Acute hepatopancreatic necrosis disease (AHPND) is a shrimp farming disease, caused by the pathogenic Vibrio parahaemolyticus carrying a plasmid encoding Vp_PirAB-like toxins. Formalin-killed cells ...of V. parahaemolyticus AHPND-causing strain D6 (FKC-VpD6) were used to select Vp_PirAB-like toxin-resistant Litopenaeus vannamei by oral administration. Stomach and hepatopancreas tissues of shrimps that survived for one week were subjected to RNA sequencing. Differentially expressed genes (DEGs) between surviving shrimp, AHPND-infected shrimp, and normal shrimp were identified. The expressions of 10 DEGs were validated by qPCR. Only one gene (a gene homologous to L. vannamei anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R)) was expressed significantly more strongly in the hepatopancreas of surviving shrimp than in the other groups. Significantly higher expression of LvALF AV-R was also observed in shrimp that survived two other trials of FKC-VpD6 selection. Recombinant ALF AV-R bound to LPS, PGN, Gram-negative bacteria, and some Gram-positive bacteria in ELISAs. ALF AV-R recombinant protein did not interact with native Vp_PirAB-like toxin in an ELISA or a Far-Western blot. For L. vannamei orally fed ALF AV-R protein for 3 days, the survival rate following challenge with VpD6-immersion was not significantly different from that of shrimp fed two control diets. These results suggest that LvALF AV-R expression was induced in the hepatopancreas of shrimp in response to the presence of Vp_PirAB-like toxin, although other factors might also be involved in the resistance mechanism.
•Genes associated with Vp_PirAB-like toxin-resistant shrimp were identified by RNA-seq.•LvALF AV-R mRNA was significantly higher in the hepatopancreas of resistant shrimp.•VpAHPND-immersion and FKC of non-VpAHPND did not induced LvALF AV-R.•Recombinant ALF AV-R bound to bacterial proteins, but not Vp_PirAB-like toxin.•Shrimp fed ALF AV-R showed higher survival at 24 hpi with VpAHPND than PBS group.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a ...piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between “full term” (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.
•Transcriptome of WSSV-infected F. merguensis hemocytes were analyzed by RNA-Seq.•The majority of differentially expressed genes upon WSSV infection were categorized as innate immunity and metabolic pathway.•The rate-limiting enzyme of the hexosamine biosynthesis pathway, FmGFAT was a WSSV-responsive gene.•FmGFAT silencing resulted in the delay of shrimp mortality and reduction of viral copy number.•FmGFAT was considered as metabolic genes whose expression was related to WSSV pathogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and ...gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is
gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from
displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and
counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.
The global aquaculture industry has suffered significant losses due to the outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticus. Since the use of antibiotics ...as control agents has not been shown to be effective, an alternative anti-infective regimen, such as phage therapy, has been proposed. Here, we employed high-throughput screening for potential phages from 98 seawater samples and obtained 14 phages exhibiting diverse host specificity patterns against pathogenic VPAHPND strains. Among others, two Chimallinviridae phages, designated Eric and Ariel, exhibited the widest host spectrum against vibrios. In vitro and in vivo studies revealed that a cocktail derived from these two nucleus-forming vibriophages prolonged the bacterial regrowth of various pathogenic VPAHPND strains and reduced shrimp mortality from VPAHPND infection. This research highlights the use of high-throughput phage screening that leads to the formulation of a nucleus-forming phage cocktail applicable for bacterial infection treatment in aquaculture.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers ...to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.
Acute hepatopancreatic necrosis disease (AHPND) is caused by a unique strain of Vibrio parahaemolyticus that has a plasmid harboring virulent genes. A rapid and accurate diagnosis method is necessary ...for surveillance of this infectious disease in aquaculture. In this study, three primer sets (TUMSAT-Vp1, Vp2 and Vp3) were designed based on the plasmid DNA sequence. We examined 98 strains of bacteria isolated from shrimp farms in different areas in Thailand. These included 48 strains of V. parahaemolyticus (AHPND strain), 38 strains of non-AHPND V. parahaemolyticus and 12 strains of non-V. parahaemolyticus. All the AHPND strains were detected by TUMSAT-Vp1, Vp2 and Vp3. However, one non-AHPND strain tested positive for TUMSAT-Vp1 and Vp2. The accuracy of AHPND detection was validated with two other primer publicly available sets (AP1 and AP2). AP1 and AP2 primers gave a few false positives in non-AHPND strains. Only TUMSAT-Vp3 primer detected the AHPND strains examined in this study with 100% accuracy.
The first isolation of Vibrio vulnificus biotype I from disease outbreaks in cultured tiger grouper Epinephelus fuscoguttatus Forsskal, 1775 in southern Thailand has been described. Gross signs of ...diseased fish included dark coloration of body, anorexia, petechial hemorrhages in the skin of the tail and fins and ulceration of the skin. Hemorrhagic septicemia was observed in intestine, body cavity and spleen. The 205-bp amplified DNA fragment of the hemolysin gene (vvhA) was detected from all of bacterial isolates, indicating V. vulnificus. Bacteria showed characteristics of biotype 1, the human clinical isolate, as indicated by their motility, positive results for indole production, ornithine decarboxylation activity, acid production from D-mannitol and growth at 42°C. The human virulence potential of V. vulnificus isolates using three biomarkers, 16S ribosomal ribonucleic acid (rRNA), vvhA gene and virulence-correlated gene (vcg), showed that genotypes of V. vulnificus was the clinical-type with two profiles. The susceptibility of tiger grouper to V. vulnificus isolate was performed by intraperitoneal injection (i.p.) with 106 CFU/mL bacterial suspensions. The experimentally injected tiger grouper had mortalities within 5 days post-injection and developed clinical signs similar to those found in disease outbreak. In a vaccination trial, the tiger grouper vaccinated with formalin inactivated whole-cell vaccine exhibited relative percent survival (RPS) of 68% following homologous isolate challenge. In conclusion, V. vulnificus biotype 1 strain that caused disease in tiger groupers is similar to V. vulnificus pathogenic to humans.
Detailed histopathological analysis (HA) and in situ DNA hybridization assays (ISH) were carried out with adjacent tissue sections from the whiteleg shrimp Penaeus vannamei that had been infected ...with decapod iridescent virus 1 (DIV1) in the laboratory. Results with two ISH methods confirmed those recently published in China indicating that unique, basophilic, cytoplasmic lesions pathognomonic for DIV1 disease were produced in the hematopoietic tissue (HPT). In addition, both methods revealed that the lymphoid organ (LO) was also a prime target for DIV1, although the lesions produced in the LO were not sufficiently unique to be considered pathognomonic for DIV1 infection. However, the HPT lesions of DIV1 often require use of a 100× objective lens to confirm their presence. In contrast, the LO can be quickly located and lesions can be recognized using a 10× objective lens and confirmed with a 40× objective lens. Once confirmed, a diagnostician can switch to the HPT (usually relatively nearby) to search for pathognomonic lesions with 40× and 100× objective lenses. This will speed up histological screening for DIV1 infections. However, the LO is unique to penaeid shrimp, so this approach would not be applicable to the many other species of crustaceans that are susceptible to DIV1 infection.
•Lesions in hematopoietic tissue (HPT) are used for histological diagnosis of DIV1 infection.•Discerning these lesions often requires use of a 100× microscope objective.•We reveal severe DIV1 lesions in the lymphoid organ (LO) visible with a 10× objective.•This can speed up preliminary screening for DIV1 infections in penaeid shrimp.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The effects of the hot water extract from
Sargassum
sp. on the growth performance, immune responses, oxidative stress, and resistance of Asian sea bass (
Lates calcarifer
) to
Streptococcus iniae
...were investigated. Four groups of fish were fed on the basal diet containing the extract at 0, 0.5, 1.0, and 2.0 g kg
−1
diet for 30 days. Thereafter, the fish from each group were divided into equal halves. The first half was injected intraperitoneally with 0.85 % NaCl, and the second half was injected with
S. iniae
(2 × 10
3
cfu fish
−1
). At the end of the 30 days feeding trial, there were no significant differences in final body weight, weight gain, feed conversion ratio, and hepatosomatic index among four groups. Plasma protein, total immunoglobulin (Ig), and lysozyme messenger RNA (mRNA) levels in fish fed on diets containing 1.0 and 2.0 g kg
−1
of
Sargassum
sp. extract, however, became increased in a non-dose-dependent manner. When fish were exposed to the bacteria, at 24 h, there were significantly (
p
< 0.05) higher levels of hematocrit, red blood cell and white blood cell, Ig, and serum lysozyme in fish fed on diet containing 2.0 g kg
−1
of
Sargassum
sp. extract than those of the fish fed the control diet, and the highest survival rate was also observed in this group. In addition, fish receiving the seaweed extract were able to suppress lipid peroxidation especially at 24-h post
S. iniae
challenge. These findings thus suggested that
Sargassum
sp. extract can be used as an immunostimulant in Asian sea bass.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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