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Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV‐2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL‐1β, and IFN‐1 in vaccinated head‐kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV‐2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV‐2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae‐displayed ORF25 could be served as a candidate vaccine to prevent CyHV‐2 infection. [ABSTRACT FROM AUTHOR]

The present work is the first comprehensive study of fungus‐like stramenopilous organisms (Oomycota) diversity in Lithuanian fish farms aimed at proper identification of saprolegniasis pathogens, which is important for water quality control, monitoring infection levels and choosing more effective treatments for this disease in aquaculture. Pathogenic to fish, Saprolegnia and other potentially pathogenic water moulds were isolated from adult fish, their eggs, fry and from water samples. All detected isolates were examined morphologically and confirmed by sequence‐based molecular methods. A total of eight species belonging to the genera Saprolegnia, Achlya, Newbya and Pythium were identified. Four species (S. parasitica, S. ferax, S. australis and S. diclina) were found to be the main causative agents of saprolegniasis in Lithuania. S. parasitica and S. ferax dominated both in hatcheries and open fishponds, accounting for 66.2% of all isolates. S. parasitica was isolated from all farmed salmonid fish species as well as from the skin of Cyprinus carpio, Carassius carassius and Perca fluviatilis. S. australis was isolated from water and once from the skin of Oncorhynchus mykiss, and S. diclina was detected only once on the skin of Salmo salar fish. In addition, Achlya ambisexualis, Saprolegnia anisospora and Newbia oligocantha isolated during this study are noted as a possible source of saprolegniasis. The results of this study are relevant for assessing the risk of potential outbreaks of saprolegniasis or other saprolegnia‐like infection in Lithuanian freshwater aquaculture. [ABSTRACT FROM AUTHOR]

In 2011 and 2015, four mass mortalities of Prussian carp (Carassius gibelio) were observed in a recreational freshwater lake and open freshwater in the western part of the Netherlands. Cyprinid herpesvirus 2 (CyHV‐2) infection was suspected in these cases, based on presumptive gross diagnosis. To elucidate the cause of the mass mortalities diagnostic PCR assays were performed for CyHV‐2, based on the helicase gene. Furthermore, the viral isolates were genotyped by sequencing the enlarged marker A and marker B sequences. Diagnostic PCR revealed that three of four samples were positive for CyHV‐2, indicating these three mass mortalities were associated with CyHV‐2 infection. The marker A sequence from one of the isolates found in this study was identical to those from different locations such as Asia and Middle East, suggesting a link among the isolates. This is the first detailed report on mass mortalities of Prussian carp associated with CyHV‐2 infection in natural aquatic environments in the Netherlands. Since 2015, additionally, in total three CyHV‐2 associated outbreaks of Dutch Prussian carp were seen in 2016 and 2020. These outbreaks in Prussian carp from lakes and open water suggest that the virus has been spreading in natural freshwaters in the Netherlands. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila can pose a great threat to the survival of farmed fish. In current study, we investigated the pathological characteristics and immune response in gut–liver axis of white crucian carp (WCC) upon gut infection. WCC anally intubated with A. hydrophila exerted a tissue deformation in damaged midgut with elevated levels of goblet cells along with a significant decrease in tight junction proteins and villi length‐to‐width ratios. In addition, immune‐related gene expressions and antioxidant properties increased dramatically in gut–liver axis of WCC following gut infection with A. hydrophila. These results highlighted the immune modulation and redox alteration in gut–liver axis of WCC in response to gut infection. [ABSTRACT FROM AUTHOR]

Cyprinid herpesvirus 2 (CyHV‐2), the etiological agent of herpesvirus haematopoietic necrosis (HVHN) in carp and goldfish, has caused significant economic losses in the aquaculture industry. During viral infection, the host initiates a series of active or passive defences to regulate the process of virus infection. Apoptosis is a key component of active cellular defence, and members of the Bcl‐2 family have been shown to play a critical role in the apoptotic process. However, the mechanism of action of the Bcl‐2 family in inducing apoptosis during CyHV‐2 infection remains unclear. In this study, we revealed the molecular mechanism of miRNA‐mediated silver crucian carp BAX (ccBax) in CyHV‐2‐induced apoptosis for the first time and demonstrated that the overexpression of miR‐124 suppressed ccBax expression and significantly down‐regulated apoptosis in caudal fin cells of Carassius auratus gibelio (GiCF), while miR‐124 inhibitors were the opposite. These studies indicated that miR‐124 inhibits CyHV‐2‐induced apoptosis by reducing the expression of ccBax. Furthermore, the fact that transfection of miR‐124 mimics promoted CyHV‐2 replication, whereas miR‐124 inhibitors inhibited CyHV‐2 replication, indicated that miR‐124 inhibited CyHV‐2‐induced apoptosis and contributed to viral replication. All these results suggested that miR‐124 suppresses virus‐induced apoptosis and promotes viral replication by targeting and regulating ccBax expression. [ABSTRACT FROM AUTHOR]

The Streptomyces lateritius Z1‐26 was isolated from soil samples which showed broad‐spectrum antibacterial activity against a broad range of fish pathogens. The In Vivo Imaging System (IVIS) monitored that strain Z1‐26 could survive and colonize in the gills and abdomen of crucian carp. The effects of dietary supplementation with strain Z1‐26 were evaluated with respect to the growth performance, antioxidant capacity, and immune response of crucian carp. The results showed that the Z1‐26‐fed fish had a significantly higher growth rate than the fish fed the control diet. The immune and antioxidant parameters revealed that the non‐specific immune indicators (AKP, SOD, and LZM) of the serum, the expression of immune‐related genes (IgM, C3, and LZM), and antioxidant‐related genes (Nrf2 and Keap1) of the immune organs were significantly increased, whereas the expression of pro‐inflammatory factors (IL‐1β, IL‐8, and TNF‐α) of the immune organs was significantly down‐regulated in crucian carp fed strain Z1‐26 compared with fish fed a control diet. Moreover, fish fed with Z1‐26 supplemented diets showed a significantly improved survival rate after Aeromonas hydrophila infection. In addition, the whole genome analysis showed that strain Z1‐26 possesses 28 gene clusters, including 6 polyketide synthetase (PKS), 4 non‐ribosomal peptide‐synthetase (NRPS), 1 bacteriocin, and 1 lantipeptide. In summary, these results indicated that strain Z1‐26 could improve the growth performance and disease resistance in crucian carp, and has the potential to be developed as a candidate probiotics for the control of bacterial diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Infections of Cyprinid herpesvirus 2 in goldfish and farmed crucian carp (Carassius auratus gibelio) are still an urgent problem worldwide. Detection and prevention are necessary for the control of haematopoietic necrosis disease caused by CyHV‐2. Although many sensitive molecular diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV‐2 are still important to CyHV‐2 study. In this experiment, purified CyHV‐2 was used as antigens to produce monoclonal antibodies (Mabs). Six Mabs bound to different proteins were selected by Dot‐blot screening and Western‐blot analysis, and no one had cross‐reactivity with closely related koi herpesvirus. Among them, Mabs 2E1‐B10, 1F5‐A3 and 4C4‐A7 belonged to IgG1 isotype, while other three Mabs 3G9‐B11, 3B4‐G5 and 4F4‐B7 belonged to IgM isotype. These six Mabs all could specifically detect CyHV‐2 in CyHV‐2 infected caudal fin of Carassius auratus gibelio (GiCF) cells by immunofluorescence assays. Then, the neutralization ability was tested in vitro, and the result showed that all six Mabs can attenuate CPE by CyHV‐2 in vitro among which 2E1‐B10 had the best neutralization ability. The virus proteins recognized by these six Mabs were identified by mass spectrometry identification, and the result showed they probably were ORF88, ORF55R, ORF115 and ORF151R. This study is the first to prepare Mabs by purifying CyHV‐2, which will provide a practical basis for the in‐depth study of CyHV‐2 virus. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is a common pathogen of freshwater fish. In this study, A. hydrophila infection was shown to cause tissue damage, trigger physiological changes as well as alter the expression profiles of immune‐ and metabolic‐related genes in immune tissues of red crucian carp (RCC). Transcriptome analysis revealed that acute A. hydrophila infection exerted a profound effect on mitochondrial oxidative phosphorylation linking metabolic regulation to immune response. In addition, we further identified cellular senescence, apoptosis, necrosis and mitogen‐activated protein kinase signal pathways as crucial signal pathways in the kidney of RCC subjected to A. hydrophila infection. These findings may have important implications for understanding modulation of immunometabolic response to bacterial infection. [ABSTRACT FROM AUTHOR]

Cyprinid herpesvirus 2 (CyHV‐2) is a viral pathogen worldwide and causing high mortality on goldfish and silver crucian carp (Carassius auratus gibelio). In order to establish a stable and sensitive immunological diagnostic approach, the recombinant ORF121 protein encoded by the CyHV‐2 ORF121 gene, was selected as a capture antigen to identify cells and tissues infected with CyHV‐2 by immunological methods in this study. Firstly, the open reading frame of CyHV‐2 ORF121 was cloned into the PGEX‐4T‐3 vector and expressed in Escherichia coli. Purified recombinant ORF121 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot‐blot assay. The resulting mAb‐3D9 was applied to detect CyHV‐2 in infected caudal fin of Carassius auratus gibelio (GiCF) cells and fish tissues by western blotting, immunofluorescence assays and immunohistological asays. The monoclonal antibody could specifically identify CyHV‐2 in infected GiCF cells and the gills, the kidney and the spleen tissues, and it could attenuate CPE by CyHV‐2 in vitro, suggesting it can be applied for CyHV‐2 detection in the crucian carp and ORF121 may be a candidate vaccine against CyHV‐2. [ABSTRACT FROM AUTHOR]

Galectin‐9, an important pathogen recognition receptor (PRR), could recognize and bind pathogen‐associated molecular patterns (PAMPs) on the surface of invading microorganisms, initiating the innate immune responses. A galectin‐9 was identified from Qihe crucian carp Carassius auratus and designated as CaGal‐9. The predicted CaGal‐9 protein contained two non‐identical carbohydrate recognition domains (CRDs), namely, N‐CRD and C‐CRD. The recombinant proteins (rCaGal‐9, rN‐CRD and rC‐CRD) were purified from Escherichia coli BL21 (DE3) and exhibited strong agglutinating activity with erythrocytes of rabbit. The haemagglutination was inhibited by D‐galactose, α‐lactose and N‐acetyl‐D‐galactose. Results of microbial agglutination assay showed that three recombinant proteins agglutinated Gram‐negative bacterium Aeromonas hydrophila and Gram‐positive bacterium Staphylococcus aureus. With regard to the binding activity, each recombinant protein could bind to LPS, PGN and the examined microorganisms (A. hydrophila and S. aureus) with different binding affinities. The integrated analyses suggested that CaGal‐9 with two CRD domains could play an important role in immune defence against pathogenic microorganisms for C. auratus. [ABSTRACT FROM AUTHOR]

Bcl2 family proteins play a critical role in cell death or survival. BAX, the death‐promoting protein of bcl2 family, mediated mitochondrial pathway inducing cells' apoptosis in mammal. MiRNAs have been implicated as negative regulators down‐regulating genes' expression after post‐transcriptional level. At present, little is known about the regulatory mechanism of miRNA on the Bcl2 family proteins during CyHV‐2 infection in silver crucian carp (Carassius auratus gibelio). In this study, the ccBAX (silver crucian carp BAX) gene was cloned and expressed, and polyclonal antibodies were raised in mouse against the purified ccBAX‐GST fusion protein. The structure analysis indicated that ccBAX protein included four conserve domains (BH1, BH2, BH3 and transmembrane domains) and the expression of ccBAX protein occurred throughout the cells. Furthermore, two miRNAs (miR‐124 and miRNA‐29b) were identified to negatively regulate ccBAX gene expression in GiCF cell. miR‐124 was found to suppress the expression of WT‐ccBAX (wild type), but not the MT‐ccBAX (mutant). Overall, the results demonstrated that the expression of the ccBAX gene was significantly down‐regulated by miR‐124 in silver crucian carp (Carassius auratus gibelio) during CyHV‐2 infection. [ABSTRACT FROM AUTHOR]

Rahnella aquatilis is an important pathogen of several aquatic organisms and is found widely distributed in the freshwater, soil, fish and human clinical samples. Our previously published study reported a novel pathogenic R. aquatilis strain KCL‐5 to crucian carp (Carassius auratus). To further investigate the characteristics and pathogenesis caused by R. aquatilis, we here report on the pathological changes, bacterial genomic and proteomic analyses of strain KCL‐5. Significantly pathological changes in liver, intestine, spleen and gills were observed in infected fish. The genome consists of one circular chromosome 5,062,299 bp with 52.02% GC content and two plasmids (506,827 bp, 52.16%; 173,433 bp, 50.00%) and predicted 5,653 genes, 77 tRNAs and 22 rRNAs. Some virulence factors were characterized, including outer membrane protein, haemolysin, RTX toxin, chemotaxis and T3SS secretion system. Antimicrobial resistance genes such as EmrAB‐TolC, MexABC‐OpmB and RosAB efflux pump were found in strain KCL‐5. KEGG analysis showed that mainly functional modules were ABC transporters, biosynthesis of amino acids, two‐component system, quorum sensing, flagellum assembly and chemotaxis, in which most of them were identified by using 2‐DE/MS analyses. To our knowledge, this was first report on the molecular characteristics of R. aquatilis by multi‐omics approaches, which will provide insights into the pathogenic mechanism of R. aquatilis infection in fish. [ABSTRACT FROM AUTHOR]

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed‐type hypersensitivity (DTH) response against culture filtrate protein‐10 (CFP‐10) and the 6‐kDa early secreted antigen target (ESAT‐6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT‐6 and CFP‐10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT‐6 and CFP‐10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon‐γ1‐1‐encoding gene (IFN‐γ1‐1). In contrast, IFN‐γ1‐1 expression accompanied by DTH response was observed only in the CFP‐10‐DNA plasmid‐injected fish. Furthermore, fish that had been prophylactically injected with CFP‐10‐DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP‐10 may facilitate diagnosis of mycobacteriosis. [ABSTRACT FROM AUTHOR]

Koi herpesvirus (KHV) infections cause high mortality in carp (Cyprinus carpio). This study compared the susceptibility of silver crucian carp (Carassius auratus langsdorfii), also called ginbuna, and koi carp to KHV infection. Silver crucian carp and koi carp were challenged with KHV by both intraperitoneal injection and immersion, respectively, and kept in tanks at 22°C. All KHV‐exposed koi carp died within 14 days post‐infection (dpi), whereas no clinics nor mortality was observed in the KHV‐exposed silver crucian carp. KHV DNA was detected in both koi and silver crucian carp shortly after infection. At 7 dpi, the copy numbers of KHV genome were increased in koi carp but decreased in silver crucian carp. Using reverse transcriptase PCR, KHV mRNA was detected in koi carp but not in silver crucian carp. Cell cultivation on common carp brain (CCB) cell samples from koi carp caused KHV‐associated cytopathic effects in CCB cells. Therefore, we concluded that KHV replicated in koi carp but not in silver crucian carp and that silver crucian carp is not susceptible to infection with KHV. [ABSTRACT FROM AUTHOR]

During a PCR‐based CEV survey in Poland in 2015–2017, the virus was detected in many farms both in clinical and asymptomatic cases and in common as well as in koi carp (Cyprinus carpio). In order to evaluate the potential carrier role of fish species that share the same habitats with carp, an experimental trial was performed. Investigations carried out on specimens of bleak (Alburnus alburnus), crucian carp (Carassius carassius), European perch (Perca fluviatilis), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) cohabited with CEV‐infected carp yielded positive results. These species of fish were experimentally cohabited with CEV‐infected common carp at a temperature of 16°C ± 1. Material from the brain, gills, spleen, kidneys, intestine and skin was investigated for the presence of CEV DNA. Similar investigations were performed with uninfected fish designated controls. Samples were tested for CEV by qPCR. [ABSTRACT FROM AUTHOR]

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited. [ABSTRACT FROM AUTHOR]

Considering the economic value of koi carps and the possible direct transmission of the infection in optimal temperature condition, I Dermocystidium i spp. infection should be considered as an important disease also in ornamental species. A new Species of the Genus Dermocystidium, D. koi sp. nov., Parasitic in Cyprinus Carpio L. Nippon Suisan Gakkaishi, 15 (12), 825 - 829. https://doi.org/10.2331/suisan.15.825 Dermocystidium koi found in skin lesions in koi carp (Cyprinus carpio). [Extracted from the article]

The article discusses the pathogenic role of Cyprinid herpesvirus 2 (CyHV-2) in the mass mortality in crucian carp population, based on the result of diagnostic examination of samples of specimens taken from Tuscany, Italy. Some of the observations of the specimen include the presence of hemorrhages and oversecretion of mucus in gills, liver cell degeneration and lymphocytic myocarditis. A comparison of CyHV-2 in crucian carp and gold fish and Prussian carp is presented.

Sixteen specimens of female crucian carp, Carassius carassius ( L.), during the breeding season, were investigated for post-mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus-like particles, subsequently identified as Cyprinid herpesvirus-2 ( Cy HV-2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to Cy HV-2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species. [ABSTRACT FROM AUTHOR]

The article focuses on flavobacterium psychrophilum which causes significant problems in aquaculture. Some of the diseases associated with the pathogen include rainbow trout fry syndrome (RTFS) and bacterial cold water disease (BCWD). The fish species which are particularly susceptible to the pathogen include rainbow trout and coho salmon. Some other fish species which reported diseases linked to the pathogen include eel, common carp, crucian carp and tench.

Hybrids of koi, Cyprinus carpio × crucian carp, Carassius carassius and koi × goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV-3) and developed KHV disease (KHVD). While hybrids of koi × goldfish were partly resistant to mortality following infection by immersion, most koi × crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi × goldfish and koi × crucian carp hybrids as well as in SPF carp. [ABSTRACT FROM AUTHOR]

Immunological methods have been developed for the diagnosis of Myxobolus rotundus but their use has been limited for the prevention and therapy of this serious parasitic pathogen. Phage display antibody libraries are a powerful technique for the development of antibodies to molecules of interest and have advantages over traditional hybridroma approaches. In the present study, four antigen fractions related to M. rotundus were prepared and a combined phage display single-chain antibody fragments (ScFv) library was constructed against this parasite. Preliminary analysis indicated that a combined antibody library of about 2.08 x 105 individual clones and high diversity was generated. After four rounds of screening (bio-panning) against soluble spore protein prepared from lysed, intact, mature M. rotundus spores, a strain monoclonal phage display ScFv, termed pCAN-6H9, with better affinity, was isolated. The pCAN-6H9 gene fragment was sequenced and analysed. The specificity of pCAN-6H9 was further demonstrated by dot-blot. In competition enzyme-linked immunosorbent assay, both the original and enriched phage-displayed ScFv repertoire showed significant inhibition of mouse anti-M. rotundus serum binding to coated antigen, while the inhibition rate of monoclonal pCAN-6H9 phage particles was only 11.83%. [ABSTRACT FROM AUTHOR]

Describes the use of monoclonal antibodies (MAb) in an antigenic study of Myxobolus rotundus, a myxosporean parasite infecting crucian carp. Reaction of MAb with spores; Stage-specificity of antigens; Antigen profile of the parasite.

Studies the occurrence of thelohanellosis caused by Thelohanellus wuhanensis in juvenile allogynogenetic silver crucian carp, Carassius auratus gibelio. Efficacy of fumagillin as a therapeutant; Seasonal changes in the mean abundance of plasmodia of Thelohanellus wuhanensis in juvenile allogynogenetic silver crucian carp.