A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cyprinid Herpesvirus 2 in Gibel Carp ( Carassius auratus gibelio

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Cyprinid herpesvirus 2 (CyHV-2) detection in gibel carp was developed. Following cloning and sequencing of the putative DNA helicase gene of CyHV-2 isolate from China, a set of four specific primers was designed based on the sequence. The MgCl2 concentration and the reaction temperature were optimized to 6 mM, 64°C, respectively. LAMP products were detected by visual inspection of a color change due to addition of SYBR Green I stain. The specificity and sensitivity of the LAMP assay were determined. No cross-reaction was observed with other fish DNA viruses including eel herpesvirus, koi herpesvirus, and Chinese giant salamander iridovirus. The LAMP assay was found to be equally sensitive as nested PCR. A comparative evaluation of 10 fish samples using LAMP and nested PCR assays showed an overall correlation in positive and negative results for CyHV-2. These results indicate that the LAMP assay is simple, sensitive, and specific and has a great potential use for CyHV-2 detection in the laboratory and field

Dietary supplementation of <em>Astragalus</em> fermentation products improves the growth performance, immunological characteristics, and disease resistance of crucian carp (<em>Carassius auratus</em>)

The fermentation products of Astragalus have been acknowledged for their ability to enhance immune functions. This study assessed the impact of incorporating Astragalus, fermented with Lactobacillus plantarum and Bacillus coagulans, on crucian carp's growth, disease resistance, and immunological characteristics. The experimental groups were fed with common feed (C), C + Astragalus (A), A + Lactobacillus plantarum (AL), A + Bacillus coagulans (AB), and AL + Bacillus coagulans (ALB). The fermented products were mixed with common feed at a 1:99 ratio, and crucian carp were fed 2% of their body weight for four weeks, with sampling conducted on days 3, 7, 14, 21, and 28. Disease resistance was evaluated using Aeromonas hydrophila (A. hydrophila) at a concentration of 0.2 mL (1.0×10^7 CFU/mL). The final weights in the AL, AB, and ALB groups significantly increased compared to the C group. The ALB group exhibited elevated serum albumin levels, alkaline phosphatase, intestinal lipase, protease enzyme, C3, and IgM gene expression compared to the C group. At the same time, alanine aminotransferase, aspartate aminotransferase, and glucose contents were significantly reduced. The survival rate significantly increased in all experimental groups after treatment with A. hydrophila. In conclusion, Astragalus products fermented with Lactobacillus plantarum and Bacillus coagulans could effectively improve crucian carp's growth, disease resistance, and immune response

Effects of Dietary <i>Enterococcus faecalis</i> YFI-G720 on the Growth, Immunity, Serum Biochemical, Intestinal Morphology, Intestinal Microbiota, and Disease Resistance of Crucian Carp (<i>Carassius auratus</i>)

Diseases of crucian carp (Carassius auratus) are closely related to intestinal parameters. Enterococcus faecalis has strong colonization ability in the intestinal tract, and produces natural antibiotics, bacteriocin, and other bacteriostatic substances, which can effectively inhibit some pathogenic bacteria and improve the intestinal microenvironment. This study aimed to assess the effects of E. faecalis YFI-G720 which was isolated from the intestinal of crucian carp on the growth, immunity, intestinal health, and disease resistance of crucian carp. Fish (48.16 ± 0.55 g) were fed four diets, commercial diet or diet containing E. faecalis at 105 CFU/g (EF1), 106 CFU/g (EF2), or 107 CFU/g (EF3) for 28 days. The results showed that supplementation of E. faecalis significantly improved the weight gain ratio (WGR) and the specific growth rate (SGR) compared with control group (p p p Aeromonas and Acinetobacter decreased while Cetobacterium increased in all experimental groups, with the greatest changes in EF2. Challenge tests showed that fish fed E. faecalis were more resistant to Aeromonas veronii (p E. faecalis YFI-G720 at 106 CFU/g can improve the health status, immune parameters, intestinal microbiota composition, and disease resistance of crucian carp

Glycosylphosphatidylinositol Mannosyltransferase &#8544; Protects Chinese Giant Salamander, Andrias davidianus, against Iridovirus

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection

Effects of Dietary Enterococcus faecalis YFI-G720 on the Growth, Immunity, Serum Biochemical, Intestinal Morphology, Intestinal Microbiota, and Disease Resistance of Crucian Carp (Carassius auratus)

Diseases of crucian carp (Carassius auratus) are closely related to intestinal parameters. Enterococcus faecalis has strong colonization ability in the intestinal tract, and produces natural antibiotics, bacteriocin, and other bacteriostatic substances, which can effectively inhibit some pathogenic bacteria and improve the intestinal microenvironment. This study aimed to assess the effects of E. faecalis YFI-G720 which was isolated from the intestinal of crucian carp on the growth, immunity, intestinal health, and disease resistance of crucian carp. Fish (48.16 &plusmn; 0.55 g) were fed four diets, commercial diet or diet containing E. faecalis at 105 CFU/g (EF1), 106 CFU/g (EF2), or 107 CFU/g (EF3) for 28 days. The results showed that supplementation of E. faecalis significantly improved the weight gain ratio (WGR) and the specific growth rate (SGR) compared with control group (p &lt; 0.05). Intestinal mucosal epithelial cells in EF2 were intact and normal, but there was obvious vacuolation in CG. Compared with CG, serum C3 and IgM in EF2 were significantly increased at the end of the experiment (p &lt; 0.05), and serum alkaline phosphatase was significantly higher in all experimental groups (p &lt; 0.05). Among studied immune-related genes, expression was detected by qPCR, C3, IgM, and IL-1&beta;were upregulated in all experimental groups to varying degrees from 14 days, with highest expression in EF2 at 28 days. Intestinal microbiota structure analyzed through high-throughput sequencing, and the results showed that the relative abundance of Aeromonas and Acinetobacter decreased while Cetobacterium increased in all experimental groups, with the greatest changes in EF2. Challenge tests showed that fish fed E. faecalis were more resistant to Aeromonas veronii (p &lt; 0.05). In conclusion, dietary E. faecalis YFI-G720 at 106 CFU/g can improve the health status, immune parameters, intestinal microbiota composition, and disease resistance of crucian carp

Four Mx Genes Identified in Andrias davidianus and Characterization of Their Response to Chinese Giant Salamander Iridovirus Infection

Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus&nbsp;Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species

Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5\u27 and 3\u27 termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL