) but lipase activity was higher (p < 0.05) in groups fed 0.5 and 1.0 g kg −1 multi-strain probiotic. Oral administration of 1.0 g kg −1 probiotic increased (p < 0.05) the level of prophenoloxidase and g-type lysozyme gene on day 30th and 60th after treatment. On day 30th and 60th, penaeidin gene expression was significantly higher in all treatment groups compared to the control group (p < 0.05). In general, findings of this study demonstrated that oral administration of 0.5 and 1.0 g kg −1 multi-strain probiotic improved the performance of the fish and increased the expression of immune related genes. [ABSTRACT FROM AUTHOR]