Cyprinid Herpesvirus Research Articles

Diagnostic case report: Disease outbreak induced by CyHV-2 in goldfish in China

A lethal disease of goldfish (Carassius auratus) afflicted ornamental fish farms in Beijing in 2018, causing severe financial losses. Morbidity and mortality are restricted to goldfish and occurred in spring and autumn, when water temperature was between 15 and 28 °C. Moribund goldfish from ornamental fish farms were tested for Cyprinid herpesvirus 2 (CyHV-2) by polymerase chain reaction (PCR) and real-time PCR using the viral polymerase gene followed by sequence analysis. The polymerase gene-specific product shared 99.10% nucleotide sequence identity to the published sequence of CyHV-2. Histology and electron microscopy showed that the moribund fish had severe necrosis in the gill and kidney and herpesvirus particles were demonstrated by Transmission Electron Microscopy(TEM). We infer, from these findings, that CyHV-2 might be transferred systemically via blood circulation system and that goldfish herpesviral hematopoietic necrosis is likely to be an important or serious infection disease for goldfish farms in this region of China.

Genetic Diversity of Cyprinid Herpesvirus 3 from Different Geographical Locations during 1999-2019 in the United States of America.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is an important pathogen in common and koi carp Cyprinus carpio, varieties. Two main genotypes of KHV have been reported worldwide that are associated with Asian and European origins. In the USA, outbreaks of KHV diseases have been reported in different states since the early 1990s; however, the diversity of KHV is unknown. In the current study, 67 DNA samples that were extracted from clinical cases of koi tissues that were submitted for diagnosis during KHV outbreaks from 10 different states in the USA from 1999 to 2019 were used to investigate their genetic diversity. The thymidine kinase gene was amplified, sequenced, and used for phylogenetic analysis. Our results showed that the KHV isolates that were collected from the different states were clustered in the two known KHV genogroups, where 31 isolates belonged to the Asian genotype branch and 36 to the European genotype branch. The spatiotemporal analysis demonstrated fluctuation of KHV genotypes in the USA, as the main KHV genotype that was detected in koi in the USA from 1999 to 2013 was the European genotype, whereas the Asian KHV genotype appeared to emerge in the USA in 2008, increasing in incidence until 2019. The current study provides evidence on the genetic diversity of KHV in the USA. Future studies that evaluate the virulence of these genetically diverse isolates is warranted to obtain a better understanding of the epidemiology of this re-emerging pathogen. This may provide an improved awareness of the current status of KHV and help to control the disease in the koi population in the USA.

First report of Cyprinid herpesvirus 2 outbreak in cultured gibel carp, Carassius auratus gibelio at low temperature

AbstractCyprinid herpesvirus 2 (CyHV‐2) is the causative agent of herpesviral hematopoietic necrosis (HVHN), an infectious disease with high morbidity and mortality worldwide. Current research indicates that CyHV‐2 only infects fish in the genus Carassius. Outbreaks of fish viral diseases are highly temperature‐dependent, and HVHN outbreaks usually occur at water temperatures of 15–25°C. In November 2018, an infectious disease occurred on cultured gibel carp farms in ZiGong, Sichuan Province, China. The water temperature at the time was approximately 10°C. The clinical signs of the diseased fish included lethargy, inappetence, severe anoxia, hemorrhage on the body surface, and pale gills. Clinical and histopathological examinations, transmission electron microscopy (TEM), and molecular analyses were used to determine the etiology of this disease. Pathological observation revealed severe necrosis of the hematopoietic organs, particularly the gills, kidney, and spleen. Spherical herpesvirus‐like virion particles were observed with TEM in the kidney and spleen tissues from moribund fish. An experimental infection demonstrated a cumulative mortality rate of 50% (10/20) at a water temperature of 13°C, whereas the mortality of the positive control group was 75% (15/20) at a water temperature of 23°C. A specific fragment of the helicase gene of CyHV‐2 was detected with polymerase chain reaction. In a phylogenetic analysis of the helicase gene, our isolate clustered with CyHV‐2 isolates in the database. Together, these results show that the putative pathogen involved in this outbreak was CyHV‐2. To the best of our knowledge, this is the first report of a gibel carp disease caused by CyHV‐2 at low temperature. These findings are extremely important in reducing the risk of HVHN outbreaks in gibel carp aquaculture.

MicroRNA expression profiling of caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection

As a member of the genus Cyprinivirus in the family Alloherpesviridae, Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss in the aquaculture industry, mainly in C. auratus gibelio and goldfish. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 remain elusive. In this study, high-throughput sequencing technology was employed to explore the miRNA expression profiles of C. auratus gibelio (GiCF) caudal fin cells in response to Cyprinid Herpesvirus-2 (CyHV-2) infection. A total of 631 novel miRNAs and 409 known miRNAs were identified. The expression levels of 7 miRNAs were found as significantly modulated (5 down-regulation and 2 up-regulation; P < 0.01, |logFC|>1, TPM>10) in CyHV-2 infected cells. 7 miRNA and their potential mRNA targets were validated by Real-time PCR (qRT-PCR), respectively. Targets prediction and functional analysis of these 7 miRNAs revealed significant enrichment for several signaling pathways, including PPAR, p53 and FoxO pathways. These studies provided more valuable basis for further study on the roles of miRNAs in CyHV-2 replication and pathogenesis.

Protective immunity against CyHV-3 infection via different prime-boost vaccination regimens using CyHV-3 ORF131-based DNA/protein subunit vaccines in carp Cyprinus carpio var. Jian

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1β, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

The First Detection of Koi Herpesvirus (CyHV3) in Migratory Wild Ducks in North America.

A viromics study on the intestinal contents of migratory wild ducks of the genera Anas, Mareca, Spatula, and Oxyura during their winter stay in Mexico showed the presence of the virus family Alloherpesviridae. The genus Cyprinivirus is part of this family and includes cyprinid herpesvirus 3 (CyHV3). This is the etiological agent of the lethal disease known as koi herpesvirosis, which affects different strains of Common Carp Cyrprinus carpio. In this study, samples of the contents of 87 wild duck intestines were analyzed by endpoint PCR, of which 7 samples were positive for the amplification of the TK gene fragment corresponding to CyHV3. These results contribute to the knowledge about the spread of this virus to other species of aquatic animals in areas where fish and ducks coexist.

Genetic relationship between koi herpesvirus disease resistance and production traits inferred from sibling performance in Amur mirror carp

Koi herpesvirus disease (KHVD) is currently the most serious threat to global carp farming. Prevention is a sensible strategy for tackling this disease and improved genetic resistance of carp strains is a desirable breeding goal. To study the potential for multitrait selection, the objective of the current study was to estimate the genetic correlations between KHVD resistance and production traits in Amur mirror carp. A total of 1500 fingerlings from four factorial crosses of five dams and ten sires were challenged with Koi herpesvirus (KHV). Juvenile growth-related traits were collected on the same individuals before the challenge test. Production traits were measured on siblings of the challenged population at different life stages (yearling to market size). The estimated heritability for resistance to KHVD was 0.43 ± 0.08 on the observed scale and 0.72 ± 0.13 on the underlying liability scale. Most genetic correlations between KHVD resistance and important production traits were insignificant, showing that selection for improved production traits would not increase susceptibility to KHV and vice versa. However, resistance to KHVD was negatively correlated with Fulton's condition factor (FC) after the second overwintering and relative head length (RHL), relative body height (RBH) and relative body width (RBW) from the second growing season to the market size, with a more prolonged body shape of Amur mirror carp (genes from Amur wild scaly carp, Cyprinus rubrofuscus) being associated with higher KHVD resistance. Intermediate favorable genetic correlations between KHVD resistance and log-log residuals of headless carcass yield (0.37 ± 0.14) and fillet yield (0.44 ± 0.13) at market size suggested that selection for improved yields of edible body parts might indirectly lead to a slight improvement in KHVD resistance and vice versa.

β-glucan and anisodamine can enhance the immersion immune efficacy of inactivated cyprinid herpesvirus 2 vaccine in Carassius auratus gibelio

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a β-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, β-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that β-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, β-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in β-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of β-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

De novo RNA-seq assembly and differential transcriptome analysis of Carassius auratus gibelio after Cyprinid herpesvirus 2 infection

Carassius auratus gibelio, in the family Cyprinidae, is one of the most popular freshwater aquaculture species in China. The infection of domestic cyprinid species by Cyprinid herpesvirus 2 (CyHV-2) causes herpesvirus hematopoietic necrosis (HVHN). Until now, the pathogenic mechanism of CyHV-2 has been unclear. In this study, we used RNA sequencing to investigate the gene expression patterns in C. auratus gibelio after CyHV-2 infection. We identified 163,365 unigenes with de novo transcriptome sequencing using the Trinity and TGICL software. In total, 8011 differentially expressed genes (DEGs) were identified in the CyHV-2-infected fish vs healthy fish, 4170 of which were upregulated and 3841 downregulated. A Gene Ontology (GO) analysis showed that most of the DEGs functioned in the cellular hyperosmotic response, inflammatory response, creatine: sodium symporter activity, voltage-gated potassium channel complex, complement activation, and oxygen binding categories. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the top five KEGG pathways enriched in upregulated genes were neuroactive ligand-receptor interaction, pyrimidine metabolism, IL-17 signaling pathway, glutathione metabolism, and arginine biosynthesis. The top five KEGG pathways enriched in downregulated unigenes were cytokine–cytokine receptor interaction, complement and coagulation cascades, Staphylococcus aureus infection, drug metabolism, and glycine, serine, and threonine metabolism. The expression of five key immunity-related genes was confirmed with real-time PCR. Specifically, the genes encoding interleukin-11 and interleukin-13 receptor, which are involved in immunity, were significantly upregulated and downregulated in CyHV-2-infected fish, respectively. Our findings not only extend our understanding of the host's response to CyHV-2, but also clarify the molecular pathogenicity mechanisms involved.

Surveillance of herpesviruses in koi carp Cyprinus carpio koi and goldfish Carassius auratus cultured in West Bengal, India

ABSTRACT Background: Viral diseases create one of the greatest challenges for the rapidly expanding ornamental fish culture sector. In recent years, the host-specific herpesviruses are receiving much attention due to the intensification in aquaculture globally. Cyprinid herpesvirus-2 (CyHV-2, goldfish hematopoietic necrosis virus), and Cyprinid herpesvirus-3 (CyHV-3, koi herpesvirus) are the lethal pathogens of koi carp Cyprinus carpio koi and goldfish Carassius auratus, respectively. In India, West Bengal is the leading state in ornamental fish production and export. Methods: The surveillance of CyHV-2 and CyHV-3 in koi carp and goldfish cultured in West Bengal was carried out between November 2014 and January 2017 as per Office International des Epizooties guidelines. Results: Both fish species were negative for CyHV-3. The CyHV-2 infection was detected in both gill and kidney tissues of apparently healthy and diseased C. auratus from December 2014 to March 2015. Severe necrosis was observed in the gills of infected C. auratus. Coinfection with members of the bacterial genera Aeromonas and Flavobacterium was also observed in the kidney of infected goldfish. The histopathological observations in the kidney of CyHV-2 infected C. auratus demonstrated the pathogenic potential of CyHV-2 and tissue tropism. Conclusions and Clinical relevance: Environmental stressors like low water temperature and the use of wastewater for culture played a vital role in the onset of CyHV-2 infection in the stressed goldfish. The spread of CyHV-2 can likely pose a certain degree of threat to aquaculture especially the unrestricted movement of goldfish. The results of the present study emphasize the necessity of an organized risk assessment to plan for the management strategies on the control and spread of CyHV-2 in Indian aquaculture.

Detection of cyprinid herpesvirus 2 by loop-mediated isothermal amplification in combination with a lateral flow dipstick

We developed a convenient technique to detect Herpesviral haematopoietic necrosis attributed to cyprinid herpes virus 2 (CyHV-2), a serious disease of Crucian carp and goldfish related to high mortality. In the present study, we employed a lateral flow dipstick (LAMP-LFD) to present a loop-mediated isothermal amplification assay. The specificity was ascertained via other six viruses, and the sensitivity was compared using PCR method, which are the reaction conditions changes for the method improved. The results revealed that CyHV-2 performance was observable at 64 °C in a separated tube within 60 min, when the samples hybridized using an FITC-labeled probe. As the LAMP-LFD method's specificity was high, with its sensitivity identical to that of traditional PCR, the overall DNA collected revealed the lowest detection limit of 0.18 pg/μl from goldfish diseased by CyHV-2. In summary, the development of LAMP-LFD's method does not require expensive instruments, and it can be regarded as a fast, simple, and reliable method for CyHV-2 detection.

Description of Gill Histopathology And Water Quality Of Koi Fish (Cyprinus Koi fishio) Infected By Koi Herpes Virus (KHV)

One of disease that attacks in common koi fish until now is caused by infection of Koi Herpes Virus. Koi Herpes Virus (KHV) is one type of DNA virus that attacks the family of Cyprinidae. Based on the preliminary test using PCR techniques gills, of koi fish infected by KHV was positive. This study aimed to determine water quality analysis and histopathological gills of koi fish (Cyprinus Koi fishio Linn) that infected KHV. Water quality measurements and gills tissue of fish observation was conducted in three repetition. Histopathological observations of gills tissue used for PCR analysis. The results of PCR analysis showed light line DNA band with size of 292 bp. The results of temperature, brightness, pH, DO, CO2 and COD are still in accordance with water quality standards, while values of ammonia showed 0.373-0.377 mg/l and BOD 3.986-4.729 mg/l that exceeding prescribed quality standards. Histopathological observations of gills tissue showed damage to the primary lamella form like edema, hyperplasia, atrophy, congestion, and necrosis than the type damage of secondary lamellae like edema, hyperplasia, fusion lamella, telangiectasis, congestion, and necrosis. Suspected KHV infection caused damage for gills of koi fish in histopathological.

Corrigendum to: Effects of fish kills on fish consumers and other water-dependent fauna: exploring the potential effect of mass mortality of carp in Australia

Although the effects of mass fish mortality (MFM) events on fish populations and water quality are frequently reported, the effects on consumers of fish and other water-dependent fauna are relatively poorly understood. Managing the effects of MFM events on other fauna is important when they occur in ecologically or culturally sensitive locations, or involve protected or significant species or ecosystems. Better understanding of the effects of such events on fish consumers and other water-dependent fauna would aid proactive management of predictable events, such as pest-fish biocontrol releases. We conducted a global literature review to synthesise the effects of unexpected MFM events on fish consumers and other water-dependent fauna in freshwater, estuarine and marine ecosystems. We then constructed two conceptual models distinguishing between the short-term (hours to weeks) and long-term (months to years) effects of such events, highlighting the importance of key factors in determining the magnitude of the response. Last, we used these models and relevant literature to explore the potential implications of the proposed release of cyprinid herpes virus 3 as a biological control agent for the invasive common carp (Cyprinus carpio) in Australia and develop recommendations to minimise adverse effects of carp mass mortality on food webs.

Development, characterization of monoclonal antibodies specific for the ORF25 membrane protein of Cyprinid herpesvirus 2 and their applications in immunodiagnosis and neutralization of virus infection

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of haematopoietic necrosis disease in farmed gibel carp (Carassius auratus gibelio). It appears to be spreading cross the world, and has resulted in massive mortality and great economic losses. Although many sensitive nucleic acid-based diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 membrane protein are still scarce to study. In this study, ORF25 gene was chemically synthesized and cloned into a pET-28a expression vector to produce rORF25 (recombinant ORF25) in E. coli BL21 (DE3). The purified rORF25 was served as an immunogen to prepare monoclonal antibodies (MAbs), and five efficient hybridoma cell lines were selected by enzyme-linked immunosorbent assay (ELISA). Among them, MAbs 7H10-1B6 and 1F8-1B6 belonged to the IgG2b isotype, while the other three MAbs designed as 8D9-1H6, 3H2-1G5 and 2C3-1E6 belonged to the IgG1 isotype. Western blotting results indicated that MAbs could specifically identify the rORF25. MAb 2C3-1E6 could specifically detect ORF25 in CyHV-2 infected Fathead Minnow cells and gible carp tissues by ELISA, indirect immunofluorescence and immunohistochemistry. In vitro neutralization assay showed that MAb 2C3-1E6 exhibited a titer-dependent neutralization effect on CyHV-2 infection in FHM cells. The results indicated that MAbs against rORF25 could serve as effective detection probes and potential neutralizing antibody for haematopoietic necrosis disease in gibel carp.

Cyprinid Herpesvirus 2 miR-C12 Attenuates Virus-Mediated Apoptosis and Promotes Virus Propagation by Targeting Caspase 8.

DNA viruses, most notably members of the herpesvirus family, generally encode miRNAs to mediate both virus and host genes expression. We previously demonstrated that Cyprinid herpesvirus 2 (CyHV-2) encodes 17 miRNAs that are involved in innate immune signaling pathways. In this study, the function of CyHV-2-encoded miRNA was further investigated in GiCF cells. We found that miR-C4 promoted CyHV-2-induced apoptosis, while miR-C12 decreased CyHV-2-induced apoptosis. miR-C12 targeted to 3′ UTR sequence of caspase 8 and suppressed the expression of caspase 8. Besides, the silencing of caspase 8 by specific siRNA led to the attenuation of CyHV-2-induced apoptosis. Furthermore, caspase 8 was downregulated in cells transfected with miR-C12 during CyHV-2 infection. Overexpression of miR-C12 significantly suppressed CyHV-2-induced apoptosis, while silencing of miR-C12 promoted CyHV-2-induced apoptosis. Finally, inhibition of miR-C12 resulted in suppression of CyHV-2 propagation, overexpression of miR-C12, and CASP8-siRNA-1 facilitated CyHV-2 propagation. Taken together, our results demonstrated that CyHV-2-encoded miR-C12 to suppress virus-induced apoptosis and promoted virus replication by targeting caspase 8.

Genetic mapping of Koi herpesvirus resistance (KHVR) in Mirror carp (Cyprinus carpio) revealed genes and molecular mechanisms of disease resistance

Koi herpesvirus is a double-stranded DNA virus that causes fatal disease and mortality in koi and common carp (Cyprinus carpio) of all ages. Koi herpesvirus disease has been added to the list of notifiable diseases of the World Organisation for Animal Health. The genetic basis of fish resistance to Koi herpesvirus remains little understood, and thus, it cannot be used in genetic breeding programs to produce high-resistance carp strains. Dissection of the molecular mechanism of resistance could help prevent or reduce losses due to Koi herpesvirus disease. Common carp is a widely distributed species cultured all around the world. Its allotetraploidy and high economic and environmental value have attracted a great deal of interest in its genetics and genomics. Here, we use both QTL mapping and GWAS in Mirror carp subjected to Koi herpesvirus infection to detect potential loci and genes related to resistance to this virus. Many immune-related genes were identified around the significant QTLs or SNPs, including tnfa, hif1a, galectin-8, rootletin, and palladin. GO and KEGG analyses were performed based on the union of the candidates. Of particular interest were the Herpes simplex infection pathway and mTOR signaling pathway, which play potential roles in the immune response. Using publicly available transcriptome data from Koi herpesvirus infected carp, the expression patterns of candidate genes were obtained, and the results indicated potential roles of homoeolog functional divergence in immune response in the allotetraploid common carp. Overall, our study of the genetic architecture of carp Koi herpesvirus resistance provides many valuable candidate genes, which could be considered in the marker-assisted selection of common carp strains with high resistance to Koi herpesvirus.

Poly-β-hydroxybutyrate (PHB) in bioflocs alters intestinal microbial community structure, immune-related gene expression and early Cyprinid herpesvirus 2 replication in gibel carp (Carassius auratus gibelio)

The aquaculture system based on biofloc technology (BFT) showed positive effects on prevention of Cyprinid herpesvirus 2 (CyHV-2) infection in gibel carp (Carassius auratus gibelio), which is detrimental to health and causes seriously economic losses to aquaculture. However, the enhancement mechanism of BFT regarding immunity and disease resistance of cultured species is scarce. Poly-β-hydroxybutyrate (PHB) has been proved as one of bioactive compounds in bioflocs. In this study, two groups (4% PHB supplementation diets and control with basal diets) with 30-day feeding were set to study the effect of PHB supplementation on immune-related gene expression by qRT-PCR, time-course CyHV-2 replication in vivo by qPCR and intestinal microbiota by illumine high-throughput sequencing. PHB supplementation significantly up-regulated transcriptional levels of eight immune-related genes, decreased cumulative mortality of gibel carp and early CyHV-2 replication in spleen in vivo (P < 0.05). Additionally, PHB changed the microbial structure but not diversity, and significantly increased beneficial bacteria such as Bacillus sp. KEGG pathway analysis by PICRUSt demonstrated that oral administration of PHB up-regulated abundances of genes responsible for seven pathways and down-regulated genes in eleven pathways. Histological structures of foregut, mindgut and hindgut were also affected. Our findings suggested that profitable effects of PHB on immunity and disease resistance might be gut microbiota-related, and regulated through pathways of enzymes secretion, replication and repair, and host immune system. This study will provide new insights into understanding the enhancing mechanism of BFT on immunity and disease resistance of cultured animals, and developing prebiotics/probiotics-based immunotherapies to improve animal health and disease resistance.

First detection of koi herpesvirus and carp oedema virus in Iraq associated with a mass mortality in common carp (Cyprinus carpio).

At the end of October 2018, a mass fish mortality occurred in Iraq, involving thousands of tons of cultured and wild common carp (Cyprinus carpio) along Euphrates and Tigris rivers. Fish were found dead or moribund along rivers coasts, showing lethargy, dyspnoea and flared gills. At necropsy, discoloration patches were noticed on the gills. Wet preparations showed rare metacercariae and Dactylogyrus spp. Samples were subjected to bacteriological tests and virological investigation through real-time PCR and nested PCR. Both were positive for koi herpesvirus (KHV) and carp oedema virus. Results obtained were confirmed by the OIE reference laboratory of KHV disease (KHVD) at Cefas (UK) and by sequence analysis. This is the first report on the detection of both viruses in Iraq.

Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance

BackgroundInfectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection.ResultsIn susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar.ConclusionsTaken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.

Development of monoclonal antibodies against serum immunoglobulins from gibel carp (Carassius auratus gibelio) and their applications in serodiagnosis of inapparent infection and evaluation of vaccination strategies

The disease outbreak caused by viral infection and bacterial pathogens has hampered the sustainable development of the gibel carp (Carassius auratus gibelio) industry, lack of monoclonal antibodies against serum immunoglobulin (Ig) of gibel carp has impeded the development of nonfatal immunoassays in detection of pathogen infection and understanding of fish immune response post vaccination. In the present study, serum Ig of gibel carp was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified Ig had an apparent molecular weight of 74 kDa and 24 kDa in SDS-PAGE. Three monoclonal antibodies (MAbs) against Ig, designed as 2F4-1G10, 3H3-1E8 and 7H11-1C8, were developed with purified Ig preparations, which were selected on the basis of the indirect enzyme-linked immunosorbent assay (ELISA). Western blotting showed that MAbs 2F4-1G10 and 7H11-1C8 reacted with the heavy chain of IgM, while MAb 3H3-1E8 showed a reaction with the light chain. MAb 7H11-1C8 could react with surface Ig-positive (sIg+) lymphocytes under indirect immunofluorescence assay. Fluorescence-activated cell sorter analysis revealed that the percentage of sIg + lymphocytes were 32.68% in peripheral blood and 12.13% in pronephros. MAb 7H11-1C8 was proven to be effective in detecting the Cyprinid Herpesvirus 2-specific serum Ig, and determining the levels of Aeromonas hydrophila specific Ig in serum and immune organs under different vaccination strategies.