Abstract:Double immunoglobulin interleukin-1 receptor-related molecule (DIGIRR) plays an important role in piscine immune response. To investigate the function of DIGIRR in immune response of large yellow croaker (Larimichthys crocea), the cDNA sequence of digirr was cloned, named Lcdigirr. Transcriptional expression levels of Lcdigirr in different tissues and immune challenged spleen, headkidney, skin, gill, intestine, and liver tissue and LCK cells (a kidney cell line from a large yellow croaker) were determined by real-time fluorescence quantitative reverse transcription PCR (qPCR). Then the recombinant plasmid pTurboGFP-DIGIRR was constructed and transfected into HEK293T cells for subcellular localization analysis. The role of LcDIGIRR in nf-κb activation was determined by transfecting the recombinant plasmid pcDNA3.1-DIGIRR into HEK293T cells using dual luciferase reporter system. Sequence analysis showed that the open reading frame (ORF) of Lcdigirr was 1,575 bp in length, encoding 524 amino acids. The LcDIGIRR protein contained two conserved N-terminal immunoglobin (Ig) domains, a transmembrane region, and a typical C-terminal Toll/IL-1 receptor (TIR) domain. Multiple alignments suggested that DIGIRR was highly conserved among the analyzed species. Phylogenetic analysis showed that LcDIGIRR was clustered with bony fish DIGIRR and closely related to spiny-head croaker (Collichthys lucidus). Transcriptional expression analysis indicated that Lcdigirr was expressed in the examined tissues with the most predominant expression in intestine, followed by liver, head-kidney, and spleen. However, the expression of Lcdigirr in heart was very weak. After challenge with pathogenic Pseudomonas plecoglossicida in vivo and LPS, flagellin and poly (I:C) in vitro, Lcdigirr transcripts in main immune tissues and LCK cells were significantly induced at the early stage after stimulation (P < 0.05). Subcellular localization revealed that LcDIGIRR mainly existed in membrane-cytoplasm. The luciferase activities of NF-κB and MyD88-mediated nf-κb were significantly suppressed by overexpression of LcDIGIRR. These findings indicated that Lcdigirr could negatively regulate nf-κb and MyD88-mediated nf-κb activation. The present study might be helpful for better understanding the function of LcDIGIRR in innate immune signaling transduction of large yellow croaker.

Abstract:In order to investigate the targeting relationship of miR-130c-5p to the potential target gene g in snakehead vesiculovirus (SHVV) infection and its effect on viral replication, the changes of viral gene and protein levels and miR-130c-5p in SHVV were determined in this study by quantitative real-time PCR (qRT-PCR) and Western blot techniques using channel catfish ovary (CCO) as experimental materials. In addition, the target sequence corresponding to miR-130c-5p on the g gene of SHVV was cloned into the plasmid pmirGLO, and the plasmid pmirGLO-G was constructed for dual luciferase reporter assay for target gene verification. The results showed that the expression levels of miR-130c-5p and g genes were significantly up-regulated with the increasing time and dose of SHVV infection. Further experiments showed that co-transfection of miR-130c-5p mimic and pmirGLO-G plasmid significantly inhibited luciferase activity, while transfection of miR-130c-5p inhibitor significantly up-regulated the fluorescence signal of pmirGLO-G reporter vector. In addition, overexpression of miR-130c-5p significantly reduced the mRNA and protein expression of the viral g gene, while inhibition of miR-130c-5p up-regulated the mRNA and protein expression levels of the g gene. The results showed that miR-130c-5p inhibited the proliferation of SHVV by targeting the g gene of SHVV and causing the degradation of G protein. The results of this study provide an important basis for understanding the pathogenic mechanism of microRNA regulation of SHVVV, and provide theoretical support for the development of anti-SHVV vaccines and other drugs.

Abstract:Incubation of Flavobacterium columnare exopolysaccharide (FC-EPS) significantly increases the expression of miR-155 in epithelioma papulosum cyprinid (EPC) cell line, leading to cell apoptosis. In order to study the mechanism of miR-155 in FC-EPS induced apoptosis, RNAi technology was used in this research. It was found that both overexpression of miR-155 and talin-1 knockout could inhibit apoptosis induced by FC-EPS incubation. Then, talin-1 was identified as the target protein of miR-155 by EMSA assay. During the process of apoptosis induced by FC-EPS incubation, the Talin-1 protein was significantly increased, and the activation of apoptosis executioner caspase-3 protein was detected. At the same time, two talin-1 isomers (about 200 ku and 250 ku, respectively) were obtained. Subsequently, the talin-1 gene inserted in plasmid pcDNA3.1 was constructed and transfected into EPC cells, the overexpressed Talin-1 protein delayed and prolonged the process of apoptosis, and two Talin-1 isomers mentioned above were detected. However, apoptosis did not occur when EPC cells were transfected with FC-EPS; on this occasion, the Talin-1 protein in EPC went down at first, then returned to normal level. Hence, in the context of apoptosis induced by FC-EPS incubation, EPC cells reduces apoptosis by regulating the level of mRNA and protein transcription of talin-1 through up-regulation of miR-155 and producing Talin-1 isomers. This paper provides a new idea for effective prevention and treatment of F. columnare diseases.

Abstract:Calcineurin (CN) is a threonine/serine protein phosphatase that plays an important role in the antimicrobial response and immune response in molluscs. In order to investigate the role of CN in the antimicrobial immune response of Hyriopsis cumingii, the α-type catalytic subunit of H. cumingii CN gene (HcCnAα) was cloned by rapid-amplification of cDNA ends (RACE) and its characteristics were analyzed by bioinformatics methods. With the pET30a-HcCnAα expression vector successfully constructed and the mice immunized after protein purification and ultrafiltration, polyclonal antibody with good specificity was obtained. The relative expression of HcCnAα in different tissues were analyzed by Western blot (WB) and quantitative real-time PCR (qPCR) in a fit state or infected by Aeromonas veronii GL2. The results showed that the full length of HcCnAα cDNA was 2 737 bp, including 131 bp of 3′ UTR, 1 154 bp of 5′ UTR, and it encoded a total of 483 amino acids. The sequence contained PP2Ac domains, which are conserved domains of protein phosphatase. WB and qPCR results showed HcCnAα was expressed in all the tissues of H. cumingii. The expression of HcCnAα was highest in adductor, higher in foot, gill, intestines, kidney, and gonad, and the lowest in blood. After infection with A. veronii GL2, the expression of HcCnAα was significantly up-regulated in gills at 3 to 24 h, and also in adductor at 6 h, in hepatopancreas, foot, blood, and mantle at 24 h, but the expression significantly decreased in adductor, gill, mantle, and hepatopancreas at 48 h. This study suggested that the HcCnAα gene of H. cumingii was highly similar to the CnAα of other species with conserved PP2Ac domains; it was expressed in all tissues, and played a role in immune responses after the bacterial infection. This study successfully accomplished the cloning of HcCnAα genes for the first time and the polyclonal antibody was obtained, supporting the involvement of HcCnAα in the immune response after the bacterial infection in H. cumingii, and thus established a solid groundwork for future investigations in immune regulation, disease prevention and control strategies for H. cumingii.

Abstract:Chinese sturgeon (Acipenser sinensis) is a critically endangered fish species found in the Yangtze River of China, for which there is scarce immunological research. Type Ⅰ interferons (IFNs) are widely recognized as pivotal cytokines in the host's antiviral immune response. Therefore, the present study aimed to investigate the immune function of interferon e2 in A. sinensis (rAsIFNe2). The recombinant A. sinensis interferon e2 protein (rAsIFNe2) was expressed through prokaryotic expression, and its effects on antiviral-related genes and its antiviral activity were analyzed. Real-time quantitative PCR (RT-qPCR) results showed that rAsIFNe2 significantly activated the expression of interferon-stimulated genes (ISGs) in A. sinensis fin cells, such as Mx, Viperin, PKR, and ADAR4 with 460.95-fold and 669.01-fold changes at 24 h, a 42.76-fold change at 6 h, and 6.72 fold change at 12 h of incubation, respectively. Furthermore, rAsIFNe2 also helped host cells establish an antiviral state by activating the expression of IRF1, IRF2, IRF3, IRF7, IFNe1, IFNe2 and IFNe3 genes with increases by 11.56, 3.08, 9.72 and 1 083.07 times at 6 h, a 2.15-fold change at 3 h, a 119.15-fold change at 12 h, and a-6.88 fold change at 3 h of incubation, respectively. In the spring viremia of carp virus (SVCV)-infected carp epithelial cells (EPC) model, rAsIFNe2 induced the expression of Mx, PKR, and Viperin in EPC cells with 13.29-fold and 14.36-fold changes at 6 h, and a 19.25-fold change at 24 h of infection. The SVCV virus G, N, and P genes in EPC cells were significantly downregulated by rAsIFNe2 with 388.50, 259.74 and 979.91-fold changes after 24 h, thereby reducing the lesions. These results indicate that AsIFNe2 plays a role in the host's antiviral innate immune response, providing a theoretical basis for understanding the interferon immune system of A. sinensis and treating viral diseases.

Abstract:With the continuous development of Siniperca chuatsi culture technology, the scale of S. chuatsi culture industry in China has been expanding. However, in recent years, outbreaks of S. chuatsi viral diseases have become more frequent, causing serious economic losses to farmers. Among them, S. chuatsi rhabdovirus (SCRV) disease is one of the most common diseases. Viruses are non-cellular organisms that rely on host cell metabolism to complete their replication and proliferation, and viruses induce host cell metabolic reprogramming to rapidly obtain the biomolecules and energy required for viral replication and proliferation, including glycolysis, glutamine metabolism, lipid metabolism, nucleotide biosynthesis and so on. In turn, c-Myc is a key transcription factor that regulates cellular metabolism, and can regulate the expression of glutaminase1 (GLS1), the rate-limiting enzyme of the glutaminase pathway, thereby regulating glutamine metabolism. Moreover, viruses can alter c-Myc protein stability or activity by encoding viral proteins that interact directly or indirectly with c-Myc, thereby regulating cellular metabolism. Therefore, in order to study the molecular mechanism of how SCRV regulates Sc-c-Myc and then regulates glutamine metabolism, the viral protein probably interacting with Sc-c-Myc was identified and analyzed by co-immunoprecipitation (Co-IP) and protein mass spectrometry in this study. SDS-PAGE results showed the specific protein bands of 45-65, 65-75, and 100-130 ku in SCRV-infected cells, and protein profiling showed that one viral protein was identified in the Sc-c-Myc IP sample, which was the nucleoprotein (N) of the SCRV, and the peptide intensity value of the hit was high. The Co-IP results showed that SCRV-N interacted with Sc-c-Myc. The SCRV-N ORF with Flag tag was obtained by PCR, and the pcDNA-N-Flag plasmid was constructed. Then pcDNA-N-Flag plasmids were transfected into Chinese perch brain cells (CPB cells), and fluorescence microscopy observation found that SCRV-N colocalized with Sc-c-Myc in the cytoplasm. Furthermore, quantitative reverse transcription PCR (RT-qPCR) and Western blot were used to detect the expression changes of Sc-c-Myc and key enzymes in the glutamine metabolism pathway (GLS1, GDH, and IDH2) in CPB cells transfected with pcDNA-N-Flag. RT-qPCR results showed that compared with the control, the expression of Sc-c-Myc and GLS1 mRNA was significantly upregulated after transfection with SCRV-N, increasing by 2.1 and 3.1 times, respectively, while the expression changes of GDH and IDH2 mRNA were not significant. Western blot results showed that the expression of Sc-c-Myc and GLS1 protein was significantly increased after transfection with SCRV-N, while the expression changes of GDH and IDH2 were not significant. In summary, it is observed that SCRV interacts with Sc-c-Myc through the N protein, thereby regulating the glutamine metabolism pathway to meet its own replication and proliferation needs. This study aimed to explore how SCRV regulates glutamine metabolism by interacting with Sc-c-Myc, investigating the role of Sc-c-Myc in SCRV-induced glutamine metabolism remodeling. The findings reveal that SCRV interacts with the Sc-c-Myc through the N protein, preliminarily uncovering the molecular mechanism of SCRV-induced glutamine metabolism remodeling. Therefore, it provides a theoretical basis and new insights for understanding the pathogenic mechanism of SCRV and for the prevention and treatment of SCRV.

Abstract:Ostreid herpesvirus 1 (OsHV-1) infection has been linked to mass mortality of cultivated mollusks of different species worldwide. In this study, we established a eukaryotic system for the expression of ORF104 and ORF33 of OsHV-1, and investigated the polymerizing characterization of ORF104 and ORF33. Firstly, the biochemical characteristics, typical domains and 3D structures were analyzed bioinformatically. Secondly, we constructed recombinant plasmids for the two genes, and individually transfected or co-transfected them into Human Embryonic Kidney Cells (HEK293t). Finally, the expression and polymerization of the two target proteins were investigated using Western blot (WB) and electron microscopy. Bioinformatic analysis demonstrated that hydrophilicity of ORF33 and ORF104 were -0.49 and 0.82, making them stable hydrophilic and hydrophobic proteins respectively. For the construction of recombinant plasmids, genes of ORF33 and ORF104 were amplified with specifically designed primers, which generated PCR products of about 1 000 bp and 3 500 bp for ORF33 and ORF104 respectively. The recombinant plasmids were transmitted into HEK293t cells using Lipo8000^TM for expression. The target bands around 140 ku and 35 ku in length were obtained by WB analysis after SDS-PAGE. However, the interaction between the two viral proteins was still undetermined. The ORF104 protein was identified by electron microscopy, which had a diameter of approximately 20 nm. Due to the small diameter of ORF33, it could not be clearly resolved with the background particles under negative staining electron microscopy in the present study. When the products of the two co-transfected plasmids were investigated, the diameter of the particles was between 10 and 30 nm. In summary, we established a method for the expression of OsHV-1 structural proteins using the eukaryotic expression system for the first time. Polymerization of the two proteins individually and together was investigated and characterized. The method described here provides a new approach for further research on the function, interaction of structural proteins, and infection mechanisms of OsHV-1.

Abstract:Lateolabrax maculatus is an important mariculture fish in China. In recent years, fishery losses caused by diseases have become more and more serious. Vibrio anguillarum is a gram-negative pathogenic bacterium widely distributed in seawater and marine organisms, and it is one of the important sources of bacterial diseases in mariculture and can cause serious infections in fish. Hemolysin is an essential component of virulence factor of V. anguillarum, which causes hemorrhagic septicemia in the host. However, very little information is available about hemolysin in L. maculatus. To explore the role of hemolysin in V. anguillarum infection of the fish, 5.0×10⁵ CFU V. anguillarum wild strain or △vah1-vah4-rtxA was immunized by intraperitoneal injection, and the colonization ability of bacteria in L. maculatus, histopathological changes and immune response of L. maculatus after deletion of hemolysis-related genes were evaluated. The results showed that the virulence of V. anguillarum decreased by 8.74 times after the deletion of hemolysis-related genes, and the LD₅₀ of wild strain or △vah1-vah4-rtxA was 2.103×10⁵ CFU/mL and 1.837×10⁶ CFU/mL, respectively. The intestine and gill were the main colonization sites of V. anguillarum, which were seriously damaged after infection. After the deletion of hemolysis-related genes, the colonizing ability of V. anguillarum and the damage degree of intestinal mucosa vacuolation and gill necrosis was reduced. The KEGG pathway enrichment analysis showed that the differentially expressed genes were significantly enriched into hematopoietic cell lineage, antigen processing and presentation, cell adhesion molecules and intestinal immune network for IgA production signal pathways related to immune response. The above results showed that deletion of hemolysis-related genes attenuated the pathogenicity of V. anguillarum to L. maculatus. The results of this study will help to further understand the pathogenic mechanism of V. anguillarum and provide a basis for the development of anti-Vibrio immune enhancer or attenuated vaccine for L. maculatus.

Abstract:The large yellow croaker (Larimichthys crocea) is the marine culture fish most affected by white spot disease, with the highest frequency of C. irritans outbreaks in China. After years of efforts from scholars at home and abroad, many research progresses have been made in C. irritans propagation, pathogen pathology and vaccine research and development, but there are fewer studies on the physiological, biochemical and immune indexes of L. crocea infected with C. irritans. In order to understand the changes in various physiological and immune indicators of L. crocea after C. irritans infection, in this study, we selected 300 experimental fish for artificial C. irritans infection experiments at the peak of hatching of C. irritans cyst. The experimental fish were randomly assigned to three experimental groups and one control group. Blood, liver, spleen, intestines, gills, head kidney, and skin tissues were collected at 0, 12, 24, 48, and 72 h after infection to monitor changes in serum cortisol and corticosterone levels, liver glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level. Transcription patterns of TNF-α, IL-8 and IL-1β in liver, spleen, intestine, gill, head kidney, and skin tissues were detected by quantitative real-time PCR experiments. During the 3 days after infection, the serum cortisol and corticosterone level increased significantly and gradually from 0 h to 72 h after C. irritans infection (P<0.05); the GSH-Px activity in the liver showed a highly significant decreasing trend (P<0.001); the SOD activity in the liver showed an overall upward trend except for a decrease at 48 h (P<0.001); the MDA level in the liver increased sharply within 12 h, and began to decrease after reaching the peak (P<0.05); the expression of TNF-α, IL-8 and IL-1β in various tissues were upregulated to varied degrees, with those in the gills, head kindey, liver, and skin being the most obvious, which indicated these organs may play important roles in the immune response against C. irritans. These results suggested that corticosteroid content and oxidative stress indexes significantly changed after C. irritans infection can reflect the degree of infection in L. crocea, facilitating the optimization of anti-parasite trait assessment. This study contributes to understanding the dynamic physiological, biochemical, and immune changes after C. irritans infection in L. crocea, offering insights for subsequent mechanistic analyses and breeding efforts.

Abstract:In recent years, the diseases of Carassius auratus gibelio have occurred frequently, especially with the decrease of water temperature in winter and the decline of fish immunity, which made the pathogenic bacteria easy to invade the fish and led to the outbreak of fish disease and the decline of culture efficiency. To determine the cause of the sudden death of C. auratus gibelio in Jiangsu during the overwintering period, high-throughput sequencing was used to compare the diversity and structure of the microbial community between healthy samples and diseased samples. The types and characteristics of bacteria in the outbreak of disease were analyzed. The results showed that the abundance of Flavobacterium was the highest in the diseased samples at the genus level. At the species level, the abundance of F. psychrophilum in the diseased samples increased significantly, reaching 63.01% and 61.31% respectively, which was significantly higher than that in the healthy group (1.55%). According to the results of the analysis of microbial community characteristics, the dominant pathogen NJ01 was isolated from the lesions on the body surface of the diseased samples. Through bacterial morphology, physiological and biochemical analysis, and 16S rDNA sequence alignment, the NJ01 strain was identified as F. psychrophilum. After 14 days of artificial infection with the NJ01 strain, the mortality rate of the 1.7×10⁶ and 1.7×10⁷ CFU/mL groups reached 100%, and the symptoms of infection were consistent with those of natural disease. Histopathological observation showed that the muscle cells of the diseased fish were necrotic and the stroma was full of lymphocytes; the large cells in the liver were lytic and necrotic, and the nucleus melted; splenocytes were found with scattered necrosis in the spleen with symptoms of hyperemia; the renal tubular epithelium fell off; there were a lot of lymphocytes in the renal interstitium. The results of the drug sensitivity test showed that the NJ01 strain was sensitive to cefoxitin, cefoperazone, gentamicin, and clarithromycin. The conclusion was that the strain NJ01 was the pathogen for the death of C. auratus gibelio, which could disturb the normal immune and metabolic function, and consequently the pathological phenomena of tissues and organs or death appeared. In this study, the pathogenicity of F. psychrophilum in C. auratus gibelio is reported for the first time, which will provide a reference basis for the study of drug prevention and the pathogenesis of “overwintering mortality syndrome" in conventional freshwater fish.

Abstract:Singapore grouper iridovirus (SGIV), a novel species of Ranavirus, caused more than 90% mortality in larval and juvenile groupers. Up to now, there is still a lack of effective prevention and control strategies for SGIV. Therefore, it is essential to develop convenient diagnostic methods for filed detection of SGIV without special equipment. In this study, to establish a rapid, sensitive and visualized method for the detection of SGIV in clinical samples, specific primers and probes were designed by targeting the SGIV ORF014L, and a recombinase polymerase amplification (RPA) technique combined with lateral flow dipstick (LFD) (RPA-LFD) was developed for the detection of SGIV. The results showed that the RPA reaction specifically detected target fragment of SGIV within 20 min at 40.1 °C with the lowest detection limit of 10² copies/μL. The RPA-LFD reaction at a constant temperature of 42 °C for 8 min was able to visualize the results on the test strips with the lowest detection limit of 10¹ copies/μL, and showed no cross-reaction with other common aquatic pathogens. The coincidence rate of positive test of clinical samples was consistent between RPA-LFD and PCR methods. Both RPA and RPA-LFD could specifically detect SGIV with lower limit than conventional PCR assay. Taken together, RPA-LFD assay developed in the present study provides a convenient, specific, sensitive and visualized method for on-site rapid detection of SGIV without special equipment.

Abstract:This study was conducted to investigate the cause of a disease outbreak in bullfrogs (Rana catesbeiana) at a breeding base in Nanning, Guangxi in May 2022. The dominant bacterial colonies were isolated from the liver, spleen, kidney and intestine tissues of diseased R. catesbeiana, and identified using morphological observation, physiological and biochemical experiments, 16S rRNA sequencing and phylogenetic analysis. To determine the pathogenic mechanism of these dominant bacteria, artificial infection experiments, histopathological observations, drug sensitivity experiments, virulence gene and resistance gene testing were conducted. The results showed that a single dominant strain was isolated from the liver, spleen, kidneys, and intestines of the critically ill R. catesbeiana. The dominant strain isolated from the intestine was named NFCF-02 and identified as Citrobacter freundii. This strain carried six virulence genes, viaB, ompX, ureE, ureD, ureG, and ureF, with a half-lethal concentration of 3.12×10⁶ CFU/mL. The R. catesbeiana infected with C. freundii presented the symptoms of liver changing to yellow and necrosis, kidney congestion and redness, blood streaks on the surface of the stomach, and blood pus in intestines. Histopathological observation revealed degeneration and necrosis of liver cells, with disordered arrangement of liver cords. Meanwhile, swelling and necrosis of renal tubular epithelial cells, severe tubular collapse and necrosis in severe areas, and splenic central vein dilation and congestion with an increase in hemosiderin and pigment cells were also observed. Besides, intestinal mucosal detachment, necrosis and detachment of intestinal epithelial cells, and necrosis of goblet cells were also the symptoms in R. catesbeiana infected with C. freundii. C. freundii was sensitive to neomycin, polymyxin B, cefradine, and doxycycline, while it has developed resistance to 14 antibiotics, including lincomycin, and carries 7 resistance genes such as gyrA and Sul1. In summary, C. freundii has high pathogenicity to bullfrogs, causing pathological damage to multiple organ tissues and even death of the R. catesbeiana. This study is the first to determine the pathogenicity of C. freundii to R. catesbeiana from the perspective of histopathological analysis and virulence factor carrying, which can provide reference for the diagnosis and drug control of C. freundii disease in farmed R. catesbeiana.

Abstract:American bullfrogs (Rana catesbeiana) are a globally distributed invasive anuran species. They are extensively farmed in aquaculture facilities, particularly in countries like China, where they are highly valued as a protein-rich delicacy. In early March 2023, an outbreak of a serious infectious disease occurred in a bullfrog breeding farm in Yubei District, Chongqing, China. To elucidate the cause of the disease and find treatment options, this study isolated predominant bacteria from the liver and intestine tissues of diseased bullfrog tadpole. Morphological observation, physiological and biochemical experiments, 16S rDNA and rpoB sequencing, and phylogenetic analysis were employed to identify the bacteria. To ascertain the pathogenic mechanism of these dominant bacteria, artificial infection experiments, histopathological observations, virulence gene detection, growth characteristic studies, and drug sensitivity testing were conducted. The results showed that a predominant strain isolated from the liver and intestine of the diseased bullfrog tadpoles, LT202303, was identified as A. johnsonii. It harbored a trio of virulence genes, OmpA, Omp34, and OmpTsx, exhibited strong salt tolerance and the ability to survive within a wide pH range, and had a half-lethal concentration of 6.8×10⁶ CFU/mL. Clinical symptoms included liver redness and swelling with large white nodules, and a transparent yellowing of the intestine. Histopathological observations indicated significant inflammation and focal necrosis in bullfrog tadpole liver and intestine. Drug sensitivity testing revealed that the isolated strain LT202303 is a β-lactamase-producing multidrug-resistant bacterium. In summary, A. johnsonii has high pathogenicity to R. catesbeiana tadpoles, causing pathological damage to multiple organ tissues including the liver and intestine, ultimately resulting in diseases or even death of bullfrog tadpoles. This study reports, for the first time, an outbreak of R. catesbeiana tadpole infectious disease caused by A. johnsonii infection and provides theoretical references for the diagnosis and prevention of this disease.

Abstract:Grass carp reovirus (GCRV) causes severe grass carp hemorrhagic disease, which is one of the main diseases that threaten grass carp culture in our country. In order to explore the transmission of grass carp reovirus type Ⅱ (GCRV-Ⅱ), we established the horizontal and vertical transmission models of GCRV-JX02 infection in rare minnow Gobiocypris rarus using molecular biology detection and real-time fluorescent quantitative (RT-qPCR) methods. We compared the transmission efficiency of GCRV-JX02 in different models, and found that immersion infection and co-culture infection could turn the rare minnow into asymptomatic carriers of GCRV-JX02 in horizontal transmission. The positive detection rates were 50% and 80%, while the mortality rates of rare minnow in co-culture infection were 8%. When the asymptomatic rare minnow in the immersion and co-culture groups were treated with heat shock, the mortality rates were 57.14% and 100%, and the overall positive detection rates were 80.95% and 100%. Copy numbers of GCRV-JX02 in the testis and ovary of asymptomatic rare minnow after being injected intraperitoneally of GCRV-JX02 were 3.64×10⁶ and 6.84×10⁶ copies/0.01g. In the vertical transmission experiment, average copy numbers of GCRV-JX02 in ova, unfertilized eggs, fertilized eggs and juvenile fish were 1.98×10³, 1.15×10⁴, 4.75×10³ and 6.74×10⁴ copies. The copy number of GCRV-JX02 in single juvenile fish was significantly higher than that of ovum and fertilized egg. The results indicated that GCRV-JX02 could not only transmit vertically in rare minnow, but also persist continuously with the development of juvenile fish. This study elucidates the transmission efficiency of GCRV-Ⅱ in multiple transmission routes, which is helpful in evaluating the epidemic risk of grass carp hemorrhagic disease and provide an effective animal model for screening drugs to block the transmission of GCRV.

Abstract:The myxosporean parasite Myxobolus honghuensis commonly infects farmed gibel carp (Cauratus auratus gibelio) in China. Heavy infections are associated with pop-eye, inflammation of pharyngeal wall and sever mortality in C. auratus gibelio and Carassius auratus var. red populations. In order to investigate the host range of M. honghuensis causing pharyngeal myxosporidiosis in pond cultured C. gibelio and the genetic differences among different strains, we collected a diverse range samples of C. auratus complex, including C. auratus, C. auratus var. red, C. auratus gibelio, C. auratus gibelio var. pengze, C. auratus gibelio var. qihe and C. auratus gibelio var. jinbei, a hybrid lineage of C. cuvieri (♀) and C. auratus var. red (♂), and a hybrid lineage of Cyprinus carpio (♀) and C. auratus var. red (♂). The infection prevalence of M. honghuensis in samples was determined using 18S rDNA PCR, and new ITS2 sequences of M. honghuensis were obtained through nested PCR (GenBank accession number: OR744899-OR744905). 18S rDNA PCR analysis revealed that all samples detected in this study were infected by M. honghuensis, and asymptomatic infection prevalence ranged from 25.0% to 88.2%. ITS2 sequences analysis showed only six distinct informative loci among the isolates of M. honghuensis from different fish host, and the average genetic distance was 0.003. Additionally, there were seven haplotypes identified among different isolates; H1, H2 and H3 were present only in goldfish and C. auratus var. red, while H5 was widely distributed in the samples of C. auratus gibelio of different breeds and localities. Phylogenetic analysis demonstrated that M. honghuensis isolated from C. auratus complex formed two distinct clades; notably, the strain found in goldfish displayed much closer relationship to M. ampullicapsulatus, which was commonly found on the gill of C. auratus gibelio. In conclusion, the results of this study provide crucial foundational data for understanding the epidemiology for pharyngeal myxosporidiosis in C. auratus gibelio as well as developing prevention and control strategies against the disease.

Abstract:Myxozoans are microscopic metazoan parasites which are recognized as a group of cnidarians that turned their free-living condition to parasitic life about 600 million years ago. There are more than 2 600 species of myxozoans, which mainly infect freshwater and marine fish, with a few infecting amphibians, reptiles, birds and mammals. Myxobolus Butschli 1882, with more than 900 species recorded to date, is the most speciose and widespread genus in Myxozoa, some of which can cause serious fish disease and bring great economic loss in fishery. Myxobolus miyairii Kudo 1919 is a pathogenic parasite of catfish Silurus asotus Linnaeus 1758, which is an economically important fish in China. This parasite was first discovered in Japan and subsequently found in China and Russia, successively. To investigate the strain divergence of M. miyairii from different parasitic sites and localities, the comparative study was carried out based on morphological characters, histotropism, geographic distribution, 18S rDNA sequence similarity, genetic distance and phylogeny. The myxospores of Chongqing strain 1 (infecting gill cavity membrane of S. asotus) of M. miyairii were ellipsoidal in shape with pointed anterior end and blunt posterior end. The myxospores of Chongqing strain 1 were 13.08±0.70 (10.56-13.83) μm in length and 6.09±0.55 (4.94-7.17) μm in width. The two polar capsules positioned at the anterior end of the spores, which were pyriform and equal, measured 4.97±0.39 (4.09-5.87) μm long and 1.34±0.18 (0.98-1.87) μm wide. The morphology of Chongqing strain 2 (infect intestine of S. asotus) was generally consistent with the Chongqing strain 1. The results of principal component analysis and Mann-Whitney U test on morphometry of the two strains further confirmed their morphological consistency. The similarity and genetic distance among Chongqing strain 1, Chongqing strain 2 and Jiangxi strain (infecting intestine of S. asotus) was 98.6%-99.9% and 0.000-0.013, respectively. Phylogenetic analysis showed that the Chongqing strain 2 and Jiangxi strain clustered together as a clade, which was a sister group to Chongqing strain 1. Results from sequence comparison and phylogenetic analysis indicated that M. miyairii did not form monophyletic lineages specific to geographic populations, but rather clustered according to the site of attachment; As far as the same host is concerned, different sites of attachment may have a greater impact on the population divergence of M. miyairii than that by geographic isolation. The comparative study of various strains of M. miyairii and its results are of great significance for people further understanding the evolution characteristics of M. miyairii.

Abstract:To determine the basic structural features of the mitochondrial genome, explore the taxonomic status of Diplozoon paradoxum, and to understand the codon usage bias in Diplozoidae species, the mitochondrial genome sequence of D. paradoxum was obtained by the second generation high-throughput sequencing technologies, annotated by MITOS Web Server, and its structure was analyzed by PhyloSuite (version1.2.2). Then, we analyzed the codon composition and usage bias of mitochondrial protein-coding genes in nine species of Diplozoidae. The results showed that the mitochondrial genome sequence of D. paradoxum was 15,713 bp in length (the large non-coding region was not fully sequenced), and the AT content of D. paradoxum was 68.7%, which had an obvious AT bias. Based on the available data, the results of phylogenetic tree analysis showed that all species of Diplozoidae were grouped into one branch, and revealed that Diplozoidae was a monophyletic group. The analysis of codon usage bias showed that the effective number of codons (ENC) ranged from 30.632 to 37.495 in Diplozoidae. The relative synonymous codon usage (RSCU) values of 18 codons were > 1 in Diplozoidae, and there was a bias for codons ending in U(T). The analysis of PR2-plot showed that the frequency of T was higher than that of A, and the frequency of G was higher than that of C. The codon usage bias might be affected by factors such as mutation and selection. The analysis of neutral mapping showed that the effects of mutation pressure on D. paradoxum and P. opsariichthydis were 61.89% and 51.31%, respectively, while the effects of mutation pressure on other seven species were below 50%. Diplozoidae species had slight differences in codon usage bias, but they were all affected by mutation pressure, natural selection, and base composition. This study can provide an important basis for follow-up phylogenetic studies.

Abstract:To improve the taxonomic characteristics of Myxobolus ampullicapsulatus and clarify its relationship with of M. honghuensis, M. ampullicapsulatus was comprehensively redescribed by morphological, histological and molecular methods, and a systematic comparison was made with molecular marker of M. honghuensis. In the present study, M. ampullicapsulatus was characterized by round or oval, white plasmodia in gills of gibel carp Carassius auratus gibelio, measuring 1.2-1.4 mm in diameter. Myxospores were pear-shaped in front view with pointed anterior end and rounded posterior, measuring 17.3-19.6 μm in length, 7.4-9.9 μm in width. Two ampullaceous polar capsules were unequal in size, with the larger polar capsule measuring 6.4-9.7 μm ×2.1-3.3 μm and the smaller one 5.3-8.9 μm ×2.0-3.3 μm. Polar filament coiled 8-11 turns. Histological analysis demonstrated that M. ampullicapsulatus developed within stratified epithelium between adjacent gill lamellae. A BLAST search conducted in this study revealed a high degree of similarity, ranging from 99.5% to 99.8%, between the obtained small subunit ribosomal DNA (SSU rDNA) sequence and those of M. ampullicapsulatus in GenBank (KC425223-KC425225, MH329620, JQ690361, JQ690373, KJ725082, MN227351, DQ339482). Furthermore, phylogenetic analysis indicated that M. ampullicapsulatus was closely related to M. honghuensis, serving as its sister species. Upon comparing the SSU rDNA sequences of M. ampullicapsulatus and M. honghuensis, it was observed that they shared a sequence similarity of 98.2% to 98.8%, a genetic distance of 0.014 to 0.018, and 27 base differences. The analysis of SSU rRNA sequences revealed that the secondary structures of different populations of M. ampullicapsulatus and M. honghuensis were found to be consistent, and there are obvious differences in the secondary structure between the two species. Moreover, notable differences were observed in the secondary structures of these two Myxobolus species, suggesting that the SSU rRNA secondary structure is expected to serve as a molecular feature to differentiate M. ampullicapsulatus and M. honghuensis. In summary, this study enhances the understanding of the detailed infection site of M. ampullicapsulatus in gills, and proposes that the SSU rRNA secondary structure may be used as an effective molecular marker for distinguishing M. ampullicapsulatus and M. honghuensis.

Abstract:To investigate the bactericidal efficacy of Thymus mongolicus essential oil, Origanum vulgare essential oil and Cinnamomum cassia essential oil on Photobacterium damselae subsp. damselae (PDD), an important pathogenic bacteria in mariculture, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these essential oils against six PDD strains with different pathogenicity were determined through double dilution method. These strains' growth curve after adding plant essential oil was detected using spectrophotometry. The expression of virulence genes and activity of extracellular products (ECP) under different essential oil concentrations in two highly pathogenic PDD strains were analyzed. Meanwhile, the bactericidal efficacy of the plant essential oils after long-time storage and metal ions addition were studied. The results showed that the three plant oils had good bactericidal effects on the six PDD strains, with MICs of 32-128 μg/mL and MBCs of 64-192 μg/mL. They had good inhibitory effects on the expression of virulence genes, phospholipase activity and hemolytic activity of ECP in two highly pathogenic PDD strains. Specifically, low concentration of plant essential oil exhibited significant inhibitory effects on the expression of virulence genes. After 35 days of storage away from light, the sterilization rates of the three essential oils remained above 99%, demonstrating excellent pharmacodynamic stability. The presence of different concentrations of Na⁺, Mg²⁺, Ca²⁺, K⁺ in the water environment did not exert a significant influence on the bactericidal efficacy of the three essential oils. The results showed the suitability of the three essential oils for the development of novel drugs or feed additives in aquaculture, with potential application in disease prevention and treatment. This study can provide reference for expanding the application of aromatic plant essential oil in the prevention and control of aquatic diseases.

Abstract:Aeromonas veronii can infect a variety of aquatic and terrestrial animals and cause motile aeromonad septicemia (MAS).To explore the immune effect of the vaccine prepared with A. veronii low pathogenic strain FS12001, Carassius auratus gibelio were immunized with live vaccine, inactivated vaccine, ISA763A-inactivated vaccine and propolis-inactivated vaccine prepared with FS12001 through intraperitoneal injection, with ISA763A- 0.65% NaCl, propolis- 0.65% NaCl and 0.65% NaCl used as controls. The gene expressions of IgM, IL-1β and LZM were detected by real-time quantitative PCR (qRT-PCR) in spleen tissues at 1, 3, 5, 7 and 14 d post immunization (dpi). Sera were collected from the tail vein at 7, 14, 21, 28, 42 and 48 dpi. The specific antibody IgM levels were detected through indirect ELISA. SOD activity and LZM activity were measured. The experimental groups were challenged with two A.veronii virulent strains at 28 dpi, and the relative percent survival (RPS) was calculated. The gene expressions of IgM, IL-1β and LZM in spleen tissues of each immunized group were higher than those of the 0.65% NaCl control, and the expressions of ISA763A-inactivated vaccine group and propolis-inactivated vaccine group were significantly higher than those of all other groups. The serum-specific antibody levels of each vaccine-immunized group were significantly higher than those of the 0.65% NaCl and adjuvant controls. LZM activity values were highest at 28 dpi in all vaccine-immunized groups, and SOD activity values were the highest at 7 dpi in the inactivated vaccine group, ISA763A-inactivated vaccine group and propolis-inactivated vaccine group. The RPSs of live vaccine group, inactivated vaccine group, ISA763A-inactivated vaccine group and propolis-inactivated vaccine group challenged with AVCA07 were 63%, 56%, 100% and 56%, respectively, and the RPSs of these immune groups challenged with YC170511 were 59%, 55%, 93% and 72%, respectively. This study shows that the several vaccines prepared with A.veronii FS12001 can increase the gene expressions of IgM, IL-1β and LZM, serum specific antibody level, SOD activity and LZM activity, and enhance the ability of C. auratus gibelio to resist A.veronii infection. This study can provide the basis for A.veronii vaccines and prevention and control of MAS.

Abstract:To study the bacteriostatic effect of triple yolk antibody (IgY) against Vibrio harveyi, V. alginolyticus and V. parahaemolyticus in vitro, the triple inactivated vaccine of V. harveyi, V. alginolyticus and V. parahaemolyticus was prepared and the laying hens were immunized. The eggs were collected for the preparation of IgY and their titer, purity and specificity were determined. By immunofluorescence, scanning electron microscopy and in vitro antibacterial experiments, the in vitro antibacterial effect of the prepared triple IgY on three species of Vibrio was preliminarily investigated. The results showed that the ELISA detected a titer of up to 1∶51 200 for the IgY, which it maintained for 5 weeks; SDS-PAGE analysis of the isolated and purified IgY showed that the protein impurity bands in the IgY were gradually reduced, and the purity increased with purification. Immunofluorescence and immunoblotting experiments demonstrated high specificity of binding of the specific IgY to the antigen. Further observation by scanning electron microscopy showed that, compared to Vibrio treated with non-specific IgY, those treated with specific IgY adhered to each other, the surface of the bacterial cells appeared rough, and the cell wall was damaged. In vitro bacterial inhibition assays showed that the specific IgY significantly inhibited Vibrio in a dose-dependent manner. In summary, the triple IgY prepared in this study was able to specifically bind to V. harveyi, V. alginolyticus and V. parahaemolyticus, and it was found through in vitro experiments that the IgY could play an inhibitory role by destroying the integrity of the bacterial cells through the agglutination and adhesion.